Hans-UIrich Bergmeyer (Eds.) - Methods of Enzymatic Analysis-Academic Press (1974) PDF

METHODS
OF ENZYMATIC
ANALYSIS
Edited by
Hans Ulrich Bergmeyer
in collaboration with Karlfried Gawehn
Second English Edition

Translated from the Third German Edition
Volume 4
Verlag Chemie Weinheim

Academic Press, Inc. New York and London
A Subsidiary of Harcourt Brace Jovanovich, Publishers
Translated by D e r m o t H. Williamson, with the editorial assistance of Patricia L u n d
Medical Research Council External Staff
Metabolic Research L a b o r a t o r y
Nuffield D e p a r t m e n t of Clinical Medicine
T h e Radcliffe Infirmary Oxford, E n g l a n d
Editor's note
T h e m e t h o d s published in this b o o k have not been checked experimentally by the editor. Sole responsibility
for the accuracy of the contents of the contributions a n d the literature q u o t e d therein rests with the a u t h o r s .
Readers are therefore requested to direct all enquiries to the a p p r o p r i a t e a u t h o r s ( a u t h o r ' s addresses are
listed on p p . X V I I - X X X I I ) .
Editions of the b o o k to d a t e :
a) G e r m a n editions with the title " M e t h o d e n der enzymatischen A n a l y s e "
1. Auflage 1962, one volume

2. neubearbeitete u n d erweiterte Auflage 1970, two volumes
3. neubearbeitete u n d erweiterte Auflage 1974, two volumes
b) English editions:
1st Edition 1963, one v o l u m e

2nd printing, revised, 1965
3rd printing, 1968
4th printing, 1971
2nd Edition (translated from the Third G e r m a n Edition), 1974, four v o l u m e s
W i t h 263 figures a n d 546 tables.
I S B N : 3-527-25370-X (Set Verlag Chemie)

3-527-25598-2 (Vol. 4 Verlag Chemie)
0-12-091304-6 (Vol. 4 A c a d e m i c Press)
Library of Congress C a t a l o g C a r d N u m b e r : 73-75657
©Verlag Chemie G m b H , Weinheim/Bergstr., 1974
All rights reserved (including those of translation into foreign languages). N o p a r t of this b o o k m a y be
reproduced in any form - by p h o t o p r i n t , microfilm, or any other m e a n s - n o r t r a n s m i t t e d or translated
into a machine language without written permission from the publishers.
Registered n a m e s , t r a d e m a r k s , etc., used in this b o o k , even without specific indication thereof, are not
to be considered unprotected by law.
C o m p o s i t i o n : H e l m u t Becker, 6232 Bad Soden, G e r m a n y

Printed in the United States of A m e r i c a
Preface
T h e first G e r m a n e d i t i o n o f t h i s b o o k a p p e a r e d in 1 9 6 2 , a n d t h e first E n g l i s h e d i t i o n in 1 9 6 3 .
In t h e m e a n t i m e t h e E n g l i s h e d i t i o n h a s b e e n t w i c e r e p r i n t e d in a r e v i s e d f o r m . T h e d e v e l o p
ment o f enzymatic analysis has been so o v e r w h e l m i n g that a completely n e w edition o f " M e t h o d s
o f E n z y m a t i c A n a l y s i s " w a s n e c e s s a r y . S o in 1 9 7 0 a s e c o n d c o m p l e t e l y r e v i s e d a n d e n l a r g e d
G e r m a n e d i t i o n a p p e a r e d . T h e t r a n s l a t i o n w a s s t a r t e d s o o n , b u t it w a s i n t e r r u p t e d b y t h e w o r k
o n a further 3rd G e r m a n e d i t i o n . S o it w a s p o s s i b l e t o edit t w o i d e n t i c a l v e r s i o n s : t h e 3rd
G e r m a n a n d t h e 2 n d E n g l i s h e d i t i o n . B o t h i n c l u d e t h e latest d e v e l o p m e n t s in a n a l y t i c a l b i o
chemistry.
T h e n u m e r o u s n e w e n z y m a t i c m e t h o d s o f a n a l y s i s , t h e a d v a n c e s m a d e in t h e v a r i o u s a s s a y
techniques, the m e t h o d s based o n completely n e w principles, the mechanization o f the labora
t o r y a n d m a n y o t h e r t o p i c s m a d e t h e n e e d for a n e w e d i t i o n o f t h i s b o o k m o r e p r e s s i n g .
A l t h o u g h m a n y o f t h e d e v e l o p m e n t s are still in p r o g r e s s a n d t h e n e w t e c h n i q u e s are j u s t
b e g i n n i n g t o b e a c c e p t e d s o t h a t a c o m p l e t e p i c t u r e c a n n o t b e g i v e n , it w a s d e c i d e d t o b r i n g
out a new edition.
L i k e its p r e d e c e s s o r , t h i s e d i t i o n is d i r e c t e d t o t h e w o r k e r in t h e a n a l y t i c a l l a b o r a t o r y . O n c e
again authors with considerable practical experience have m a d e available their m e t h o d s . T h e
individual contributions therefore contain the important details from a practical standpoint.
T h e division o f the w o r k into four parts has been maintained, b e c a u s e this h a s p r o v e d suc
cessful. T h e c o m p o s i t i o n o f t h e i n d i v i d u a l c h a p t e r s is e s s e n t i a l l y t h e s a m e . T h e p r i n c i p l e s o n
w h i c h t h e m e a s u r e m e n t s are b a s e d c a n b e e a s i l y s e e n i n t h e e q u a t i o n s g i v e n in e a c h c h a p t e r .
A p r o m i n e n t f e a t u r e o f e a c h m e t h o d is t h e p i p e t t i n g p r o t o c o l , w h i c h is p r i n t e d a s a T a b l e w i t h
data o n the " C o n c e n t r a t i o n in assay mixture". T h e accepted abbreviations for e n z y m e s a n d
m e t a b o l i t e s n o w r e q u i r e n o further e x p l a n a t i o n s (a list o f a b b r e v i a t i o n s is t o b e f o u n d o n
p. X X X V ) . I n a d d i t i o n , e v e r y c i t e d e n z y m e is d e f i n e d b y its s y s t e m n a m e a n d n u m b e r o f t h e
Enzyme Commission o f t h e International Union of Biochemistry, R e c o m m e n d a t i o n s 1972. With
a f e w n e c e s s a r y e x c e p t i o n s o n l y International Units are u s e d ( a b b r e v i a t i o n U ; for t h e "Inhibitor
Unit", t h e s y m b o l is I U ) .
M e t h o d s w h i c h have in the m e a n time b e c o m e obsolete or o u t o f date have been o m i t t e d ;
n e w m e t h o d s have t a k e n their place.
A n i n n o v a t i o n is t h e i n f o r m a t i o n o n t h e a s s a y s for e n z y m e a c t i v i t y i n S e c t i o n B " B i o c h e m i c a l
R e a g e n t s " . T h e i n c l u s i o n o f t h e s e is at t h e r e q u e s t o f m a n y b i o c h e m i s t s , w h o h a v e p r e v i o u s l y
l o o k e d in v a i n f o r t h e s e m e t h o d s i n S e c t i o n C .
In spite o f strict e d i t i n g o f t h e i n d i v i d u a l c h a p t e r s t h e w h o l e b o o k is far m o r e t h a n t w i c e the s i z e
o f t h e first e d i t i o n . I n p a r t i c u l a r t h e S e c t i o n o n " E x p e r i m e n t a l T e c h n i q u e s " h a d t o b e e x t e n d e d
c o n s i d e r a b l y . N e w b u t e s s e n t i a l s e c t i o n s h a v e b e e n i n c l u d e d in t h e i n d i v i d u a l c h a p t e r s , t h o u g h
this h a s n o t b e e n w i t h o u t risk, a s for e x a m p l e , t h e i n f o r m a t i o n o n t h e " A c c u r a c y a n d P r e c i s i o n
of M e t h o d " . N o n e o f the authors were aware of the content o f the large chapter reviewing the
" S t a t i s t i c a l A n a l y s i s , C o n t r o l a n d A s s e s s m e n t o f E x p e r i m e n t a l R e s u l t s " . T h a t is w h y t h e
p r e s e n t a t i o n o f t h e d a t a i n t h e i n d i v i d u a l m e t h o d s is n o t u n i f o r m . T h e v a l u e s p r e s e n t e d m o s t l y
c o n c e r n " P r e c i s i o n in t h e S e r i e s " . T h e p r e s e n t a t i o n o f t h e d a t a o n t h e reliability o f e n z y m a t i c
a n a l y t i c a l m e t h o d s in a s t a n d a r d w a y o n l y r e c e n t l y h a s b e c o m e a c c e p t e d as a r o u t i n e .
The b o o k had to be divided into four v o l u m e s and therefore the order o f sections A to D had
t o b e c h a n g e d . T o m a k e it e a s i e r for t h e r e a d e r e a c h v o l u m e c o n t a i n s t h e c o m p l e t e i n d e x , t h e
VI Preface
list o f a u t h o r s a n d a b b r e v i a t i o n s a n d t h e c o m p l e t e list o f c o n t e n t s . It is h o p e d that t h e s e

v o l u m e s will c o n t i n u e t o b e true l a b o r a t o r y b o o k s .
I w i s h t o p a r t i c u l a r l y t h a n k all t h e c o n t r i b u t o r s for their u n d e r s t a n d i n g a n d w i l l i n g c o - o p e r a t i o n
in c o m p l y i n g w i t h the strict l a y o u t o f t h e t e x t . I t h a n k t h e r e v i e w e r s o f t h e first e d i t i o n for t h e
w e l l - m e a n i n g a n d c o n s t r u c t i v e c r i t i c i s m s , w h i c h I h a v e t a k e n n o t e o f in this e d i t i o n . T h a n k s
are a l s o d u e t o m y c o l l e a g u e s for n u m e r o u s d i s c u s s i o n s , t o D r . D . H . W i l l i a m s o n for t h e
e x c e l l e n t t r a n s l a t i o n a n d for m a n y s u g g e s t i o n s d u r i n g this w o r k , t o m y c o - w o r k e r s for their
h e l p a n d t o Verlag C h e m i e for t h e fruitful c o l l a b o r a t i o n . I a m e s p e c i a l l y o b l i g e d t o M r . K.
G a w e h n . W i t h o u t h i s h e l p it w o u l d h a v e b e e n i m p o s s i b l e t o p r o d u c e t h i s E n g l i s h e d i t i o n in
a f o r m i d e n t i c a l t o t h e 3rd G e r m a n e d i t i o n .
Tutzing/Oberbayern (Germany), M a r c h 1974 H a n s Ulrich Bergmeyer.
From the Preface to the 1st Edition

T o d a y e n z y m e s are m u c h m o r e w i d e l y u s e d a s a n a l y t i c a l t o o l s t h a n in t h e p a s t . N e w m e t h o d s
h a v e b e e n w o r k e d o u t for t h e u s e o f t h o s e e n z y m e s w h i c h are n o w a v a i l a b l e in a h i g h state o f
purity, a n d e x i s t i n g t e c h n i q u e s h a v e b e e n i m p r o v e d .
T h i s l a b o r a t o r y m a n u a l c o n t a i n s t h e w o r k i n g d i r e c t i o n s for c a r e f u l l y t e s t e d p r o c e d u r e s .
T h e a n a l y t i c a l m e t h o d s h a v e b e e n c o n t r i b u t e d by a u t h o r s w h o h a v e h a d m a n y y e a r s o f e x
p e r i e n c e in their p a r t i c u l a r field o f s t u d y . C o n s e q u e n t l y , t h e r e a d e r is c e r t a i n t o h a v e reliable
e x p e r i m e n t a l d i r e c t i o n s w h i c h r e p r e s e n t t h e latest a d v a n c e s in t h i s b r a n c h o f s c i e n c e .
A n y t y p e o f l a b o r a t o r y c a n m a k e g o o d u s e o f this b o o k , s i n c e it is d e s i g n e d o n strictly p r a c t i c a l
lines. T h e i n d i v i d u a l c h a p t e r s are a r r a n g e d a c c o r d i n g t o t h e s u b s t a n c e s t o b e d e t e r m i n e d ( n o t
a c c o r d i n g t o t h e e n z y m e s u s e d ) . G r o u p i n g b y s u b s t r a t e s is e m p l o y e d s i n c e t o d a y t h e r e a g e n t s
are c o m m e r c i a l l y a v a i l a b l e ( w i t h t h e e x c e p t i o n o f a f e w s p e c i a l e n z y m e s ) . F o r t h e s e e x c e p t i o n s
a s h o r t r e s u m e o f i s o l a t i o n t e c h n i q u e s is i n c l u d e d . T h e p o s s i b i l i t y o f a t t e m p t i n g t h e p r e p a r a t i o n
o f t h e s e e n z y m e s is t h e n e a s i l y j u d g e d b y t h e reader, b e a r i n g in m i n d t h e facilities a v a i l a b l e t o
him
Tutzing/Oberbayern (Germany), M a r c h 1963 Hans Ulrich Bergmeyer

VI Preface
list o f a u t h o r s a n d a b b r e v i a t i o n s a n d t h e c o m p l e t e list o f c o n t e n t s . It is h o p e d that t h e s e

v o l u m e s will c o n t i n u e t o b e true l a b o r a t o r y b o o k s .
I w i s h t o p a r t i c u l a r l y t h a n k all t h e c o n t r i b u t o r s for their u n d e r s t a n d i n g a n d w i l l i n g c o - o p e r a t i o n
in c o m p l y i n g w i t h the strict l a y o u t o f t h e t e x t . I t h a n k t h e r e v i e w e r s o f t h e first e d i t i o n for t h e
w e l l - m e a n i n g a n d c o n s t r u c t i v e c r i t i c i s m s , w h i c h I h a v e t a k e n n o t e o f in this e d i t i o n . T h a n k s
are a l s o d u e t o m y c o l l e a g u e s for n u m e r o u s d i s c u s s i o n s , t o D r . D . H . W i l l i a m s o n for t h e
e x c e l l e n t t r a n s l a t i o n a n d for m a n y s u g g e s t i o n s d u r i n g this w o r k , t o m y c o - w o r k e r s for their
h e l p a n d t o Verlag C h e m i e for t h e fruitful c o l l a b o r a t i o n . I a m e s p e c i a l l y o b l i g e d t o M r . K.
G a w e h n . W i t h o u t h i s h e l p it w o u l d h a v e b e e n i m p o s s i b l e t o p r o d u c e t h i s E n g l i s h e d i t i o n in
a f o r m i d e n t i c a l t o t h e 3rd G e r m a n e d i t i o n .
Tutzing/Oberbayern (Germany), M a r c h 1974 H a n s Ulrich Bergmeyer.
From the Preface to the 1st Edition

T o d a y e n z y m e s are m u c h m o r e w i d e l y u s e d a s a n a l y t i c a l t o o l s t h a n in t h e p a s t . N e w m e t h o d s
h a v e b e e n w o r k e d o u t for t h e u s e o f t h o s e e n z y m e s w h i c h are n o w a v a i l a b l e in a h i g h state o f
purity, a n d e x i s t i n g t e c h n i q u e s h a v e b e e n i m p r o v e d .
T h i s l a b o r a t o r y m a n u a l c o n t a i n s t h e w o r k i n g d i r e c t i o n s for c a r e f u l l y t e s t e d p r o c e d u r e s .
T h e a n a l y t i c a l m e t h o d s h a v e b e e n c o n t r i b u t e d by a u t h o r s w h o h a v e h a d m a n y y e a r s o f e x
p e r i e n c e in their p a r t i c u l a r field o f s t u d y . C o n s e q u e n t l y , t h e r e a d e r is c e r t a i n t o h a v e reliable
e x p e r i m e n t a l d i r e c t i o n s w h i c h r e p r e s e n t t h e latest a d v a n c e s in t h i s b r a n c h o f s c i e n c e .
A n y t y p e o f l a b o r a t o r y c a n m a k e g o o d u s e o f this b o o k , s i n c e it is d e s i g n e d o n strictly p r a c t i c a l
lines. T h e i n d i v i d u a l c h a p t e r s are a r r a n g e d a c c o r d i n g t o t h e s u b s t a n c e s t o b e d e t e r m i n e d ( n o t
a c c o r d i n g t o t h e e n z y m e s u s e d ) . G r o u p i n g b y s u b s t r a t e s is e m p l o y e d s i n c e t o d a y t h e r e a g e n t s
are c o m m e r c i a l l y a v a i l a b l e ( w i t h t h e e x c e p t i o n o f a f e w s p e c i a l e n z y m e s ) . F o r t h e s e e x c e p t i o n s
a s h o r t r e s u m e o f i s o l a t i o n t e c h n i q u e s is i n c l u d e d . T h e p o s s i b i l i t y o f a t t e m p t i n g t h e p r e p a r a t i o n
o f t h e s e e n z y m e s is t h e n e a s i l y j u d g e d b y t h e reader, b e a r i n g in m i n d t h e facilities a v a i l a b l e t o
him
Tutzing/Oberbayern (Germany), M a r c h 1963 Hans Ulrich Bergmeyer

Contributors
Abeles, Robert H. Ashwell, Gilbert
G r a d u a t e D e p a r t m e n t of Biochemistry N a t i o n a l Institutes of H e a l t h
Brandeis University D e p a r t m e n t of H e a l t h , E d u c a t i o n a n d
Waltham, M a s s a c h u s e t t s 02154, U S A Welfare
p. 2200 Bethesda, M a r y l a n d 20014, U S A
p. 1365, 1368
Aebi, H u g o
A w , Swee E.
Medizinisch-Chemisches Institut der
Universitat Bern D e p a r t m e n t of Biochemistry
Buhlstrasse 28 Faculty of Medicine
CH-3000 Bern, Switzerland p. 673 Singapore 3, Republic of Singapore p. 909
Bachrach, Uriel
A n g g a r d , Erik
D e p a r t m e n t of Bacteriology
D e p a r t m e n t of P h a r m a c o l o g y
The Hebrew University-Hadassah
K a r o l i n s k a Institute
Medical School
S-10401 S t o c k h o l m 60, Sweden
Jerusalem, Israel p. 1740,1744
A n d e r s o n , N o r m a n G. Bassler, K a r l - H e i n z
Molecular A n a t o m y P r o g r a m Physiologisch-Chemisches Institut der
Oakridge National Laboratory Johannes Gutenberg-Universitat
Oakridge, Tennessee 37830, U S A D-6500 M a i n z , G e r m a n y p. 1381
a n d the M o l e c u l a r A n a t o m y Institut,
P . O . Box 17
B a g i n s k i , E u g e n e S.
Oakridge, Tennessee 37830, U S A p. 213
St. J o s e p h Mercy H o s p i t a l
Pontiac, Michigan 48053, U S A p. 876
A p p e l , Walter
Z e n t r a l l a b o r a t o r i u m der Beaucamp, Klaus
St.-Vincentius-Krankenhauser Boehringer M a n n h e i m G m b H
Sudendstrasse 32 Biochemica Werk Tutzing
D-7500 K a r l s r u h e 1, G e r m a n y p. 949, D-8132 Tutzing/Obb., G e r m a n y
950, 954, 958, 964, 967, 978, 986, 1041, 1058 p. 523, 1656
Bechtler, G u n t e r
Aprison, M. H. Eppendorf Geratebau
Section of N e u r o b i o l o g y Netheler & H i n z G m b H
T h e Institute of Psychiatric Research Barkhausenweg 1
I n d i a n a University Medical Center D-2000 H a m b u r g 63, G e r m a n y
Indianapolis, I n d i a n a 46202, U S A p. 1690 p. 611, 733, 758, 1106
XVIII Contributors
Bergmeyer, H a n s Ulrich Boulanger, Paul

Boehringer M a n n h e i m G m b H L a b o r a t o i r e d e Chimie Biologique
Biochemica Werk Tutzing Faculte de Medicine et P h a r m a c i e
D-8132 Tutzing/Obb., G e r m a n y p. 94,95, Lille, F r a n c e p. 1648
103, 121, 131, 158, 221, 308, 417, 425, 523,
557, 574, 579, 590, 613, 624, 727, 735, 739, Brand, Karl
742, 752, 760, 764, 784, 864, 1100, 1149, Max-Planck-Institut
1172, 1176, 1196, 1205, 1222, 1233, 1243, fur Ernahrungsphysiologie
1304, 1323, 1492, 1496, 1506, 1517, 1520, R h e i n l a n d d a m m 201
1528, 1538, 1577, 1643, 1696, 1704, 1772, D-4600 D o r t m u n d , G e r m a n y p. 396,
1786, 1791, 1813, 1919, 1951, 1967, 2008, 399, 710
2078, 2097, 2127, 2132, 2246
Brin, M y r o n
D e p a r t m e n t of Biochemical N u t r i t i o n
Bernt, Erich
Hoffmann-La R o c h e Inc.
Boehringer M a n n h e i m G m b H Nutley, N e w Jersey 07110, U S A p. 703
Biochemica Werk Tutzing
D-8132 Tutzing/Obb., G e r m a n y p. 158, B r o c k , D a v i d J. H .
308, 557, 574, 579, 590, 613, 624, 727, 735,
D e p a r t m e n t of H u m a n Genetics
739, 742, 752, 760, 764, 774, 784, 864, 868,
Western G e n e r a l Hospital
1100, 1172, 1176, 1196, 1201, 1205, 1215,
E d i n b u r g h 4, Scotland p. 1844
1304, 1499, 1506, 1577, 1643, 1696, 1704,
1772, 1890, 1951, 2246
B r o s n a n , J o h n T.
D e p a r t m e n t of Biochemistry
M e m o r i a l University of
Beutler, H a n s - O t t o
Newfoundland,
Boehringer M a n n h e i m G m b H St. J o h n s ,
Biochemica Werk Tutzing Newfoundland, Canada p. 2266
D-8132 Tutzing/Obb., G e r m a n y p. 523
1314, 1708
Brown, David H.
Bevill, R a r d o n D . D e p a r t m e n t of Biological Chemistry

School of Medicine
D e p a r t m e n t of Molecular Biology
Washington University
Albert Einstein College of Medicine
St. Louis, Missouri 63110, U S A
1300 M o r r i s P a r k Avenue
p. 1251, 1257
Bronx, N e w Y o r k 10461, U S A
p. 2209, 2217 B r o w n , Joseph G.
D e p a r t m e n t of P h a r m a c o l o g y
Birchmeier, Heidi School of Medicine
L a b o r a t o i r e Central Washington University
Hopital C a n t o n a l St. Louis, Missouri 63110, U S A p. 1565
CH-1011 L a u s a n n e , Switzerland p. 721
Bucher, T h e o d o r
Institut fur Physiologische Chemie u n d
Bodansky, Oscar Physikalische Biochemie der
Sloan Kettering Institute Universitat M u n c h e n
for Cancer Research Goethestrasse 33
New Y o r k , N . Y. 10021, U S A p. 768 D-8000 M u n i c h , G e r m a n y p. 254
Contributors XIX
Buttner, H a n n e s Dahl, Katharina von

Medizinische H o c h s c h u l e H a n n o v e r Boehringer M a n n h e i m G m b H
Institut fur Klinische Chemie Abt. Stoffwechsel
R o d e r b r u c h s t r a s s e 101 Sandhofer Strasse
D-3000 Hannover-Kleefeld, G e r m a n y D-6800 M a n n h e i m 31, G e r m a n y p. 819
p. 318
Cerioiti, G i o v a n n i Dahlqvist, Arne

L a b o r a t o r i o Centrale di Analisi D e p a r t m e n t of N u t r i t i o n ,
Ospedale Civile di P a d o v a Chemical Center
1-35100 P a d o v a , Italia p. 691 University of L u n d
S-22007 L u n d 7, Sweden p. 916
Chase, James F. A . *
D e p a r t m e n t of Biochemistry Decker, Karl
University of C a m b r i d g e Albert Ludwigs-Universitat F r e i b u r g
Tennis C o u r t R o a d Medizinische F a k u l t a t
C a m b r i d g e , England p. 1758 Biochemisches Institut
Hermann-Herder-Strasse 7
D-7800 F r e i b u r g i. Br., G e r m a n y p. 1127,
Coddington, Alan
1228, 1988, 2001, 2017, 2022, 2172, 2221,
School of Biological Sciences 2225
University of East Anglia
N o r w i c h , N O R 88 C, England
p. 1928, 1932 D u b a c h , Ulrich C.
Medizinische Universitatspoliklinik
C o h e n , P a t r i c i a S. A b t . fur Innere Medizin
Medical L a b o r a t o r y Assoc. Hebelstrasse 1
Birmingham, A l a b a m a 35233, U S A CH-4056 Basel, Switzerland p. 699
p. 793
C o o p e r m a n , Jack M.
Eberhard, A r n o l d
D e p a r t m e n t of Pediatrics Klinisch-Chemisches Institut der
Hematology and Nutrition Laboratories
Rhein.-Westf. Techn. Hochschule
N e w Y o r k Medical College
D-5100 A a c h e n , G e r m a n y p. 1165
N e w Y o r k , N . Y. 10029, U S A p. 1556
Czok, Rudolf Egami, Fujio
Sandoz Forschungsinstitut G m b H Mitsubishi-Kasei Institute

B n m n e r s t r a s s e 59 of Life Sciences
A-1235 Wien-Liesing, Austria 11, M i n a m i o o y a
p. 1424, 1446 Machida-shi,
Tokyo,Japan p. 2260
Dagley, Stanley
University of M i n n e s o t a Eggleston, Leonard V.*
St. Paul, M i n n e s o t a 55101, U S A M e t a b o l i c Research L a b o r a t o r y
p. 1562 Nuffield D e p a r t m e n t of Clinical Medicine
T h e Radcliffe Infirmary
* deceased Oxford, England p. 1308
XX Contributors
Eggstein, Manfred Friebe, Ursula

Medizinische Universitatsklinik (IV) Biochemisches Institut der
Otfried-Muller-Strasse Universitat F r e i b u r g
D-7400 Tubingen, G e r m a n y p. 1825 Hermann-Herder-Strasse 7
D-7800 F r e i b u r g i. Br., G e r m a n y
p. 1935
E i s e n b e r g , jr., F r a n k
Fried, Lygia W.
T h e N a t i o n a l Institutes of Health
Public H e a l t h Service
Creighton University
United States D e p a r t m e n t of Health,
School of Medicine
Education a n d Welfare
O m a h a , N e b r a s k a 68131, U S A
Bethesda, M a r y l a n d 20014, U S A
p. 644, 1945
p. 1337
Fried, Rainer
Fasold, H u g o D e p a r t m e n t of Biochemistry
Creighton University
Institut fur Biochemie der
School of Medicine
J. W. Goethe-Universitat Frankfurt
O m a h a , N e b r a s k a 68131, U S A
Sandhofstrasse
p. 644, 1945
D-6000 F r a n k f u r t / M . - N i e d e r r a d , G e r m a n y
Friedmann, Herbert C.
p. 1625, 1640
T h e University of Chicago
Fishman, William H. D e p a r t m e n t of Biochemistry
School of Medicine Chicago, Illinois 60637, U S A p. 824,
Tufts University 1963, 2179, 2182
Boston, Massachusetts 02111, U S A
p. 929 Fritsch, Wolf-Peter
I. Medizinische Klinik der Universitat
F o a , P i e r o P. Moorenstrasse 5
D e p a r t m e n t of Research D-4000 Diisseldorf 1, G e r m a n y p. 1046
Sinai Hospital
Detroit, Michigan 48235, U S A p. 876 Fritz, H a n s
Institut fur Klinische Chemie u n d
Klinische Biochemie der Universitat
Forster, Edith
N u s s b a u m s t r a s s e 20
II. Med. Universitatsklinik der
D-8000 M u n i c h , G e r m a n y p. 1064
Johannes-Gutenberg-Universitat
Langenbeckstrasse 1
F r o m m , H e r b e r t J.
D-6500 M a i n z , G e r m a n y p. 1923
Iowa State University of Science
a n d Technology
Forster, G e o r g D e p a r t m e n t of Biochemistry a n d
Schweizerische Pflegerinnenschule Biophysics
Samariterstrasse 5 A m e s , Iowa 50010, U S A p. 1354
CH-8032 Zurich, Switzerland p. 784
Gale, Ernest F.
University of C a m b r i d g e
Frei, J o r g D e p a r t m e n t of Biochemistry
L a b o r a t o i r e Central Sub.-Dept. of Chemical Microbiology
Hopital C a n t o n a l C a m b r i d g e , CB 2 1 Q W , England
CH-1011 L a u s a n n e , Switzerland p. 721 p. 1662
Contributors XXI
Garland, Peter Bryan Goldberg, Nelson D .
D e p a r t m e n t of Biochemistry D e p a r t m e n t of P h a r m a c o l o g y
University of D u n d e e , University of M i n n e s o t a
D u n d e e , Scotland p. 1981,1993, 2015 Medical School
Minneapolis, M i n n e s o t a 55455, U S A
p. 1573, 1600, 1608
G a w e h n , Karlfried
Boehringer M a n n h e i m G m b H G r a h a m , Jr., L. T .
Biochemica Werk Tutzing Institute of Psychiatric Research
D-8132 Tutzing/Obb., G e r m a n y p. 158, I n d i a n a University Medical Center
425, 1263, 1492, 1496, 2172, 2234, 2239 Indianapolis, I n d i a n a 46207, U S A
p. 1690
Gerlach, Ulrich Grassl, Marianne

Medizinische Klinik u n d Poliklinik der Boehringer M a n n h e i m G m b H
Westfalischen Wilhelms-Universitat Biochemica Werk Tutzing
D-4400 Minister/Westf., G e r m a n y D-8132 Tutzing/Obb., G e r m a n y p. 308,
p. 31, 569, 871 425, 1268, 1296, 1331, 1682, 1686, 2073,
2145, 2149, 2153, 2158, 2162, 2166, 2168
Giang, Paul A.
Analytical Chemistry L a b o r a t o r y
Greenberg, Elaine
Agr. E n v i r o n m e n t a l Quality Institute
University of California
Beltsville, M a r y l a n d 20705, U S A
p. 2249
a n d Biophysics
Gibbs, Martin Davis, California 95616, U S A p. 2204
D e p a r t m e n t of Biology
Brandeis University Greiling, H e l m u t
Waltham, M a s s a c h u s e t t s 02154, U S A
Klinisch-Chemisches Institut der
p. 409, 881, 1385
Rhein.-Westf. Techn. H o c h s c h u l e
Gitzelmann, Richard D-5100 Aachen, G e r m a n y p. 1157,1165
L a b o r fur Stoffwechselforschung der
Universitats-Kinderklinik Gruber, Wolfgang
Steinwiesstrasse 75 Boehringer M a n n h e i m G m b H
CH-8032 Zurich, Switzerland p. 1291 Biochemica Werk Tutzing
D-8132 Tutzing, O b b . , G e r m a n y p. 1323,
Giusti, Guiseppe 1890, 2078, 2097, 2127
Clinica Delle M a l a t t i e Infetive

dell'Universita Gundlach, Gerd
I ' F a c o l t a di Medicina e Chirurgia Universitatsklinik, Urologie
Via D . C o t u g n o , 1 ( O s p . G e s u e M a r i a ) Landeskrankenhaus
1-80135 N a p o l i , Italia p. 1086,1092 D-6650 H o m b u r g / S a a r , G e r m a n y
p. 1625
Goedde, Heinz W. Gutmann, Ingeborg

Institut fur H u m a n g e n e t i k der Boehringer M a n n h e i m G m b H
Universitat Biochemica Werk Tutzing
Butenfeld 32 D-8132 Tutzing/Obb., G e r m a n y p. 774,
D-2000 H a m b u r g 54, G e r m a n y 1149, 1172, 1185, 1323, 1464, 1499, 1517,
p. 1394, 1514 1585, 1791
XXII Contributors
Hagen, Alexander Heinz, Fritz

Boehringer M a n n h e i m G m b H Medizinische H o c h s c h u l e H a n n o v e r
Biochemica Werk Tutzing Institut fur Klinische Biochemie
D-8132 Tutzing/Obb., G e r m a n y p. 283 u n d Physiologische Chemie
R o d e r b r u c h s t r a s s e 101
D-3000 H a n n o v e r , G e r m a n y p. 1777
H a i d , Erich
Boehringer M a n n h e i m G m b H
Biochemica Werk Tutzing Hess, Benno
M a x - P l a n c k - I n s t i t u t fur
Ernahrungsphysiologie
R h e i n l a n d d a m m 201
Haindl, Hans D-4600 D o r t m u n d , G e r m a n y
Institut fur Klinische Biochemie p. 3, 396, 399, 778
u n d Physiologische Chemie
der Medizinischen Hochschule
Heuckenkamp, Peter-Uwe
Osterfeldstrasse 5
D-3000 H a n n o v e r , G e r m a n y p. 1886 Medizinische Poliklinik der
Universitat M u n c h e n
Pettenkoferstrasse 8 a
Halliwell, Geoffrey D-8000 M u n i c h 2, G e r m a n y p. 1288
S u b - D e p a r t m e n t of Microbiology
D e p a r t m e n t of Botany
University College of Swansea H i b y , Walter
Swansea, Wales, England p. 1132, 1143 Medizinische Klinik u n d Poliklinik der
Westfalischen Wilhelms-Universitat
Westring 3
Hansert, Erwin D-4400 Minister/Westf., G e r m a n y
Abteilung fur Biostatistik p. 569, 871
Max-Planck-Institut fur Psychiatrie
Kraepelinstrasse 10
Hildebrand, John G.
D-8000 M u n i c h 40, G e r m a n y p. 318
D e p a r t m e n t of N e u r o b i o l o g y
H a r v a r d Medical School
25 S h a t t u c k Street
Hasegawa, Shin
Boston, M a s s a c h u s e t t s 02115, USA
Fruit & Vegetable Chemistry-
p. 1819
Laboratory
U S D e p a r t m e n t of Agriculture
Hillmann, Giinther
263 South Chester Avenue
Pasadena, California 91106, U S A Chemisches Institut der
p. 1299 Stadtischen K r a n k e n a n s t a l t e n
D-8500 N u r n b e r g , G e r m a n y p. 903
Hazen, George G.
Merck, Sharp & D o h m e
Research L a b o r a t o r i e s Hjelm, Magnus
Division of M e r c k & C o . , Inc. D e p t . of Clinical Chemistry University
R a h w a y , N e w Jersey 07065, U S A Hospital
p. WOO S-75014 U p p s a l a 14, Sweden p. 1282
Contributors XXIII
H o b b s , J o h n R. Holzer, Helmut
D e p a r t m e n t of Chemical P a t h o l o g y Biochemisches Institut der
Westminster Medical School, Universitat F r e i b u r g
17, Page Street, Hermann-Herder-Strasse 7
L o n d o n , S. W. 1, E n g l a n d p. 909 D-7800 F r e i b u r g i. Br., G e r m a n y p. 1419
Hochella, N o r m a n Joseph H o r e c k e r , B e r n h a r d L.
The University of N o r t h C a r o l i n a R o c h e Institute of Molecular Biology
T h e School of Medicine Nutley, N e w Jersey 07110, U S A
D e p a r t m e n t of Medicine p. 1193,1350, 1371
Chapel Hill, N o r t h C a r o l i n a 27514, U S A
p. 1479
Horikoshi, Koki
T h e Institute of Physical a n d
Hopner, Thomas Chemical Research
Universitat O l d e n b u r g D e p a r t m e n t of Microbiology
Fachbereich Naturwissenschaften Wako-shi, S a i t a m a Pref., J a p a n p. 1271
Postfach 243
D-2900 O l d e n b u r g , G e r m a n y p. 1551
Hurlbert, R o n a l d E.
D e p a r t m e n t of Bacteriology a n d
Public H e a l t h
Hofner, Helmut
Washington State University
Institut fur Physiologische C h e m i e
Pullman, Washington 99163, U S A
u n d Physikalische Biochemie der
p. 1397
Goethestrasse 33
Hutzler, Joel
D e p a r t m e n t of Pediatrics
N e w Y o r k University School of Medicine
550 First A v e n u e
Holldorf, A u g u s t W.
N e w Y o r k , N . Y. 10016, U S A p. 1669
Institut fur Physiologische Chemie
Ruhr-Universitat Bochum
D-4630 B o c h u m , G e r m a n y p. 1419
1457, 1916, 1923, 1935 I s s e l b a c h e r , K u r t J.
Massachusetts G e n e r a l H o s p i t a l
Boston, M a s s a c h u s e t t s 02114, U S A
p. 802
H o l z , Gtinter
Biochemica Werk Tutzing Jagow-Westermann, Barbara v o n
D-8132 T u t z i n g / O b b . , G e r m a n y Gotthelfstrasse 97
p. 87, 1528, 1786 D-8000 M u n i c h 27, G e r m a n y p. 1483
XXIV Contributors
Jakoby, William B. K e a r n e y , E d n a B.
Section on Enzymes University of California
N a t i o n a l Institute of Arthritis San F r a n c i s c o Medical Centre
a n d M e t a b o l i c Diseases School of Medicine
N a t i o n a l Institutes of H e a l t h D e p a r t m e n t of P h a r m a c o l o g y
Bethesda, M a r y l a n d 20014, U S A San F r a n c i s c o , California 94122, U S A
p. 1346. 1397, 1542, 1622 p. 1802
Keppler, Dietrich
Jaworek, Dieter Biochemisches Institut der
Boehringer M a n n h e i m G m b H Universitat F r e i b u r g
Biochemica Werk Tutzing Hermann-Herder-Strasse 7
D-8132 Tutzing/Obb., G e r m a n y D-7800 F r e i b u r g i. Br., G e r m a n y p. 1127,
p. 2097, 2127 1228, 2088, 2172, 2221, 2225
Jones, Mary Ellen King, John
D e p a r t m e n t of Biochemistry R o y a l Infirmary
School of Medicine D e p a r t m e n t of Biochemistry
T h e University of S o u t h e r n California Glasgow C 4, Scotland p. 607,
2025 Z o n a l A v e n u e 627, 632, 656, 798, 1113
L o s Angeles, California 90033, U S A
p. 1749
Klein, Bernard
D e p a r t m e n t of Diagnostic Research
J0rgensen, S0ren Hoffmann-La R o c h e Inc.
Anaestesiologisk afdeling Nutley, N e w Jersey 07110, U S A p. 582
Odense A m t s og Bys Sygehus
Odense, D e n m a r k p. 1941 Klingenberg, Martin
Institut fur Physiologische Chemie der
Kaiser, Wolfram Goethestrasse 33
Medizinische Poliklinik D-8000 M u n i c h 15, G e r m a n y p. 2045
der Universitat M u n c h e n
Pettenkoferstrasse 8 a Klose, Siegmar
D-8000 M u n i c h 12, G e r m a n y p. 1151 Boehringer M a n n h e i m G m b H
Kaltwasser, Heinrich
Mikrobiologie K l o t z s c h , H e l m u t R.
Universitat Saarbriicken Boehringer M a n n h e i m C o r p .
D-6600 Saarbriicken, G e r m a n y p. 1081 219 East 44th Street
N e w York, N . Y. 10017, U S A p. 557
Kattermann, Reinhard Klungs^yr, Leiv

Abteilung Klinische Chemie D e p a r t m e n t of Physiology
Medizinische Klinik u n d Poliklinik der University of Bergen
Universitat G o t t i n g e n Aarstadveien 19
D-3400 G o t t i n g e n , G e r m a n y p. 1419 N-5000 Bergen, N o r w a y p. 1275
Contributors XXV
Knappe, Joachim Kusche, Jurgen

Universitat Heidelberg Institut fur Klinische Chemie u n d
Institut fur Biologische C h e m i e Klinische Biochemie der
Berliner Strasse 23, Universitat M u n c h e n
D-6900 Heidelberg, G e r m a n y N u B b a u m s t r a s s e 20
p. 1551, 2026 D-8000 M u n i c h 2, G e r m a n y p. 660
Kohn, Leonard D .
L a b o r a t o r y of Biochemical P h a r m a c o l o g y Lachenicht, Rudolf
N a t i o n a l Institute of Arthritis, Boehringer M a n n h e i m G m b H
M e t a b o l i s m u n d Digestive Diseases A b t . A u s b i l d u n g u n d Training
N a t i o n a l Institutes of H e a l t h Sandhofer Strasse,
Bethesda, M a r y l a n d 20014, U S A D-6800 M a n n h e i m 3 1 , G e r m a n y
p. 1397 p. 864, 1201, 1215
Koss, Friedrich-Wilhelm
Lamprecht, Walther
Institut fur Klinische Biochemie u n d
Medizinische H o c h s c h u l e H a n n o v e r
Physiologische C h e m i e der
Institut fur Klinische Biochemie u n d
Medizinischen H o c h s c h u l e
Physiologische Chemie
R o d e r b r u c h s t r a s s e 101
Karl-Wiechert-Allee 9
D-3000 Hannover-Kleefeld, G e r m a n y
p. 1446, 1777, 2101
Krakow, Gladys
St. Joseph's H o s p i t a l Lang, Gunter
Milwaukee, Wisconsin 53210, U S A Boehringer M a n n h e i m G m b H
p. 1963 Biochemica Werk Tutzing
D-8132 Tutzing/Obb., G e r m a n y
Kuhlmann, Elisabeth
p. 1238, 1415
Medizinische Universitatsklinik
Olfried-Muller-Strasse
Langenbeck, Ulrich
D-7400 Tubingen, G e r m a n y p. 1825
Institut fur H u m a n g e n e t i k der
Universitat
K u n , Ernest N i k o l a u s b e r g e r Weg 5 a
University of California D-3400 G o t t i n g e n , G e r m a n y
San Francisco Medical Center p. 1394, 1514
School of Medicine
D e p t . of P h a r m a c o l o g y Latzko, Erwin
a n d Biochemistry
Chemisches Institut
San Francisco, California 94122, U S A
Technische Universitat M u n c h e n
p. 1460, 1802
D-8050 Freising-Weihenstephan, G e r m a n y
Kurz, Gerhart p. 82, 409, 881, 1385
Lehrstuhl Biochemie
Chemisches L a b o r a t o r i u m der Laudahn, Gerhard
Universitat Schering A G
D-7800 F r e i b u r g i. Br., G e r m a n y Miillerstrasse 1 7 0 - 1 7 2
p. 1180, 1279 D-1000 Berlin 65, G e r m a n y p. 37
XXVI Contributors
Leuthardt, Franz Lowry, Oliver H.

Biochemisches Institut der Washington University
Universitat School of Medicine
Zurichbergstrasse 4 D e p a r t m e n t of P h a r m a c o l o g y
CH-8032 Zurich, Switzerland 1109 St. Louis, Missouri 63110, U S A
p. 135, 1452, 2059
Levine, Jacob B. Luhby, A. Leonard

Biochemistry
D e p a r t m e n t of Pediatrics
Technicon C o r p o r a t i o n Hematology and Nutrition Laboratories
511 Benedict A v e n u e N e w York Medical College
Tarrytown, N e w York 10591, U S A N e w York, N . Y. 10029, U S A p. 1556
p. 851
Linker, Alfred
Lund, Patricia
D e p a r t m e n t of Biological Chemistry
M e t a b o l i c Research L a b o r a t o r y
University of U t a h
Nuffield D e p t . of Clinical Medicine
College of Medicine
T h e Radcliffe Infirmary
Salt Lake City, U t a h 84112, U S A
Oxford, E n g l a n d p. 1719
p. 944
LonTer, G e o r g
Institut fur Diabetesforschung Lundquist, Frank
Stadt. K r a n k e n h a u s Schwabing D e p a r t m e n t of Biochemistry
Kolner Platz University of C o p e n h a g e n
D-8000 M u n i c h 23, G e r m a n y 2100 C o p e n h a g e n , D e n m a r k
p. 228,1611 p. 1509, 1532
Lohr, Georg Wilhelm Lynen, Feodor

Medizinische Universitatsklinik Max-Planck-Institut fur Biochemie
Hugstetter Strasse 55 D-8033 Martinsried near M u n i c h , G e r m a n y
D-7800 F r e i b u r g i. Br., G e r m a n y p. 636 „ 2034
Mattenheimer, Hermann
Loschenkohl, Karin
D e p a r t m e n t of Biochemistry,
Institut fur Klinische Chemie u n d
Rush-Presbyterian-St. L u k e ' s Medical
Klinische Biochemie der
Center,
1753 Congress P a r k w a y
NuBbaumstrasse 20
Chicago, Illinois 60612, U S A p. 62
D-8000 M u n i c h 2, G e r m a n y
p. 1731, 1736
Lorenz, Wilfried Matthaei, Heinrich

Institut fur Klinische Chemie u n d Arbeitsgruppe Biochemie
Klinische Biochemie der Max-Planck-Institut fur
Universitat M u n c h e n experimentelle Medizin
NuBbaumstrasse 20 Hermann-Rein-Strasse 3
D-8000 M u n i c h 2, G e r m a n y p. 660 D-3400 G o t t i n g e n , G e r m a n y p. 1901
Contributors XXVII
Maurer, Claus Nagel, Charles W.

Chirurgische Universitatsklinik Washington State University
Klinisch-Chemische Abteilung F o o d Science P r o g r a m
Kirschnerstrasse 1 D e p a r t m e n t of H o r t i c u l t u r e
D-6900 Heidelberg, G e r m a n y p. 1472 P u l l m a n , Washington 99163, U S A
p. 1299
Narins, Robert G.
Mayer, Dieter
University of Pennsylvania
Institut fur Klinische Biochemie
D e p a r t m e n t of Medicine
u n d Physiologische Chemie der
Philadelphia, Pennsylvania 19104, U S A
Medizinischen H o c h s c h u l e
p. 1580
D-3000 H a n n o v e r , G e r m a n y p. 1886 Negelein, Erwin
Lindenberger Weg 74
1115 Berlin-Buch, D D R p. 1429
Mecke, Dieter
Biochemisches Institut der
Netheler, Heinrich G.
Universitat F r e i b u r g
Eppendorf Geratebau
D-7800 F r e i b u r g i. Br., G e r m a n y p. 1716 Netheler & Hinz G m b H
Barkhausenweg 1
D-2000 H a m b u r g 63, G e r m a n y p. 181,
Mellanby, Jane 184, 191, 193, 202, 203, 205
D e p a r t m e n t of Experimental Psychology
South P a r k s R o a d N e w s h o l m e , Eric A .
Oxford, England p. 1836, 1840
D e p a r t m e n t of Biochemistry,
South Parks Road
Oxford, England p. 283,1409, 2144
Michal, Gerhard
Biochemica Werk Tutzing Noll, Franz
D-8132 Tutzing/Obb., G e r m a n y p. 136, A k a d e m i e der Wissenschaften der D D R
144, 158, 308, 1233, 1238, 1314, 1415, 1433, Zentralinstitut fur Molekularbiologie
1708, 1967,2008, 2136 Abteilung Zellphysiologie
1115 Berlin-Buch, D D R p. 1475
Mollering, Hans Ohlenbusch, Hans-Dieter

Boehringer M a n n h e i m G m b H Abteilung Physiologische Chemie der
Biochemica Werk Tutzing Medizinischen F a k u l t a t a n der
D-8132 Tutzing/Obb., G e r m a n y p. 136 Rhein.-Westf. Technischen Hochschule
1222, 1243, 1520, 1538, 1589, 1696, 1772, D-5100 A a c h e n , G e r m a n y p. 923
1813, 1919, 1959, 2073, 2078, 2132
Osteux, Roger*
L a b o r a t o i r e de Biochemie
Naher, Gotthilf
Pharmaceutique
Faculte de Medecine et P h a r m a c i e
Lille, F r a n c e p. 1648
D-8132 Tutzing/Obb., G e r m a n y
p. 814, 1909 * deceased
XXVIII Contributors
Otto, Peter Preiss, Jack

Medizinische Hochschule H a n n o v e r University of California
Medizinische Klinik D e p a r t m e n t of Biochemistry
Abteilung fur Gastroenterologie a n d Biophysics
Podbielskistrasse 380 Davis, California 95616, U S A
D-3000 H a n n o v e r , G e r m a n y p. 50 />. 2204, 2213
Putter, J o h a n n
O u d h e u s d e n , A n t o n i u s P. M . v a n
Farbenfabriken Bayer A . G .
Sint Josef Ziekenhuis D-5600 Wuppertal-Elberfeld, G e r m a n y
Slingelaan 1 p. 685
Doetinchem, N e t h e r l a n d s p. 971
R a b i n o w i t z , Jesse C.
Packmann, Paul M. University of California

D e p a r t m e n t of P h a r m a c o l o g y Psychiatry
Berkeley, California 94720, U S A
Washington University
p. 1546, 2110
School of Medicine
St. Louis, Missouri 63110, U S A p. 1346
Racker, Efraim
Cornell University
Passonneau, Janet V. Division of Biological Sciences
Section on Cellular N e u r o c h e m i s t r y Section of Biochemistry arid Molecular
Bethesda, M a r y l a n d 20014, U S A p. 135, Biology
1452, 1468, 1565, 1573, 1580, 1600,1608, Ithaca, N e w York 14850, U S A p. 1189,
2059, 2229 1320, 1342, 1359, 1362, 1377, 1391, 1439
P e a r s o n , D a v i d J.
R a u s c h e r , Elli
University of C a m b r i d g e
C a m b r i d g e , England p. 1758
Pfleiderer, G e r h a r d
Rick, Wirnt
Ruhr-Universitat B o c h u m
Abteilung fur Chemie I. Medizinische Klinik der Universitat
D-4630 Bochum/Westf., G e r m a n y Moorenstrasse 5
p. 1696 D-4000 D u s s e l d o r f 1, G e r m a n y />. 824,
885, 1006, 1013, 1046, 1864
Pilz, W o l f g a n g
Institut fur klinische Chemie
u n d analytische Chemie der arztlichen Rimbach, Erwin
Abteilung
Universitats-Frauenklinik
D-5090 Leverkusen-Bayerwerk, G e r m a n y
D-7400 T u b i n g e n , G e r m a n y p. 56
p. 806, 831
Poppendiek, Brunhilde
Chirurgische Universitatsklinik R o s c h l a u , Peter
Klinisch-Chemische Abteilung Boehringer M a n n h e i m G m b H
Kirschnerstrasse 1 Biochemica Werk Tutzing
D-6900 Heidelberg, G e r m a n y p. 1472 D-8132 T u t z i n g / O b b . , G e r m a n y p. 1890
Contributors XXIX
Rosano, Carmen Louis Schmid, Ella

Basic Science Research L a b o r a t o r y Laboratoire Central
Veterans A d m i n i s t r a t i o n H o s p i t a l Hopital Cantonal
Albany, N e w York 12208, U S A p. 1723 CH-1011 L a u s a n n e , Switzerland p. 721
Rouayrenc, Jean-Francois
Schmidt, Ellen
Institut fur Physiologische C h e m i e
u n d Physikalische Biochemie der
Medizinische Klinik
Abteilung fur Gastroenterologie
Goethestrasse 33
D-3000 Hannover-Kleefeld, G e r m a n y
p. 6, 14, 650
Samuelsson, Bengt
D e p a r t m e n t of Chemistry Schmidt, Felix H.
K a r o l i n s k a Institutet Boehringer M a n n h e i m G m b H
S-10401 S t o c k h o l m 60, Sweden p. 1877 Abteilung Stoffwechsel
Sandhofer Strasse
Schaiberger, G e o r g e E. D-6800 M a n n h e i m 3 1 , G e r m a n y
p. 819,1196
D e p a r t m e n t of Microbiology
University of M i a m i School of Medicine
Schmidt, Friedrich W.
P. O. Box 875, Biscayne A n n e x
M i a m i , F l o r i d a 33152, U S A p. 1701
Medizinische Klinik
Abteilung fur G a s t r o e n t e r o l o g i e
Scheibe, Peter
Podbielskistrasse 380
Boehringer M a n n h e i m G m b H D-3000 H a n n o v e r , G e r m a n y p. 6, 14
Schmidt, Helmuth
Scher, William II. Medizinische Universitatsklinik
Center for E x p e r i m e n t a l Cell Biology Martinistrasse 52
M o u n t Sinai School of Medicine D-2000 H a m b u r g 20, G e r m a n y p. 1848
Fifth A v e n u e a n d 100th Street
N e w York, N . Y. 10029, U S A p. 1622
Schoner, Wilhelm
Schievelbein, H e l m u t Institut fur Biochemie

und Endokrinologie
Institut fur Klinische Chemie
Universitat Giessen
u n d Klinische Biochemie der
Universitat M u n c h e n F r a n k f u r t e r Strasse 110
N u B b a u m s t r a s s e 20 D-6300 Giessen, G e r m a n y p. 1596, 1994
D-8000 M u n i c h 2, G e r m a n y p. 1731, 1736
Schormiiller, Josef
Schlegel, Hans-Giinter Technische Universitat Berlin
Institut fur M i k r o b i o l o g i e Institut fur Lebensmittelchemie
der Universitat G o t t i n g e n u n d Lebensmitteltechnologie
Grisebachstrasse 8 Strasse des 17. J u n i 135
D-3400 G o t t i n g e n , G e r m a n y p. 1081 D-1000 Berlin 12, G e r m a n y p. 71
XXX Contributors
Schreiber, Gerhard Staib, Wolfgang

Biochemisches Institut Physiologisch-Chemisches Institut II
der Universitat F r e i b u r g der Universitat Dusseldorf
Hermann-Herder-Strasse 7 Moorenstrasse 5
D-7800 F r e i b u r g i. Br., G e r m a n y p. 2194 D-4000 Dusseldorf 1, G e r m a n y
p. 1858, 1868
Schutt, Christian Stamm, Dankwart
Stadtische Kliniken D a r m s t a d t Abteilung fur Klinische Chemie
Institut fur L a b o r a t o r i u m s d i a g n o s t i k Max-Planck-Institut fiir Psychiatrie
D-6100 D a r m s t a d t , G e r m a n y Kraepelinstrasse 10
p. 856, 860 D-8000 M u n i c h 23, G e r m a n y p. 318
Schulz, D e m o y W .
D e p a r t m e n t of N e u r o s u r g e r y Stegbauer, Hans-Peter
University of C o l o r a d o K r a n k e n h a u s der Barmherzigen Bruder
Medical School D-8400 Regensburg, G e r m a n y p. 885
Denver, C o l o r a d o 80220, U S A p. 2229
Stein, Philipp
Schwartz, M o r t o n K. Behringwerke A G
M e m o r i a l Hospital for C a n c e r D-3550 M a r b u r g / L a h n , G e r m a n y
and Allied Diseases p. 1777
N e w York, N . Y 10021, U S A p. 768
Stork, Harald
Schweitzer, Gertraud Abteilung Stoffwechsel
Medizinische Hochschule H a n n o v e r Sandhofer Strasse
Abteilung fur Klinische Biochemie D-6800 M a n n h e i m 3 1 , G e r m a n y
R o d e r b r u c h s t r a s s e 101 p. 819, 1196
Street, H a r o l d V .
D e p a r t m e n t of Forensic Medicine
S e u b e r t , Werner Medical School
Physiologisch-Chemisches Institut University of E d i n b u r g h
der G e o r g - A u g u s t Universitat E d i n b u r g h E H 8 9 A G , Scotland p. 898
Humboldtallee 7
D-3400 G o t t i n g e n , G e r m a n y
Strehler, B e r n a r d L .
p. 1994, 2010
University of S o u t h e r n California
Siebert, G u n t h e r
D e p a r t m e n t of Biological Sciences
Lehrstuhl fur Biologische Chemie L o s Angeles, California 90007, U S A
u n d Ernahrungswissenschaft p. 2112
Universitat H c h e n h e i m
Garbenstrasse 30 Siidhof, H e i n r i c h *
D-7000 Stuttgart 70, G e r m a n y p. 1570 R o b e r t - K o c h - K r a n k e n h a u s des
Landkreises H a n n o v e r
Siegel, A b r a h a m L. Medizinische Klinik
D-3011 G e h r d e n b . H a n n o v e r , G e r m a n y
University of A l a b a m a
p. 1025
Medical Center
Birmingham, A l a b a m a 35233, U S A p. 793 * deceased
Contributors XXXI
Szasz, G a b o r Voigt, Klaus-Dieter

Institut fur Klinische C h e m i e Klinisch-chemische Abteilung
an den Universitatskliniken Giessen II. Medizinische Universitatsklinik
Klinikstrasse 32 b Martinistrasse 52
D-6300 Giessen, G e r m a n y p. 715,1798 D-2000 H a m b u r g 20, G e r m a n y p. 1848
Wahlefeld, A u g u s t Wilhelm
Taniguchi, Shigehiko
University of H i r o s h i m a
D-8132 Tutzing/Obb., G e r m a n y p. 136,
School of Dentistry
894, 1464, 1585, 1604, 1786, 1831
H i r o s h i m a City, J a p a n p. 2260
Wallenfels, Kurt
Trautschold, Ivar
Lehrstuhl Biochemie
Medizinische H o c h s c h u l e H a n n o v e r Chemisches L a b o r a t o r i u m der
Abteilung fur Klinische Biochemie Universitat F r e i b u r g
R o d e r b r u c h s t r a s s e 101 Albertstrasse 21
D-3000 H a n n o v e r , G e r m a n y D-7800 F r e i b u r g i. Br., G e r m a n y
p. 228, 1031, 1064, 2101 p. 1180, 1279
Tubbs, Philip K. Waller, H a n s D i e r c k

D e p a r t m e n t of Biochemistry Medizinische Universitatsklinik
University of C a m b r i d g e H u g s t e t t e r Strasse 55
Cambridge, England p. 1758 D-7400 Tubingen, G e r m a n y p. 636
Walter, H a n s Elmar
Ullrich, Johannes
Universitat Regensburg
Biochemisches Institut der
F a c h b e r e i c h Biologie
Universitat F r e i b u r g
Universitats-Strasse 32
D-8400 R e g e n b u r g , G e r m a n y p. 1656
p. 2186
Walter, K l a u s
V a g e l o s , P. R o y Institut fur L a b o r a t o r i u m s d i a g n o s t i k
Washington University Grafenstrasse 9
School of Medicine D-6100 D a r m s t a d t , G e r m a n y
D e p a r t m e n t of Biological Chemistry p. 856, 860
St. Louis, Missouri 63110, U S A Warburg, O t t o *
p. 2005, 2031, 2038
Max-Planck-Institut
fur Zellphysiologie
Verdier, C a r l - H e n r i e d e
Garystrasse 32
Dept. of Clinical Chemistry University
D-1000 Berlin 33 - D a h l e m , G e r m a n y
Hospital
p. 248
S-75014 U p p s a l a 14, Sweden p. 1282
Weissbach, Arthur
N a t i o n a l Institutes of H e a l t h
Vogele, Peter Bethesda, M D . 20014, U S A p. 1333
Henkel & Cie, G m b H
D-4000 Dusseldorf, G e r m a n y p. 923 * deceased
XXXII Contributors
Weisser, H e r w i g Williamson, D e r m o t H.
Medizinische Hochschule H a n n o v e r M e t a b o l i c Research L a b o r a t o r y
Institut fur Klinische Biochemie u n d Nuffield D e p a r t m e n t
Physiologische Chemie of Clinical Medicine
Karl-Wiechert-Allee 9 T h e Radcliffe Infirmary
D-3000 Hannover-Kleefeld, G e r m a n y Oxford, England p. 1679
p. 1777 1727, 1836, 1840, 1844, 2041, 2266
Werle, E u g e n
Institut fur Klinische Chemie
W i l l i a m s o n , J o h n R.
u n d Klinische Biochemie der
Universitat J o h n s o n Research F o u n d a t i o n
N u s s b a u m s t r a s s e 20 University of Pennsylvania
D-8000 M u n i c h 2, G e r m a n y Philadelphia, Pennsylvania 19174, U S A
p. 660, 1031, 1064 /?. 1616
Wharton, H. Whitney
Wilmanns, Wolfgang
T h e Procter a n d G a m b l e C o .
Medizinische Klinik der
W i n t o n Hill Technical Center
Universitat Tubingen
Cincinnati, O h i o 45224, U S A p. 1807
Auf d e m Schnarrenberg
D-7400 Tubingen, G e r m a n y p. 666, 1118
Wieker, Hans-Joachim
Max-Planck-Institut fur
Ernahrungsphysiologie Witt, Irene
R h e i n l a n d d a m m 201
Universitats-Kinderklinik
D-4600 D o r t m u n d , G e r m a n y p. 778 Mathildenstrasse 1
Wieland, Otto p. 1442, 1502, 1593, 1713
Klinisch-chemisches Institut des
Stadtischen K r a n k e n h a u s e s Wolf, H a n s - P e t e r
Munchen-Schwabing
Klinische F o r s c h u n g der
u n d F o r s c h u n g s g r u p p e Diabetes
E. M e r c k A . G .
D-8000 M u n i c h 23, G e r m a n y
D-6100 D a r m s t a d t , G e r m a n y p. 1109
p. 1404, 1442, 1483, 1611
Wieme, Roger Jozef

Clinical Biochemistry Wunderwald, Peter
D e p a r t m e n t Internal Medicine Boehringer M a n n h e i m G m b H
Academic Hospital Biochemica Werk Tutzing
D e Pintelaan 115 D-8132 Tutzing/Obb., G e r m a n y p. 2136
G h e n t , Belgium p. 261, 593, 618, 745
W i l k i n s o n , J. H e n r y Zachau, Hans Georg

D e p a r t m e n t of Chemical Pathology Institut fur Physiologische Chemie u n d
Charing Cross Hospital Physikalische Biochemie der
F u l h a m Palace R o a d Universitat M u n c h e n
London W6 8 R F Goethestrasse
England p. 603 D-8000 M u n i c h , G e r m a n y p. 1894
Contributors XXXIII
Zak, Bennie Ziegenhorn, Joachim

Pathology D e p a r t m e n t Boehringer M a n n h e i m G m b H
Wayne State University Biochemica Werk Tutzing
School of Medicine D-8132 Tutzing/Obb., G e r m a n y p. 2034
Detroit, Michigan 48207, U S A p. 876
Zollner, N e p o m u k
Medizinische Poliklinik der
Zankl, Gottfried Universitat M u n c h e n
Boehringer M a n n h e i m G m b H Pettenkoferstrasse 8 a
Sandhofer Strasse D-8000 M u n i c h 2, G e r m a n y
D-6800 M a n n h e i m 3 1 , G e r m a n y p. 557 p. 1151, 1288
Abbreviations
Abbreviations of Units of Mass and Constants
m. M e t r e [m.] g. G r a m [g.]
cm. C e n t i m e t r e [ 1 0 ~ m.]
2
mg. Milligram [10 ~ g.] 3
mm. Millimetre [10 ~ m.] 3

fig. M i c r o g r a m [ 1 0 ~ g.] 6
nm. N a n o m e t r e [ 1 0 ~ m.]9
ng. N a n o g r a m [ 1 0 ~ g.] 9
hr. Hour 1. Litre [1.]

min. Minute ml. Millilitre [ 1 0 ~ 1 . ]
3
sec. Second /il. Microlitre [10 " 1 . ] 6
t T i m e [hr.] [min.] [ s e c ]
T T e m p e r a t u r e [° Kelvin]
V Volume (usually v o l u m e of assay mixture) [ml.]
v Volume (usually v o l u m e of sample in assay mixture) [ml.]
v R a t e of reaction, e.g. [jumole/min.]
MW M o l e c u l a r weight [g.]
c C o n c e n t r a t i o n [g./l.]; [mole/1.]
% Percentage
%(v/v) Percentage, v o l u m e related t o v o l u m e
%(v/w) Percentage, v o l u m e related t o weight
%(w/v) Percentage, weight related t o v o l u m e
%(w/w) Percentage, weight related t o weight
d Light p a t h [cm.]
sp. gr. Specific gravity at 20 °C relative t o w a t e r at 4 °C
Ci Curie
[a] ) 2 0
Specific r o t a t i o n (D-line at 20 °C)
cpm C o u n t s per m i n u t e [min. ~ ] 1
rpm Revolutions per minute [ m i n . ] - 1
g Acceleration [cm./sec. ] 2
£ Extinction coefficient [ c m . / m o l e ] , [ c m . / ^ m o l e ]
2 2
E Extinction (absorbance)
OD Optical density (Extinction)
F Fluorescence
I Light b e a m
k Reaction constant
K Equilibrium c o n s t a n t
K A p p a r e n t equilibrium c o n s t a n t
K m Michaelis c o n s t a n t [M]
Kj I n h i b i t o r c o n s t a n t [M]
XXXVI Abbreviations
pH H y d r o g e n ion c o n c e n t r a t i o n ( - log)
pK Acid dissociation c o n s t a n t (— log)
x M e a n value
s or SD S t a n d a r d deviation
CV Coefficient of variation
M M o l a r [mole/1.]
mM Millimolar [mmole/1.], [ 1 0 ~ mole/1.] 3
nM M i c r o m o l a r [/imole/1.], [ 1 0 ~ mole/1.] 6
nM N a n o m o l a r [nmole/1.], [ 1 0 ~ mole/1.] 9
pM P i c o m o l a r [pmole/1.], [ 1 0 ~ 1 2
mole/1.]
U I n t e r n a t i o n a l U n i t (for enzymes)
mU I n t e r n a t i o n a l milliunit [10 " 3
U]
IU I n t e r n a t i o n a l inhibitor unit
ImU I n t e r n a t i o n a l inhibitor milliunit [ 1 0 ~ I U ] 3
DN Dibucaine number
FN Fluoride number
RZ Reinheitszahl (of peroxidase)
Abbreviations for Chemical and Biochemical Compounds

It is unavoidable with the n u m e r o u s abbreviations in use t h a t one a b b r e v i a t i o n occasionally is used for
different c o m p o u n d s . In such cases the correct m e a n i n g can be o b t a i n e d from the text. Only the u n e q u i
vocal abbreviations are used in the b o o k w i t h o u t further explanation.
AA A m i n o acid arylamidase (micro AK A c e t a t e kinase

somal) ALD F r u c t o s e - 1 , 6 - d i p h o s p h a t e aldolase
y-ABA-T y - A m i n o b u t y r i c acid t r a n s a m i n a s e Ammediol 2 - A m i n o - 2 - m e t h y l - p r o p a n e - l ,3-
ABTS® 2,2'-Azino-di-(3-ethylbenzthi- diol
azoline)-6'-sulphonate AMP Adenosine-5 '-monophosphate
AcAc-CoA Acetoacetyl-coenzyme A A-2-MP Adenosine-2'-monophosphate
Ac-CoA Acetyl-coenzyme A A-3-MP Adenosine-3 '-monophosphate
AChE Acetylcholinesterase A-3,2-MP Adenosine-3'(2')-monophosphate
Ac-P Acetyl p h o s p h a t e A-3:5-MP Adenosine-3': 5'-monophosphate,
ACT C a r n i t i n e acetyltransferase cyclic
ADA Adenosine deaminase A-5-MP Adenosine-5 ' - m o n o p h o s p h a t e
ADH Alcohol dehydrogenase D-AOD D - A m i n o acid oxidase
ADP Adenosine-5'-diphosphate L-AOD L - A m i n o acid oxidase
A-2,5-DP Adenosine-2',5'-diphosphate AP Alkaline p h o s p h a t a s e
A-3,5-DP Adenosine-3', 5 '-diphosphate APAD Acetylpyridine-adenine dinucleo-
ADPG Adenosine-5 '-diphosphoglucose tide
AGS Amyloglucosidase APADH Acetylpyridine-adenine dinucleo-
AGT A c y l p h o s p h a t e : D-glucose-6- tide, r e d u c e d
phosphotransferase ARS Aryl s u l p h a t a s e
Abbreviations XXXVII
ATCase Aspartate transcarbamylase E-4-P D-Erythrose-4-phosphate

ATP Adenosine-5'-triphosphate ETF Electron transferring flavoprotein
ATPase Adenosine-5 '-triphosphatase
FAD Flavin-adenine dinucleotide
BAEE Benzoyl-L-arginine ethyl ester
FDH Formate dehydrogenase
BAPNA N-Benzoyl-arginine-p-nitroanilide
FDNB l-Fluoro-2,4-dinitrobenzene
BMTD 6-Benzamido-4-methoxy-m-
FDP D-Fructose-l,6-diphosphate
t o l u i d i n e - d i a z o n i u m chloride
(F-l,6-P ) 2
Bz-CoA Benzoyl-coenzyme A
FDPase F r u c t o s e - 1,6-diphosphatase
FH 2 Dihydrofolate
CA Carbonic anhydrase
FH 4 Tetrahydrofolate
CAA Carbamyl-L-aspartate
FIGLU N-Formimino-L-glutamate
CAP Carbamyl phosphate
FMN Flavin m o n o n u c l e o t i d e
CCE C i t r a t e cleavage enzyme
FNR F o r m a t e nitrate reductase
CCPN N-3-(carboxypropionyl)-L-
F-l-P D-Fructose-1-phosphate
phenylalanine-p-nitroanilide
F-l,6-P 2 D-Fructose-1,6-diphosphate
CDP Cytidine-5'-diphosphate
F-6-P D-Fructose-6-phosphate
CDPG Cytidine-5 '-diphosphoglucose
F-6-PK F r u c t o s e - 6 - p h o s p h a t e kinase
CE Citrate c o n d e n s i n g enzyme
FUM Fumarase
Cellosolve Ethylene glycol m o n o m e t h y l ether
CHA Cyclohexylammonium
CHE, ChE Cholinesterase GAD G e n e r a l acyl-CoA d e h y d r o g e n a s e
CHTR Chymotrypsin Gal-DH Galactose dehydrogenase
C K (CPK) C r e a t i n e kinase Gal-OD G a l a c t o s e oxidase
CL C i t r a t e lyase Gal-l-P D-Galactose-1-phosphate
C-2-MP Cytidine-2'-monophosphate Gal-6-P D-Galactose-6-phosphate
C-2 : 3 - M P Cytidine-2': 3'-monophosphate, GAP D-Glyceraldehyde-3-phosphate
cyclic GAPDH D-Glyceraldehyde-3-phosphate
C-3-MP Cytidine-3'-monophosphate dehydrogenase
C-3,2-MP Cytidine-3'(2')- m o n o p h o s p h a t e GDH L-Glycerol-3-phosphate dehydro
C-5-MP Cytidine-5'-monophosphate genase (glycerol-1-phosphate
CoA, CoA-SH Coenzyme A d e h y d r o g e n a s e ; a-glycerophos-
CP Creatine phosphate phate dehydrogenase)
CPK C r e a t i n e kinase ( C K ) GDP Guanosine-5'-diphosphate

CS C i t r a t e synthase GK Glycerokinase
CTP Cytidine-5 ' - t r i p h o s p h a t e Gl-I Glyoxalase I
Cyt-c Cytochrome c G1DH L-Glutamate dehydrogenase
(GluDH)
DAO D i a m i n e oxidase Gly-R Glyoxylate reductase (glycerate
DAP Dihydroxyacetone phosphate dehydrogenase)
DEAE Diethylaminoethyl GMP Guanosine-5'-monophosphate
DFP Diisopropyfluorophosphate G-2-MP Guanosine-2'-monophosphate
-DH -Dehydrogenase G-3-MP Guanosine-3'-monophosphate
DIA Diaphorase G-3,2-MP Guanosine-3'(2')-monophosphate
DNase Deoxyribonuclease G-3 : 5 - M P Guanosine-3 :5-monophosphate,
DNP Dinitrophenylhydrazine cyclic
DTNB 5:5-Dithiobis-(2-nitrobenzoic acid) G-5-MP Guanosine-5'-monophosphate
GOD G l u c o s e oxidase
EDTA Ethylenediaminetetra-acetate GOT Glutamate-oxaloacetate trans
ENOL, ENO Enolase aminase
XXXVIII Abbreviations
G-l-P D-Glucose-1-phosphate NBT N i t r o - B T - t e t r a z o l i u m salt, 2,2'-

G-l,6-P 2 D-Glucose-l,6-diphosphate di-p-nitrophenyl-5,5 '-diphenyl-
G-6-P D-Glucose-6-phosphate 3.3 '-(-dimethoxy-4,4'-dipheny lene)-
G6Pase Glucose-6-phosphatase d i t e t r a z o l i u m chloride
G6P-DH(ZF) G l u c o s e - 6 - p h o s p h a t e dehydrogen NBTH N-Methyl-2-benzothiazolone
ase (Zwischenferment) hydrazone
GPT Glutamate-pyruvate transaminase NDPK N u c l e o s i d e d i p h o s p h a t e kinase
GR G l u t a t h i o n e reductase NMN Nicotinamide mononucleotide
GRD ^-Glucuronidase NMPK N u c l e o s i d e m o n o p h o s p h a t e kinase
GSH Glutathione NP N u c l e o s i d e phosphorylase
GSSG G l u t a t h i o n e , oxidized
GT Glucuronyltransferase OA Oxaloacetate
y-GT, G G T P y - G l u t a m y l transpeptidase OCT O r n i t h i n e - c a r b a m y l transferase
GPT Guanosine-5'-triphosphate ODTG Octadehydro-tetraguaiacol
3-OH-A 3 - H y d r o x y a n t h r a n i l i c acid
Hb Haemoglobin 3-OH-K 3-Hydroxykynurenine
HBDH D-3-Hydroxybutyrate dehydrogen 1 5 - O H - P G D H 1 5 - H y d r o x y p r o s t a g l a n d i n de
ase hydrogenase
HK Hexokinase OxoG 2-Oxoglutaric acid
HMG-CoA 3-Hydroxy-3-methylglutaryl- OxoG-DH 2-Oxoglutarate dehydrogenase
coenzyme A
HOADH 3-Hydroxyacyl-CoA dehydrogen PALP Pyridoxal-5-phosphate
ase PAMP Pyridoxamine-5-phosphate
HXM Hypoxanthine PChE Pseudocholinesterase
PDC P y r u v a t e decarboxylase
ICDH Isocitrate d e h y d r o g e n a s e PDE Phosphodiesterase
IDP Inosine-5'-diphosphate PEP Phosphoenol pyruvate
I-5-MP Inosine-5'-monophosphate PFA F r u c t o s e - 1 - p h o s p h a t e aldolase,
INT 2-(p-Iodophenyl)-3-(p-nitro- 1 -phosphofructoaldolase
phenyl)-5-phenyltetrazolium PFK Phosphofructo-kinase, fructoses-
chloride p h o s p h a t e kinase
ISN Inosine PG Prostaglandin
ITP Inosine-5'-triphosphate 2-PG 2-Phosphoglycerate, D-glycerate-2-
phosphate
3-PG 3-Phosphoglycerate, D-glycerate-3-
LAP Leucine a m i n o p e p t i d a s e
phosphate
LDH L-Lactate dehydrogenase
6-PG 6-Phosphogluconate, D-gluconate-
D-LDH D-Lactate dehydrogenase
6-phosphate
MDH L-Malate dehydrogenase
6-PG-DH 6 - P h o s p h o g l u c o n a t e dehydrogen
MK M y o k i n a s e , adenylate kinase
ase
MPDH Mannitol-l-P dehydrogenase
PGI, PHI Phosphoglucose-isomerase,
phosphohexose-isomerase
NAD N i c o t i n a m i d e - a d e n i n e dinucleotide PGK 3-Phosphoglycerate kinase
NADH N i c o t i n a m i d e - a d e n i n e dinucleo PGluM Phosphoglucomutase
tide, reduced PGM Phosphoglycerate mutase
NADP N i c o t i n a m i d e - a d e n i n e dinucleo 1,3-PGP D-Glycerate-1,3-diphosphate,
tide p h o s p h a t e 1,3-Diphosphoglycerate
NADPH N i c o t i n a m i d e - a d e n i n e dinucleo 2,3-PGP D-Glycerate-2,3-diphosphate,
tide p h o s p h a t e , reduced 2,3-Diphosphoglycerate
Abbreviations XXXIX
Pi Inorganic phosphate TA Transamidinase

PK P y r u v a t e kinase TA Transaldolase
PL-A Phospholipase A dTDP Deoxythymidine-5 '-diphosphate
PL-D Phospholipase D dTDPG Deoxythymidine-5 '-diphospho-
PMI P h o s p h o m a n n o s e isomerase glucose
PMS Phenazine methosulphate THF Tetrahydrofolate
POD Peroxidase TIM T r i o s e p h o s p h a t e isomerase
Poly-A Polyadenylic acid TK Thiokinase
Poly-C Polycytidylic acid TK Transketolase
Poly-I Polyinosylic acid TPP Thiamine pyrophosphate
Poly-U Polyuridylic acid TR Trypsin
DM-POPOP 1,4-bis-(4-methyl-5-phenyl- TRA Triethanolamine
oxazolyl)-benzene Tris Tris-hydroxymethyl-amino-
PPase P y r o p h o s p h a t a s e , inorganic methane
PPi Inorganic pyrophosphate dTTP Deoxythymidine triphosphate
PPO 2,5-Diphenyloxazole
PTA Phosphotransacetylase
RDH Ribitol dehydrogenase UDP Uridine-5 ' - d i p h o s p h a t e
RNA R i b o n u c l e i c acid UDPG Uridine-5'-diphosphoglucose
sRNA Soluble ribonucleic acid UDPAG Uridine-5 '-diphospho-N-acetyl-
tRNA Transfer ribonucleic acid glucosamine
RNase Ribonuclease UDPGA Uridine-5 ' - d i p h o s p h o g l u c u r o n a t e
R-l-P D-Ribose-1 -phosphate UDPGal Uridine-5'-diphosphogalactose
R-l,5-P 2 D-Ribose-1,5-diphosphate UDPG-DH Uridine-5 ' - d i p h o s p h o g l u c o s e
R-5-P D-Ribose-5-phosphate dehydrogenase
R5P-I R i b o s e - 5 - p h o s p h a t e isomerase UDPGP UDPG-pyrophosphorylase
Ru-l-P D-Ribulose-1 -phosphate UMP U ridine-5 ' - m o n o p h o s p h a t e
Ru-1,5-P 2 D-Ribulose-1,5-diphosphate U-2-MP Uridine-2'-monophosphate
Ru-5-P D-Ribulose-5-phosphate U-2:3-MP Uridine-2': 3'-monophosphate,
cyclic
SBI Soya b e a n inhibitor U-3-MP Uridine-3 ' - m o n o p h o s p h a t e
SD Succinate d e h y d r o g e n a s e U-3,2-MP Uridine-3 '(2 ^ - m o n o p h o s p h a t e
SDH Sorbitol d e h y d r o g e n a s e , polyol U-5-MP Uridine-5 ' - m o n o p h o s p h a t e
dehydrogenase UT Uridyltransferase
SDPase Sedoheptulose-1,7-diphosphatase UTP Uridine-5 ' - t r i p h o s p h a t e
S-l,7-P 2 D-Sedoheptulose-1,7-diphosphate
S-7-P D-Sedoheptulose-7-phosphate
SSA-DH Succinate semialdehyde d e h y d r o XOD X a n t h i n e oxidase
genase Xu-5-P Xylulose-5-phosphate
20-StDH 3a,20/?-Hydroxysteroid d e h y d r o
genase
SUPHEPA N-Succinyl-L-phenylalanine-p- Z F ( G 6 P - D H ) Zwischenferment, glucose-6-
nitroanilide phosphate dehydrogenase
Characterization of Peptides and Proteins with Enzymes
H u g o Fasold and Gerd Gundlach
Since the fundamental work of Fred Sanger on the structure of i n s u l i n 1 - 8

, an increasing amount o f
research has been undertaken to determine the amino acid sequences in proteins and peptides. It is useful
to degrade the proteins to be analysed with proteolytic enzymes, since the specificity of enzymes permits
the isolation of uniform fragments. The "spectrum" of the peptides obtained in this way is sometimes
characteristic of a certain protein. The action of peptidases on individual peptides allows some determin
ation of amino acid sequences.
Application of Method: In biochemistry for the elucidation of protein structures and for the isolation and
characterization of active centres in enzymes and hormones.
Hydrolysis of Proteins to Peptides

1. Method with Trypsin
Principle
The high specificity of this enzyme permits the isolation o f defined f r a g m e n t s 9,10
. Trypsin (EC 3.4.21.4)
splits peptide bonds wherever the basic amino acids lysine and arginine occur; the cleavage is such that
the carboxyl group of these amino acids is liberated. In this way peptides with a basic amino acid at the
carboxyl end are obtained. Only two exceptions are k n o w n : trypsin does not hydrolyse peptide linkages
if 1. the a-amino group of lysine or arginine is free or 2. lysine is followed by proline in the peptide c h a i n ' . 11 12
The cleavage of the peptide is slow if the basic amino acid is adjacent to an acidic amino acid or c y s t i n e ' 6 4 6 5 , 1 1
.
Carbamoylation 66,67
or trifluoroacetylation 68
on the e-amino group of lysine prevents the cleavage by
trypsin, so that degradation occurs only at the arginine residues. On the other hand, addition o f ethylenimine
to thiol groups of cysteine gives new basic groups that allow cleavage by t r y p s i n . The "action spectrum"
69
of the cleavage by trypsin can thus be specifically narrowed or extended.

If a protein cannot be degraded directly, or if it can be degraded only incompletely by enzymatic means,
it must be prepared for the enzymatic hydrolysis by denaturation in solutions o f 6 - 8 M u r e a * or 2 M 13
guanidine hydrochloride , and by splitting the disulphide bridges. The latter is accomplished 1. by
14
oxidation with performic a c i d 1 5 1 6

(tryptophan is destroyed in the oxidation) or 2. by reduction with
thiols, such as thioglycol , with sodium borohydride
17 18
(followed by reaction of the resulting thiol groups
to carboxymethyl residues) or with s u l p h i t e 19,20
(formation of thiosulphonic acids).
The proteins are usually hydrolysed at r o o m temperature in a buffer solution at the p H optimum o f trypsin
(pH 7 - 8 ) or if it is desired to work in salt-free conditions, in an autotitrator. The peptides formed are
separated either on ion exchange columns (e. g. D o w e x 50 X 2 ) 1 1
or chromatographically or electrophoretic-
ally on paper. The most elegant method is two-dimensional separation on paper, in which one dimension
is run electrophoretically and the other dimension chromatographically ("fingerprint") ' . 21 22
* It is not essential to remove the urea in this case, since the cleavage by trypsin takes place even in 2 M
urea .
70
1626 Metabolites: Protein Metabolism
a ) H y d r o l y s i s in Buffer S o l u t i o n 1 1
Reagents
1. S o d i u m d i h y d r o g e n p h o s p h a t e , 7. A c e t i c a c i d , A . R.
N a H P 0 H 0 , A . R.
2 4 2 8. E t h y l e n e g l y c o l m o n o m e t h y l e t h e r
2. D i s o d i u m h y d r o g e n p h o s p h a t e , (Cellosolve)
N a H P 0 , a n h y d r o u s , A . R.
2 4
9. E t h a n o l , c a . 5 0 % ( w / v )
3. H y d r o c h l o r i c a c i d , A . R . , 2 N 10. T r y p s i n
4. N i n h y d r i n recrystallized repeatedly, salt-free or contain
5. S o d i u m a c e t a t e - 3 H 0 , A . R.
2 ing 50 % magnesium sulphate. Commercial prep
6. H y d r i n d a n t i n ( p r e p a r a t i o n , s e e 2 3
) arations, see p. 515.
Purity of Enzyme Preparation
Commercially available trypsin usually contains small amounts o f chymotrypsin and elastase. It is therefore
necessary in special cases to start with chromatographically pure trypsinogen, which is activated with
a small amount of trypsin before the experiment and recrystallized . 24
Most of the chymotrypsin activity can be eliminated by inactivation with diphenylcarbamoyl chloride 71
or N-tosylphenylalanine chloromethyl k e t o n e , without appreciable loss of trypsin activity.

72
Preparation of Solutions
I. P h o s p h a t e buffer ( 0 . 2 M ; p H 7 . 0 ) :
D i s s o l v e 8 . 2 8 g. N a H P 0 H 0
2 4 2 a n d 1 9 . 8 8 g. N a H P 0
2 4 ( a n h y d r o u s ) in distilled w a t e r
and m a k e u p to 1 0 0 0 ml.
II. N i n h y d r i n r e a g e n t 2 3
D i s s o l v e 2 0 g. n i n h y d r i n a n d 3 g. h y d r i n d a n t i n i n 7 5 0 m l . p e r o x i d e - f r e e e t h y l e n e g l y c o l
m o n o m e t h y l e t h e r a n d a d d 2 5 0 m l . 4 M s o d i u m a c e t a t e buffer, p H 5.5 ( 5 4 4 . 0 g. s o d i u m
a c e t a t e - 3 H 0 a n d 1 0 0 m l . g l a c i a l a c e t i c a c i d in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l . ) .
2
T h e r e d d i s h s o l u t i o n is s t a b l e u n d e r n i t r o g e n in t h e d a r k . D o n o t p r e p a r e m o r e t h a n
a week's supply.
III. T r y p s i n ( 0 . 0 5 % p r o t e i n ) :
D i s s o l v e 5 0 m g . salt-free t r y p s i n in p h o s p h a t e buffer ( s o l u t i o n I) a n d m a k e u p t o 100 m l .
P r e p a r e t h e s o l u t i o n freshly for e a c h h y d r o l y s i s .
Procedure
Collection, Treatment and Stability of Sample
D i s s o l v e t h e m a t e r i a l t o b e h y d r o l y s e d i n p h o s p h a t e buffer ( s o l u t i o n I) t o g i v e a final c o n c e n t r a
t i o n o f 1% ( w / v ) . A d j u s t t h e p H if n e c e s s a r y . In v i e w o f t h e p o s s i b i l i t y o f a u t o l y s i s , u s e o n l y
freshly p r e p a r e d s o l u t i o n s . ( T h e d i l u t e d t r y p s i n s o l u t i o n w i t h o u t p r o t e i n t o b e h y d r o l y s e d
k e e p s for o n l y a f e w h o u r s ) . T h e e n z y m a t i c h y d r o l y s i s is f o l l o w e d w i t h t h e a i d o f t h e n i n h y d r i n
reaction.
C h a r a c t e r i z a t i o n of Peptides a n d Proteins with E n z y m e s 1627
Assay System
F o r the n i n h y d r i n r e a c t i o n a n d t h e d e t e r m i n a t i o n o f a u t o l y s i s p r o d u c t s o f t r y p s i n , p r e p a r e
a b l a n k w i t h p h o s p h a t e buffer ( s o l u t i o n I) i n s t e a d o f s a m p l e .
Enzymatic Hydrolysis
I n c u b a t i o n v o l u m e : 2 0 m l . ; t e m p e r a t u r e : 38 ° C .
P i p e t t e i n t o a 50 m l . r o u n d - b o t t o m e d flask
C o n c e n t r a t i o n in mixture*
(37 ° C w a t e r b a t h )
Sample 10 m l . 5.00 m g . / m l .
Trypsin solution (III) 10 m l . 0.25 m g . / m l .
M i x ; r e m o v e 0.1 m l . s a m p l e s after 0, 2 0 , 4 0 , 6 0 , 1 2 0 ,
240, a n d 3 6 0 m i n . C a r r y o u t t h e n i n h y d r i n r e a c t i o n
o n t h e s e s a m p l e s . * * W h e n the c o n t e n t o f n i n h y d r i n -
positive substances no longer increases, stop the
enzymatic reaction with HC1.
2NHC1 ca. 2 ml.
T h e p H s h o u l d b e 2 . 2 . T h e m i x t u r e c a n b e d i r e c t l y transferred t o a n i o n e x c h a n g e c o l u m n
for the s e p a r a t i o n o f t h e p e p t i d e s 1 1
.
Ninhydrin Reaction
Wavelength: 570 n m ; light p a t h : 1 c m . M e a s u r e against the blank (see a b o v e ) .

P i p e t t e i n t o s t o p p e r e d test t u b e s :
0.1 m l . s a m p l e f r o m t h e e n z y m a t i c r e a c t i o n m i x t u r e
1.0 m l . n i n h y d r i n r e a g e n t ( s o l u t i o n II)
H e a t for e x a c t l y 15 m i n . in a b o i l i n g w a t e r b a t h , a d d
5.0 m l . 5 0 % e t h a n o l ,
m i x a n d a l l o w t o c o o l t o r o o m t e m p e r a t u r e . M e a s u r e t h e e x t i n c t i o n . If t h e e x t i n c t i o n s a r e
very h i g h , the s o l u t i o n s c a n b e d i l u t e d w i t h 5 0 % e t h a n o l . T h e e n z y m a t i c h y d r o l y s i s is f o l l o w e d
through the formation o f ninhydrin-positive substances. W h e n these n o longer increase the
h y d r o l y s i s is c o m p l e t e ( p l o t t h e c o l o u r i n t e n s i t y a g a i n s t t h e t i m e ) .
b) H y d r o l y s i s in " S a l t - F r e e " S o l u t i o n
If the peptides m u s t be w o r k e d u p by m e t h o d s in which non-volatile salts interfere a n d if an a u t o m a t i c

titration a p p a r a t u s is available, the hydrolysis is carried out in a solution t h a t c o n t a i n s only volatile s a l t s . 10
The tryptic hydrolysis is followed by m e a n s of the c o n s u m p t i o n of alkali with time.
* T h e weight ratio of substrate to enzyme in the "digestion m i x t u r e " is 20 : 1. In m a n y cases the enzyme
concentration in the mixture can be reduced to 0.025 mg./ml.
** T h e enzymatic reaction is stopped o n mixing with the ninhydrin reagent.
Reagents
1. T r i e t h y l a m i n e , r e d i s t i l l e d , b . p . 8 9 . 4 ° C / 3. Trypsin
760 m m . s e e p . 515.
2 . F o r m i c a c i d , A . R.
I. T r i e t h y l a m i n e (0.1 M ) :
M i x 1.00 g. t r i e t h y l a m i n e w i t h d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
II. T r y p s i n ( 0 . 0 5 % p r o t e i n ) :
D i s s o l v e 5 0 m g . salt-free t r y p s i n in d i s t i l l e d w a t e r , adjust t o p H 8 w i t h 0.1 M triethyl
a m i n e s o l u t i o n (I) a n d d i l u t e t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r . P r e p a r e freshly for e a c h
experiment.
Procedure
D i s s o l v e t h e salt-free p r o t e i n t o b e h y d r o l y s e d in d i s t i l l e d w a t e r t o g i v e a final c o n c e n t r a t i o n
o f 0.5 t o 2 % ( w / v ) , a n d a d j u s t t h e p H o f t h e s o l u t i o n t o 8.0 w i t h f o r m i c a c i d o r 0.1 M tri
e t h y l a m i n e s o l u t i o n (I). T h e t r y p s i n s o l u t i o n k e e p s for o n l y a f e w h o u r s . T h e h y d r o l y s e d
s a m p l e s h o u l d b e t r e a t e d further as s o o n a s p o s s i b l e , o r s h o u l d at least b e k e p t f r o z e n after
the c o m p l e t i o n o f the experiment.
Assay System
Incubation v o l u m e : 2 0 m l . ; temperature: 37 °C.

P r e p a r e a b l a n k w i t h w a t e r at p H 8 ( a d j u s t e d w i t h t r i e t h y l a m i n e ) i n s t e a d o f s a m p l e . T h i s is
u s e d t o d e t e r m i n e t h e c o n s u m p t i o n o f alkali d u e t o a b s o r p t i o n o f C 0 2 f r o m the air d u r i n g
the experiment.
In t h e t i t r a t i o n v e s s e l o f t h e C o n c e n t r a t i o n in
a u t o t i t r a t o r ( e q u i l i b r a t e d at 3 7 ° C ) assay mixture
Sample 10 m l . 2 . 5 - 1 0 mg./ml.
Trypsin solution (II) 10 m l . 0.25 m g . / m l .
Mix.
Triethylamine solution (I) x ml.
M a i n t a i n a u t o m a t i c a l l y at p H 8.0 w i t h s o l u t i o n (I).
Record c o n s u m p t i o n every 30 min. S t o p experiment
w h e n alkali c o n s u m p t i o n c o r r e s p o n d s t o t h e a b s o r p t
ion of C 0 2 f r o m t h e air ( b l a n k ) ( F i g . 1).
A f t e r f r e e z e - d r y i n g , t h e p e p t i d e s f o r m e d c a n b e s e p a r a t e d directly b y p a p e r c h r o m a t o g r a p h y ;
t h e salts p r e s e n t in t h e r e a c t i o n m i x t u r e are v o l a t i l e .
Characterization of Peptides and Proteins with Enzymes 1629
Fig. 1. Hydrolysis of a protein with trypsin

(in an autotitrator).
Curve A : Blank ( C 0 absorption from the
2
atmosphere)
Curve B: Extrapolation to t = 0
Portion C: Alkali consumption equivalent to
the hydrolysis of the protein (net alkali
consumption).
2. Method with Chymotrypsin

Principle
The specificity of chymotrypsin (EC 3.4.21.1) is not quite so high as that of trypsin. Generally, chymotrypsin
hydrolyses proteins at the carboxyl group of aromatic amino acids such as phenylalanine, tyrosine and
tryptophan. However, the enzyme is also capable of hydrolysing amino acid residues that have a space
similar to that of the aromatic amino acids. Thus peptides have been obtained with leucine, valine, meth
ionine, glutamic acid, asparagine, glutamine and histidine as carboxyl end groups. Although the aromatic
residues are always hydrolysed, providing they are not N-terminal residues, the other group of residues
are only occasionally h y d r o l y s e d 1 0 , 2 6 , 2 7
. The p H optimum of chymotrypsin is at 8.0, but the hydrolysis
can also be carried out at p H 7 and 9.
The concentrations of substrate and of enzyme in the assay mixture may be varied within wide limits,
depending on the intended purpose. The technique used with trypsin is recommended for the hydrolysis
in the first experiments.
The reaction can be carried out in buffer or "salt-free" solution. In addition to phosphate buffer, 0.2 M
ammonium acetate/ammonia buffer 27
(pH 8.5) has been successfully used. This buffer has the advantage
that the salt can be sublimed off in vacuo and a salt-free peptide mixture is obtained. In this case, however,
the hydrolysis cannot be followed by the ninhydrin method because of the presence of ammonia.
Reagents
See under "Trypsin", p. 1626. U s e chymotrypsin instead of trypsin: recrystallized repeatedly,

salt-free; specific a c t i v i t y a n d c o m m e r c i a l p r e p a r a t i o n s , see p . 4 4 0 .
Purity of the Enzyme Preparation
Chymotrypsin usually contains a small amount of tryptic activity. The occurrence of "tryptic" peptides
must therefore be reckoned with. For this reason the addition of trypsin inhibitor from soya beans may
be useful . For special cases, the chromatographic purification of c h y m o t r y p s i n
73 28
or c h y m o t r y p s i n o g e n 29
(followed by activation to chymotrypsin ) is recommended. 24
See under "Trypsin", p. 1626 or p. 1628.

1630 M e t a b o l i t e s : Protein M e t a b o l i s m
Procedure
See under "Trypsin", p. 1626 or p. 1628.
3. Method with Pepsin

Principle
C o m p a r e d with trypsin the specificity of pepsin is n o t very high (EC 3.4.23.1). It hydrolyses synthetic
substrates at a r o m a t i c a m i n o acids so that the a m i n o g r o u p of these acids is liberated. M o s t of the proteins
so far studied are n o t completely hydrolysed by pepsin. Often only peptides from the a m i n o or carboxyl
end are split off. T h e reaction is usually n o t quantitative. Very often " u n e x p e c t e d " peptide linkages that
contain n o a r o m a t i c a m i n o acids are rapidly a n d quantitatively hydrolysed, while peptide linkages in
volving a r o m a t i c a m i n o acids are n o t a t t a c k e d at all. In spite of this, pepsin can be used for structural
studies 3 0 , 3 1
, since every new peptide that clarifies an a m i n o acid sequence is of value. T h e p H o p t i m u m for
the hydrolysis of proteins is at p H 2, while for m a n y synthetic substrates it is at p H 4 . 3 2
T h e enzymatic hydrolysis of proteins in the acidic range is particularly useful if the disulphide groups are
to be preserved in the native state. Exchange is decreased u n d e r these c o n d i t i o n s . 14
Reagents
1. Citric a c i d , A . R. 7. A c e t i c a c i d , A . R.
2. S o d i u m h y d r o x i d e , A . R. 8. E t h y l e n e g l y c o l m o n o m e t h y l e t h e r
3. H y d r o c h l o r i c acid, cone, 37% (w/w), (Cellosolve)
A . R. 9. P r o p a n o l
4. N i n h y d r i n 10. P e p s i n
5. Hydrindantin crystalline, salt-free; specific activity and com
6. S o d i u m a c e t a t e - 3 H 0 , A . R.
2 mercial p r e p a r a t i o n s , see p . 493.
Purity of Enzyme Preparations
T h e purity of the crystalline commercial p r e p a r a t i o n s is generally satisfactory.
I. Citrate buffer ( 0 . 2 M ; p H 2 . 2 ) :
D i s s o l v e 2 1 . 0 g. citric a c i d a n d 8.4 g. N a O H in distilled w a t e r , a d d 16.0 ml. c o n e . HC1 a n d
d i l u t e t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r . If n e c e s s a r y , adjust t h e p H t o 2.2 (glass e l e c t r o d e ) .
II. S o d i u m h y d r o x i d e (0.1 N ) :
D i s s o l v e 4 g. N a O H in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
III. N i n h y d r i n r e a g e n t :
P r e p a r a t i o n , see p . 1 6 2 6 .
IV. H y d r o c h l o r i c acid (0.01 N ) :
D i l u t e 1 ml. c o n e . HC1 t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r a n d s t a n d a r d i z e a g a i n s t s o l u t i o n II
d i l u t e d 1 : 10.
V . P e p s i n (ca. 0 . 1 % w / v p r o t e i n ) :
D i s s o l v e 5 0 m g . p e p s i n in citrate buffer ( s o l u t i o n I) a n d m a k e u p t o 50 ml. P r e p a r e freshly
before each experiment.
Procedure
D i s s o l v e the m a t e r i a l t o b e h y d r o l y s e d in c i t r a t e buffer ( s o l u t i o n I) o r in 0.01 N H C 1 ( s o l u t i o n

IV) t o g i v e a final c o n c e n t r a t i o n o f 1% ( w / v ) .
I m m e d i a t e further t r e a t m e n t is a d v i s a b l e t o a v o i d e x c h a n g e o f d i s u l p h i d e g r o u p s . T h e p e p s i n
s o l u t i o n r e q u i r e d for t h e h y d r o l y s i s k e e p s for a f e w h o u r s at r o o m t e m p e r a t u r e .
Assay System
I n c u b a t e t h e b l a n k a n d t h e " d i g e s t i o n m i x t u r e " at 37 ° C a s d e s c r i b e d f o r t r y p s i n , p . 1 6 2 7 . A t
the e n d o f t h e h y d r o l y s i s a d j u s t t h e m i x t u r e t o p H 7.0 w i t h 0.1 N N a O H ( s o l u t i o n II).
4. Methods with Other Proteinases
Other proteolytic enzymes are used for special purposes. F o r example, when subtilisin is allowed to act
for a short time on ribonuclease it hydrolyses only one peptide l i n k a g e . P a p a i n ( E C 3.4.22.2) hydrolyses
33
n u m e r o u s b o n d s (benzoylarginine a m i d e is used as assay s u b s t r a t e ) . In the elucidation of the structure

34
of ribonuclease, p a p a i n was used to hydrolyse a p e p t i d e 35

t h a t was n o t a t t a c k e d by trypsin, c h y m o t r y p s i n
or pepsin.
Papain also hydrolyses peptide linkages in solutions in which urea is present in c o n c e n t r a t i o n s of u p to
8 M . This m e t h o d is particularly suitable for hydrolysis in the cores, which are difficult t o r e a c h . P r o 74
teolysis with " i m m o b i l e " enzymes ( t r y p s i n , p a p a i n ) coupled to p o l y m e r s h a s been found useful in

75 7 6
recent years. T h e possibility of using elastase ( E C 3.4.21.11) h a s been investigated with the B chain of insulin
as the s u b s t r a t e . T h e enzyme p r o n a s e from Streptomyces
36
griseus has a very b r o a d specificity . 77
Stepwise Degradation of Peptides and Proteins with

Carboxypeptidase and Leucine Aminopeptidase
Peptides (e. g. obtained by the tryptic hydrolysis of a protein) as well as proteins can be degraded stepwise
from either the carboxyl or the amino end by enzymes. Carboxypeptidase (Peptidyl-L-amino-acid hydrolase,
EC 3.4.12.2) was introduced into protein chemistry by Waldschmidt-Leitz , 31
Grassmann , 38
Lens 39
and others.
Leucine aminopeptidase (a-Aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) has only been used
more recently, particularly by Bergmann* , 0
Smith* , 1
HilP1
and Spackmann* . 3
Principle
As these two enzymes are typical exopeptidases, they split off successive amino acids from the carboxyl
and the amino end respectively of the protein.
R 1
O R 2
O
(1) —NH—C—C—NH—C—C—OH + H 0 2
c a r b o x y p e p t l d a s e
>
H H
R 1
O R 2
O
I II Ml
—NH—C—C—OH + H N—C—C—OH
I I
2
H H
R 3
O R 4
O
(2) H
I II I II
N—C—C—NH—C—C—NH— + H 0
, - "»"°P'P"*""S
•
l c u c i n e a
2 2
I I
H H
R 3
O R 4
O
I II Ml
H N—C—C—OH + H N—C—C—NH—
I I
2 2
H H
Conclusions about the amino acid sequence of a peptide or protein can be drawn if the stepwise enzymatic
hydrolysis is followed as a function o f time. Samples are removed from the reaction mixture and the amino
acids are determined qualitatively and semi-quantitatively. The terminal amino acid appears first, followed
by the second, then the third, etc. The semi-quantitative determination of the concentration of the frag
ments at various times confirms the results.
Reagents
For the enzymatic reaction
1. D i s o d i u m h y d r o g e n p h o s p h a t e , 5. H y d r o c h l o r i c a c i d , A . R . , 0.1 N
N a H P 0 1 2 H 0 , A . R.
2 4 2 6. S o d i u m h y d r o x i d e , A . R . , 0.1 N
2. S o d i u m d i h y d r o g e n p h o s p h a t e , 7. M a g n e s i u m c h l o r i d e , M g 0 - 6 H 0 , A . R . 2 2
NaH P0 H 0,
2 4 2 A.R. 8. M a n g a n o u s c h l o r i d e , M n C l - 4 H 0 , 2 2
3. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris A.R.
4. Hydrochloric acid, c o n e , ca. 36% (w/w), 9. S o d i u m h y d r o g e n c a r b o n a t e , NaHC0 , 3
A.R. A . R . , 1% s o l u t i o n
Characterization of Peptides and Proteins with Enzymes 1633
10. L i t h i u m c h l o r i d e , L i C l , A . R . . Leucine aminopeptidase, L A P

11. T r i c h l o r o a c e t i c a c i d , A . R . , 1 0 % s o l u t i o n isolated from pig kidney according to 4 6
« 4 6 a
,
12. D i i s o p r o p y l f l u o r o p h o s p h a t e , DFP ca. 100 U/mg. (25 °C). The enzyme solution is
13. I s o p r o p a n o l , A . R . , a b s o l u t e usually about 3 x 1 0 ~ M (estimated molecular
5
14. Carboxypeptidase weight about 300000). Commercial prepara

crystalline, from p a n c r e a s 44,45
, suspension in tions, see p. 482.
water, ca. 150 U / m g . (25 °C); commercial prep
arations, see p. 436.
For the ninhydrin reaction and paper chromatography
16. P o l y s t y r e n e s u l p h o n i c a c i d i o n e x c h a n g e 1 9 . A m m o n i a s o l u t i o n , A . R . , ca. 15 N
resin 20. Ninhydrin, pure
H - f o r m , particle size 30 mesh or smaller, 2 1 . n - B u t a n o l , r e d i s t i l l e d
+
8 - 1 2 % cross-linked, e.g. D o w e x 50 X 8 or 2 2 . C o l l i d i n e , r e d i s t i l l e d
Nalcite H C R . 4 7
23. Chromatography paper
17. A c e t i c a c i d , c a . 9 6 % A . R . e.g. Schleicher & Schull 2 0 4 3 b ; Whatman
18. F o r m i c a c i d , A . R . N o . 1 or 4 ; Macherey & Nagel 621.
Purity of the Enzyme Preparations
Carboxypeptidase: Carbobenzoxyglycylphenylalanine is used as substrate to assay the activity; the pro

teolytic coefficient Cf should be at least 10. Before the start of the experiment the enzyme crystals should be
washed four times with distilled water (centrifuge) to remove free amino acids and resuspended in 1%
N a H C 0 solution to give a final concentration of 1 mg. protein/ml. Contaminants such as trypsin, chymo
3
trypsin, etc., are inhibited with diisopropyl fluorophosphate (DFP).

Leucine aminopeptidase: Leucinamide is used for the assay of the activity . 46
I. P h o s p h a t e buffer (1 M ; p H 8 . 0 ) :
D i s s o l v e 1 7 9 . 0 8 g. N a H P 0 - 2 H 0 in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2 4 2
A d j u s t t h i s s o l u t i o n t o p H 8.0 ( g l a s s e l e c t r o d e ) w i t h 6 0 m l . o f a s o l u t i o n o f 138 g.
N a H P 0 H 0 in 1 0 0 0 m l . d i s t i l l e d w a t e r .
2 4 2
II. Tris buffer ( 0 . 2 M ; p H 8 . 3 ) :

D i s s o l v e 2 4 . 3 g. t r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e in 8 0 0 m l . d o u b l y d i s t i l l e d w a t e r ,
adjust t o p H 8.3 ( g l a s s e l e c t r o d e ) w i t h c a . 2 0 m l . c o n e . HC1 a n d d i l u t e t o 1 0 0 0 m l . w i t h
d o u b l y distilled water.
III. P h o s p h a t e buffer (0.1 M ; p H 8 . 3 ) :
D i s s o l v e 3 5 . 8 g. N a H P 0 1 2 H 0 in 8 0 0 m l . d o u b l y d i s t i l l e d w a t e r , a d j u s t t o p H 8.3
2 4 2
w i t h ca. 2 0 m l . c o n e . H C 1 a n d d i l u t e t o 1 0 0 0 m l . w i t h d o u b l y d i s t i l l e d w a t e r .
I V . A m m o n i a (5 N ) :
D i l u t e c a . 2 5 m l . c o n e , a m m o n i a t o 100 m l . w i t h d o u b l y d i s t i l l e d w a t e r ; s t a n d a r d i z e
with 1 N HC1.
* C x where E = amount of enzyme in mg. and k = reaction constant (first order). Refer to t
E
"Peptidases", p. 950. The substrate concentration should be 50 m M . The initial rate of the reaction
is used for the calculations.
V . Tris buffer (ca. 5 m M ; p H 8 . 0 ; 5 m M M g C l ) : 2
D i s s o l v e 6 0 7 m g . tris a n d 1.016 g. M g C l • 6 H 0 in 9 0 0 m l . b o i l e d , d o u b l y distilled w a t e r ,

2 2
adjust t o p H 8.0 w i t h 0.1 N H C 1 a n d d i l u t e t o 1 0 0 0 m l . w i t h d o u b l y distilled w a t e r .

V I . D i i s o p r o p y l f l u o r o p h o s p h a t e , D F P (0.1 M ) :
B e w a r e o f i n h a l i n g e v e n s m a l l a m o u n t s o f t h e v a p o u r . A n t i d o t e : a t r o p i n e . A d d 1 g. D F P
t o 54.5 m l . a b s o l u t e i s o p r o p a n o l w i t h a n a u t o m a t i c p i p e t t e a n d m i x .
V I I . M a n g a n o u s c h l o r i d e (1 M ) :
D i s s o l v e 19.8 g. M n C l - 4 H 0 in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
2 2
V I I I . Tris buffer (0.5 M ; p H 8 . 5 ) :

D i s s o l v e 6.05 g. tris in 5 0 m l . d o u b l y d i s t i l l e d w a t e r , a d j u s t t o p H 8.5 ( g l a s s e l e c t r o d e )
w i t h c a . 5 m l . c o n e . H C 1 a n d d i l u t e t o 100 m l . d o u b l y d i s t i l l e d w a t e r .
IX. Spray reagent:
D i s s o l v e 2 5 0 m g . n i n h y d r i n in 95 m l . w a t e r - s a t u r a t e d n - b u t a n o l a n d a d d 5 m l . c o l l i d i n e .
X . C a r b o x y p e p t i d a s e (6 m g . p r o t e i n / m l . ) :
T h e c r y s t a l s f r o m 1 m l . s u s p e n s i o n in N a H C 0 3 solution dissolve on addition of 0 . 0 5 - 0 . 1
ml. 0.1 N N a O H ( p H —• 10) w i t h g e n t l e s t i r r i n g . A d d 0.1 m l . p h o s p h a t e buffer ( s o l u
10
t i o n III) o r 0.1 N H C 1 t o g i v e p H 7 . 8 - 8 . 3 ( p H - m e t e r ) . F o r e a c h m g . e n z y m e a d d 0 . 0 1 5 m l .
D F P s o l u t i o n ( V I ) , a n d after s t a n d i n g for o n e h o u r at 25 ° C t h e s o l u t i o n is r e a d y for u s e . 4 8
Stir larger a m o u n t s o f c r y s t a l s w i t h c o l d 1 0 % L i C l s o l u t i o n ; a r r a n g e t h e v o l u m e s o t h a t
the L i C l c o n c e n t r a t i o n in t h e e x p e r i m e n t s is 0 . 5 - 1 % . A d d 1 0 " 8
mole D F P (undiluted)
for e a c h m l . o f t h e clear s o l u t i o n a n d s t o r e at 0 ° C . 4 9
X I . Leucine aminopeptidase, L A P (30 mg. protein/ml.):

A d d 0.015 ml. D F P solution (VI) per ml. e n z y m e solution (50-fold m o l a r excess).
C o n t a m i n a t i n g p r o t e i n a s e s are i n a c t i v a t e d . D i a l y s e a g a i n s t tris buffer ( s o l u t i o n V ) t o
r e m o v e free a m i n o a c i d s t h a t h a v e b e e n h y d r o l y s e d f r o m c o n t a m i n a t i n g p r o t e i n s .
Activation of the e n z y m e : to 0 . 1 - 1 . 5 ml. of the dialysed solution (usually 1 to 3% pro
tein) a d d 0 . 0 2 5 m l . M n C l 2 s o l u t i o n ( V I I ) a n d 0.5 m l . tris buffer ( s o l u t i o n V I I I ) , d i l u t e t o
2.5 ml. w i t h d o u b l y d i s t i l l e d w a t e r a n d i n c u b a t e for 3 0 m i n . at 4 0 ° C .
Stability of Solutions
Carboxypeptidase dissolved in N a H C 0 solution or buffer should be used within a few days. The solut
3
ion in 10% LiCl solution is stable for several weeks.

Leucine aminopeptidase in 5 m M tris-MgCI buffer loses n o n e of its activity over several years, if stored
2
in the cold under toluene. As the enzyme is very unstable at p H < 7, it is r e c o m m e n d e d that the p H of
the stock solution should be occasionally checked.
Procedure
P r o t e i n is d i s s o l v e d in 0.001 N H C 1 a n d d i a l y s e d for 2 4 h o u r s at 4 ° C a g a i n s t t h e s o l v e n t t o
r e m o v e a d s o r b e d a m i n o a c i d s , p e p t i d e s o r c o n t a m i n a t i n g salts. F r e e z e - d r y t h e d i a l y s e d s o l u
t i o n . F o r the p r e l i m i n a r y e x p e r i m e n t s , d i s s o l v e w e i g h e d a m o u n t s in p h o s p h a t e buffer ( s o l u
t i o n III) or tris buffer ( s o l u t i o n II), w h i l e for t h e m a i n e x p e r i m e n t s u s e 1% N a H C 0 3 solution
as the s o l v e n t .
Assay System
A s the rate o f t h e e n z y m a t i c h y d r o l y s i s varies f r o m s u b s t r a t e t o s u b s t r a t e , t h e o p t i m u m c o n

ditions must be determined beforehand. T h e substrate and e n z y m e concentrations, reaction
t e m p e r a t u r e a n d s a m p l i n g t i m e s are v a r i e d in p r e l i m i n a r y e x p e r i m e n t s . T h e a m i n o g r o u p s
liberated are d e t e r m i n e d o n s m a l l a m o u n t s o f t h e h y d r o l y s i s m i x t u r e w i t h n i n h y d r i n o r b y
p a p e r c h r o m a t o g r a p h y . T h e latter a l s o g i v e s a g e n e r a l i d e a o f t h e a m i n o a c i d s f o r m e d .
Preliminary Experiments
Enzymatic hydrolysis: P i p e t t e i n t o a 10 m l . c e n t r i f u g e t u b e (in a 2 5 ° C w a t e r b a t h ) :
2 . 0 0 m l . s a m p l e ( 0 . 2 jumole p r o t e i n * ) ,
0.01 m l . e n z y m e s o l u t i o n ( X o r X I ) ( 6 0 /ig. c a r b o x y p e p t i d a s e or 30 îg. L A P ) .
M i x , a n d s t o p p e r t h e t u b e . O v e r a p e r i o d o f 8 h o u r s , r e m o v e 0.1 m l . s a m p l e s for t h e n i n h y d r i n
r e a c t i o n a n d 0 . 0 1 - 0 . 0 3 m l . s a m p l e s for p a p e r c h r o m a t o g r a p h y . T i m e s : 5, 10, 3 0 , 6 0 , 120, 3 0 0 ,
420 min., etc.
Variations:
C a r b o x y p e p t i d a s e s o l u t i o n ( X ) : 0 . 0 0 1 - 0 . 5 m l . (6 f.ig.-3 mg. protein)

L A P solution (XI): 0 . 0 0 2 - 0 . 2 ml. (30 / £ g . - 6 m g . protein)
Temperature: 8 - 4 0 °C
Duration of reaction: 4 hours to 3 days
F o r e x p e r i m e n t s l a s t i n g m o r e t h a n 12 h o u r s c o v e r t h e r e a c t i o n s o l u t i o n w i t h a 5 m m . l a y e r o f
t o l u e n e . W i t h r e a c t i o n v o l u m e s o v e r 1.5 m l . t a k e 0.2 ml. s a m p l e s i n s t e a d o f 0.1 m l . ' . 2 3 5 0
Ninhydrin reaction: T o d e p r o t e i n i z e , a d d t o a 10 m l . c e n t r i f u g e t u b e :
0.1 m l . 1 0 % t r i c h l o r o a c e t i c a c i d s o l u t i o n * *
0.1 m l . s a m p l e after t h e e n z y m a t i c r e a c t i o n .
M i x , c e n t r i f u g e in t h e c o l d a n d carry o u t t h e n i n h y d r i n r e a c t i o n ( a c c o r d i n g t o p . 1 6 2 7 ) o n 0.1 m l .
o f the s u p e r n a t a n t fluid. P l o t t h e jumole a m i n o a c i d ( o r d i n a t e ) a g a i n s t t h e s a m p l i n g t i m e
(abscissa).
Preparation for paper chromatography: P i p e t t e i n t o a s m a l l test t u b e :
0.01 - 0 . 0 3 m l . s a m p l e after t h e e n z y m a t i c h y d r o l y s i s
0.05 m l . 0.1 N H C 1 ( i c e - c o l d )
or 0.10 ml. c o n e , formic acid (ice-cold).
F r e e z e the m i x t u r e u n t i l r e a d y for p a p e r c h r o m a t o g r a p h y (see " M a i n E x p e r i m e n t " ) .

T h e s u p e r n a t a n t fluid after t r i c h l o r o a c e t i c a c i d d e p r o t e i n i z a t i o n c a n a l s o b e u s e d for p a p e r
c h r o m a t o g r a p h y . H o w e v e r , t h e t r i c h l o r o a c e t i c a c i d m u s t first b e r e m o v e d w i t h a n a n i o n
e x c h a n g e resin ( e . g . A m b e r l i t e I R 4 B ) . 5 1
* i. e. 14 mg. for a protein with a molecular weight of 70000.

** Same volume as the sample, i. e. 0.2 ml. for 0.2 ml. sample.
Main Experiment
Enzymatic hydrolysis: U s e the o p t i m u m reaction temperature, a m o u n t of e n z y m e and time

o f s a m p l i n g ( 0 . 2 m l . ) d e t e r m i n e d in t h e p r e l i m i n a r y e x p e r i m e n t s . T o t a l v o l u m e o f t h e r e a c t i o n
m i x t u r e : 1 - 5 m l . ; 0 . 5 - 2 jumole p r o t e i n s a m p l e d i s s o l v e d in 1 % b i c a r b o n a t e s o l u t i o n . O t h e r
w i s e t h e m i x t u r e is a s for t h e p r e l i m i n a r y e x p e r i m e n t s . p H 8 . 0 - 8 . 3 .
C h e c k t h e p H o f t h e m i x t u r e ( g l a s s e l e c t r o d e ) w h e n e a c h s a m p l e is r e m o v e d a n d adjust w i t h
0.1 N N a O H f r o m a m i c r o - b u r e t t e .
Preparation of samples for paper chromatography: P i p e t t e i n t o 2 0 m l . test t u b e s w i t h g r o u n d -

glass stoppers:
s u s p e n s i o n o f D o w e x 5 0 x 8* ( H +
-form)
(25 m g . o f i o n e x c h a n g e resin p e r 0.1 / m i o l e a m i n o a c i d c a l c u l a t e d a c c o r d i n g t o
the preliminary experiments)
o r 0.1 m l . s a m p l e after t h e e n z y m a t i c h y d r o l y s i s ( p r e l i m i n a r y e x p e r i m e n t s ) . T h e p e p t i d a s e
r e a c t i o n is s t o p p e r e d b y l o w e r i n g t h e p H t o c a . 3. S t o p p e r t h e t u b e s a n d s h a k e m e c h a n i c a l l y
for 1 h o u r . D e c a n t t h e s u p e r n a t a n t a n d d i s c a r d . W a s h t h e resin 4 t i m e s w i t h
5 m l . w a t e r p e r g r a m resin
a n d d i s c a r d t h e w a s h i n g s . T o e l u t e t h e a m i n o a c i d s s h a k e for 10 m i n . w i t h
4 m l . 5 N N H O H p e r g r a m resin,
4
l e a v e t h e c o m b i n e d s u p e r n a t a n t s o v e r n i g h t in a d e s i c c a t o r o v e r c o n e . H S 02 4 and then lyophilize

o r dry c o m p l e t e l y o v e r H S0 .
2 4
Paper chromatography . * * A s c e n d i n g , l e n g t h o f r u n 3 0 - 4 5 c m . S o l v e n t s y s t e m s : a) n - b u t a n o l :
glacial a c e t i c a c i d : w a t e r 4 : 1 : 5 ; b) s e c - b u t a n o l : f o r m i c a c i d : w a t e r 7 0 : 15 : 5 ; c) m e t h y l
ethyl k e t o n e .-pyridine : w a t e r 7 0 : 15 : 5.
F o r t h e rapid s e l e c t i o n o f a s u i t a b l e s y s t e m , p r e p a r e trial t w o - d i m e n s i o n a l chromatograms
according t o 5 5
o r . F o r t h e s e m i - q u a n t i t a t i v e d e t e r m i n a t i o n * * * c h r o m a t o g r a p h 1 - 1 5 pg.
5 6
of
e a c h a m i n o a c i d in t h e b e s t s o l v e n t s y s t e m . S p o t t h e t r e a t e d s a m p l e s f r o m t h e m a i n a n d p r e l i m i n
ary e x p e r i m e n t s o n t h e p a p e r at 2 c m . i n t e r v a l s (5 m m . d i a m e t e r s p o t s ) . A l s o s p o t test m i x t u r e s
c o n t a i n i n g k n o w n a m i n o a c i d s in v a r i o u s c o n c e n t r a t i o n s (serial d i l u t i o n s ) .
D r y the c h r o m a t o g r a m s w h i c h have been d e v e l o p e d with solvent, spray with the spray reagent
( s o l u t i o n I X ) until t h e p a p e r s h o w s a " s i l k y l u s t r e " a n d t h e n d e v e l o p in a d r y i n g o v e n at
100 °C for 6 m i n . . +
* O r Nalcite H C R or other suitable polystyrenesulphonic acid ion exchange resin.

'* High voltage electrophoresis at p H 6 . 5 , 3 . 5 or 1.8 can be used to identify the hydrolysis p r o d u c t s ,
52 53 54
especially in studies on peptides. This can be followed by paper c h r o m a t o g r a p h y for the second
dimension.
:
**The carboxyl end g r o u p of a protein can be determined quantitatively after the reaction with carboxy
peptidase ( p H 9; 16 hours) by the dinitrofluorobenzene m e t h o d . 57
+
The addition of collidine to the spray reagent facilitates the identification of the a m i n o acids by the
formation of different colours.
Evaluation
Cut the c h r o m a t o g r a m of the m a i n experiment into strips. Classify the a m i n o acid spots of the experiment
by subjective c o m p a r i s o n with the c o r r e s p o n d i n g dilutions of the k n o w n a m i n o acids. This a p p r o x i m a t i o n
is liable to an error of 1 0 % . T h e strips can also be evaluated colorimetrically. T h e c h r o m a t o g r a m s of the
serial dilutions of the k n o w n a m i n o acids serve as the s t a n d a r d s . Plot the fig. a m i n o acid (ordinate)
against the sampling time (abscissa). T h e type of curve obtained with each a m i n o acid indicates the a m i n o
sequence (Fig. 2).
Fig. 2. Schematic representation of the action

of carboxypeptidase on a protein. Semi
quantitative p a p e r c h r o m a t o g r a p h i c deter
mination of the a m i n o acids 1 - 4 . It follows
from the figure that the sequence at the
carboxyl end of the protein is 1, 2, 3 and 4.
Time
S p e c i f i c i t y and L i m i t s
Carboxypeptidase: F o r the action of carboxypeptidase the free carboxyl g r o u p and the peptide linkage
on the a m i n o g r o u p of the second a m i n o acid (b) are essential.
O R' O R"
II I II
—C—NH—C—C—NH—C—COOH
(b) (a) |
H
The residues R " and R ' determine the rate of the hydrolysis. F r o m the action of carboxypeptidase on
synthetic peptides it follows that the a r o m a t i c a m i n o acids are hydrolysed most easily, and then a m i n o
acids with aliphatic R " . T h e ease of hydrolysis is greater for long chain a m i n o acids t h a n for short chain.
A n a m i n o acid at the carboxyl end carrying a n ionized g r o u p in the chain (e. g. arginine o r aspartic acid)
considerably retards the enzymatic reaction, while proline and hydroxyproline are not attacked at all.
T h e hydrolysis therefore comes to a stop at these points in a peptide c h a i n 5 8 , 5 9
. T h e residue R ' also exerts
an influence: a glutamyl or prolyl residue in the adjacent position retards the hydrolysis of the b o n d 6 0 , 6 1
.
Leucine aminopeptidase: T h e action of this enzyme h a s been tested with the amides of the c o m m o n
a m i n o a c i d s . T h e activity with leucinamide, which is hydrolysed best, serves as a point of reference
62
(100 % ) . T h e best substrates a p a r t from leucine are norvaline, isoleucine, phenylalanine, t r y p t o p h a n , tyrosine,
histidine and valine ( 8 4 - 1 6 % ) . A m i d e s of a m i n o acids with charged side chains are hydrolysed m o r e
slowly ( 7 - 2 % ) , as are amides of alanine and glycine (3 and 0.13%) and of proline and hydroxyproline
(0.7 and 0.5%). However, an imino acid obviously forms no obstacle to the hydrolysis of a peptide chain.
In general, the hydrolysis with leucine a m i n o p e p t i d a s e does not come to a stop so rapidly as that with
carboxypeptidase; the hydrolysis of 24 a m i n o acid residues has been d e s c r i b e d ' . 4 2 6 3
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53 F. Rodbell & R. Frederickson, J. biol. C h e m . 234, 562 [1959].
54 H. N. Atfield & C. / . O. R. Morris, J. 74, 37 P [I960].
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56 F. Turba: C h r o m a t o g r a p h i s e h e M e t h o d e n in der Proteinchemie. Springer, Heidelberg 1954. p . 174.
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58 E. L. Smith in G. E. W. Wolstenholme & M. P. Cameron: Chemical Structure of Proteins. Little
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64 A. Tsugita, D. T. Gish, J. Young, H. Fraenkel-Conrat, C. A. Knight, & W. M. Stanley, P r o c . N a t . Acad.
Sci. U . S. 46, 1463 [I960].
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66 D. G. Smyth, W. H. Stein & S. Moore, J. biol. C h e m . 237, 1845 [1962].
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Determination of Acetyl Groups in Proteins
Hugo Fasold
Proteins consist of linear sequences of a m i n o acids joined to one a n o t h e r by peptide linkages. They
must therefore always have at least one free a-amino g r o u p a n d one free a - C O O H g r o u p . There is a
series of m e t h o d s for the identification of b o t h terminal a m i n o acids (end groups). T h e a m i n o end g r o u p
in proteins is frequently acetylated. In addition to o t h e r m e t h o d s , such as hydrazinolysis or hydrolysis 1
and gas c h r o m a t o g r a p h y , an enzymatic m e t h o d h a s been developed for the detection a n d quantitative

2
determination of such acetylated end g r o u p s . 3 , 4
Application of Method: Qualitative detection of acetyl groups, where evidence points to an acetylated end
g r o u p a n d the usual a m i n o end g r o u p determinations give negative results. If the molecular weight of the
protein is k n o w n , the quantitative d e t e r m i n a t i o n gives the n u m b e r of acetylated groups, a n d helps to
determine the n u m b e r of peptide chains per molecule. It can also be useful in molecular weight determina
tions.
Principle
The protein is treated with concentrated sulphuric acid, a n d the acetic acid liberated is extracted a n d
assayed enzymatically with the aid of acetate kinase, citrate synthase, a n d m a l a t e dehydrogenase (for
principle of the acetate determination, see p . 1520).
O p t i m u m C o n d i t i o n s for M e a s u r e m e n t s
Since the spectrophotometric assay gives the best results in the range between 0.02 a n d 0.07 jumole acetate,
and since several parallel d e t e r m i n a t i o n s are desirable, it is advisable t o use 0.1 t o 0.2 //mole protein,
i. e. 6 to 12 mg. of protein having a molecular weight of 60000. F o r o p t i m u m conditions for the deter
mination of acetate, see p . 1521.
Equipment
H y d r o l y s i s t u b e s 10 t o 15 c m . l o n g , J e n a g l a s s t u b e s h a v i n g a n i n s i d e d i a m e t e r o f 0 . 4 t o 0.5 c m .
a n d a w a l l t h i c k n e s s o f a b o u t 0.7 m m . , w i t h o n e e n d s e a l e d a n d a n n e a l e d . P h o t o m e t e r a n d e q u i p
m e n t for e n z y m a t i c d e t e r m i n a t i o n o f a c e t a t e a s d e s c r i b e d o n p. 1 5 2 1 .
Reagents
1. C o n e , s u l p h u r i c a c i d , A . R., s p . g r . 1.8 6. Tris-hydroxymethyl-aminomethane,

2. Purified n i t r o g e n , steel c y l i n d e r tris, p u r e
3. S o d i u m s u l p h a t e , A . R., a n h y d r o u s 7. H y d r o c h l o r i c a c i d , A . R., 3 6 % ( w / v )
4. P o t a s s i u m h y d r o x i d e , A . R. 8. H y d r o c h l o r i c a c i d , A . R., 1 N a n d 2 N
5. t - B u t y l e t h y l e t h e r o r i s o p r o p y l e t h e r o r 9. P o t a s s i u m h y d r o x i d e s o l u t i o n , A . R., 0.5 N
i s o a m y l nitrite and2N
R e a g e n t s for t h e d e t e r m i n a t i o n o f a c e t a t e , s e e p . 1 5 2 1 .
Acetyl Groups in Proteins 1641
Purity of Reagents
Before use, the organic solvents, of which t-butyl ethyl ether is the most suitable, are extracted with an
equal volume of 0.5 N potassium hydroxide solution to remove traces of acetic acid, and are then washed
with an equal volume of distilled water.
I. T r i s - b u f f e r / K O H m i x t u r e (1 M tris, 1 N K O H ) :
D i s s o l v e 2 4 3 g. tris i n 6 0 0 m l . d i s t i l l e d w a t e r , a d j u s t p H t o 8.2 ( g l a s s e l e c t r o d e ) w i t h c a .
200 ml. cone. HC1, a n d m a k e u p to 1 0 0 0 ml. with distilled water. F o r use, a d d 5 ml. 2 N K O H
(titrate) t o 5 m l . tris buffer in a 10 m l . g r a d u a t e d flask a n d m a k e u p a c c u r a t e l y w i t h d i s t i l l e d
water.
S o l u t i o n s for d e t e r m i n a t i o n o f a c e t a t e , s e e p . 1 5 2 2 .
It is recommended that the tris buffer be kept cold and that a hydrolysis tube or test tube filled with toluene
be placed in the bottle in such a way that the mouth is above the surface of the liquid, so that the two
liquids cannot mix. (Prevent growth o f micro-organisms.)
Procedure
Collection of Samples
W e i g h t h e p u r e p r o t e i n t o b e i n v e s t i g a t e d , a s l y o p h i l i z a t e , i n t o a h y d r o l y s i s t u b e , a n d dry t o
c o n s t a n t w e i g h t in a d r y i n g p i s t o l at 6 0 ° C . A 5 - 1 0 % s o l u t i o n o f t h e p r o t e i n m a y b e u s e d if
a n a l i q u o t is l y o p h i l i z e d after a c c u r a t e d e t e r m i n a t i o n o f t h e p r o t e i n c o n t e n t in t h e t u b e o r
u s e d directly for t h e h y d r o l y s i s in q u a n t i t i e s o f u p t o 0.1 m l .
Assay System
U s e b l a n k s c o n t a i n i n g all t h e r e a g e n t s ( b u t n o p r o t e i n ) t o d e t e r m i n e t h e a c e t a t e c o n t e n t o f t h e
reagents.
T o rule o u t free a c e t a t e i n t h e p r o t e i n , s u s p e n d t h e u n h y d r o l y s e d p r o t e i n s a m p l e in 0 . 2 m l .
1 N H C 1 , d i a l y s e for 2 4 hr. a g a i n s t 1 m M H C 1 , a n d l y o p h i l i z e ; t h e n t r e a t a s for s a m p l e .
P i p e t t e 0.2 m l . c o n e , s u l p h u r i c a c i d o n t o t h e w e i g h e d p r o t e i n s a m p l e ; s l o w r o t a t i o n a n d t i l t i n g
in t h e ice b a t h l e a d s t o s u s p e n s i o n a n d s o l u t i o n o f t h e s a m p l e in a p p r o x . 2 m i n . ; l o n g e r t i m e s
are u n f a v o u r a b l e .
F l u s h t h e c o o l e d h y d r o l y s i s t u b e w i t h n i t r o g e n for 3 0 sec. b y m e a n s o f a g l a s s c a p i l l a r y ( d o n o t
t o u c h s o l u t i o n ) a n d s t o p p e r w i t h a p l u g o f c o t t o n w o o l ; fuse t h e t u b e . T h e d a r k s o l u t i o n
s h o u l d o c c u p y o n l y o n e t h i r d o r less o f t h e t o t a l v o l u m e .
S u s p e n d t h e h y d r o l y s i s t u b e in a 105 ° C oil b a t h for 1 2 0 m i n . T h e n c o o l in a n ice b a t h a n d c e n t r i
f u g e for a s h o r t t i m e t o c o l l e c t all t h e l i q u i d at t h e b o t t o m . S c r a t c h t h e c o l d t u b e , o p e n a s n e a r
the t o p as p o s s i b l e , a n d transfer t h e c o n t e n t s i n t o a test t u b e s t a n d i n g in a n ice b a t h b y m e a n s
o f a g l a s s t u b e d r a w n i n t o a c a p i l l a r y a n d fitted w i t h a r u b b e r b u l b ( P e l e u s b a l l ) . R i n s e h y d r o
lysis t u b e t w i c e w i t h 0.1 m l . i c e - c o l d d i s t i l l e d w a t e r .
A d d 150 m g . a n h y d r o u s s o d i u m s u l p h a t e a n d 0.5 m l . t - b u t y l e t h y l e t h e r ( o r o n e o f t h e o t h e r
o r g a n i c s o l v e n t s ) a n d m i x t h o r o u g h l y b y s h a k i n g in t h e ice b a t h . L e a v e in t h e ice b a t h for s e v e r a l
m i n u t e s until t h e p h a s e s s e p a r a t e . S y p h o n off o r g a n i c s o l v e n t a n d i n t r o d u c e i n t o a c o o l e d
test t u b e c o n t a i n i n g 0.3 m l . tris b u f f e r / K O H m i x t u r e ( s o l u t i o n I). S h a k e in ice b a t h a n d s y p h o n
off o r g a n i c p h a s e . R e p e a t e x t r a c t i o n o f t h e a c i d w i t h 0.5 m l . o r g a n i c s o l v e n t a n d r e - e x t r a c t i o n
i n t o tris b u f f e r / K O H m i x t u r e t h r e e t i m e s .
A f t e r t h e last e x t r a c t i o n , r e m o v e t r a c e s o f o r g a n i c s o l v e n t a s f o l l o w s . A r e v e r s e d , b o r e d r u b b e r
s t o p p e r t h a t is t o o large for t h e test t u b e is c o n n e c t e d t o a w a t e r - j e t p u m p b y a g l a s s t u b e .
T h e test t u b e is s u c k e d o n t o t h i s s t o p p e r . T h e t u b e is s h a k e n in t h e ice b a t h , a n d t h e s t o p p e r
c a n b e r e m o v e d as s o o n as f o a m i n g b e g i n s .
A d d 0.1 m l . 1.0 N H Q (titrate) a n d 0.1 m l . w a t e r ; t h e p H is a r o u n d 8 a n d t h e v o l u m e is 0.5 m l .
U s e 0 . 0 5 or 0.1 m l . a l i q u o t s o f t h i s s o l u t i o n , c o r r e s p o n d i n g t o b e t w e e n 0 . 0 2 a n d 0.08 / i m o l e
a c e t a t e a c c o r d i n g t o a p r e l i m i n a r y e x p e r i m e n t , f o r t h e e n z y m a t i c d e t e r m i n a t i o n in a c c o r d a n c e
w i t h p. 1 5 2 0 .
A c c u r a c y and P r e c i s i o n
0 . 9 - 1 . 0 /imole acetic acid is quantitatively a n d reliably recovered by the e x t r a c t i o n ; larger quantities

of acetylated protein should n o t be used. F o r reproducibility of t h e acetate d e t e r m i n a t i o n , see p 1527.
S o u r c e s of Error
The loss of v o l u m e on brief evacuation of t h e tris b u f f e r / K O H m i x t u r e t o r e m o v e the organic solvent

is negligible. F o r sources of error in t h e very specific enzymatic assay, see p . 1527.
A distinction between o-acyl a n d N-acyl derivatives a m o n g the acetyl g r o u p s detected is possible by
careful incubation with h y d r o x y l a m i n e ; only the o-acetyl groups are r e m o v e d from the protein.
5
References
1 H. Hormann, K. T. Joseph & M. v. Wilm, Hoppe-Seylers Z. physiol. C h e m . 341, 284 [1965].

2 R. E. Alving & K. Laki, Biochemistry 5, 2597 [1966].
3 L. D. Stegink, Analyt. Biochemistry 20, 502 [1967].
4 L. D. Stegink & C. S. Vestling, J. biol. C h e m . 241, 4923 [1966].
5 P. M. Gallop, S. Seifter, M. Lukin & E. Meilman, J. biol. C h e m . 235, 2619 [I960].
Glutathione
Erich Bernt and H a n s Ulrich Bergmeyer
T h e non-enzymatic m e t h o d s used previously for the d e t e r m i n a t i o n of glutathione, e.g. the colour reaction
with nitroprusside ,5,5'-dithiobis-2-nitrobenzoic a c i d or p h o s p h o t u n g s t i c a c i d , give results which are not
1 2 3
reproducible when applied to biological material. O t h e r m e t h o d s , e.g., the iodometric determination are
less specific. Enzymatic m e t h o d s are preferable for biological material because of their specificity (for a
review, see ). G l u t a t h i o n e ( G S H ) reacts quantitatively with methylglyoxal in the presence of glyoxalase I,
4
Gl-I (S-Lactoyl-glutathionemethylglyoxal-lyase, isomerizing, E C 4.4.1.5) to give S - l a c t o y l - G S H . Oxidized 5,6
glutathione ( G S S G ) is quantitatively reduced to G S H 7

by reduced nicotinamide-adenine dinucleotide
p h o s p h a t e ( N A D P H ) a n d glutathione reductase, G R ( N A D ( P ) H : oxidized glutathione oxidoreductase,
E C 1.6.4.2.).
Application of Method: In biochemistry a n d clinical biochemistry.
Principle
(1) G S H + Methylglyoxal - S ^ U S-Lactoyl-GSH
(2) GSSG + NADPH + H +

— ^ 2 GSH + NADP +
L a c t o y l - G S H is m e a s u r e d directly at 240 n m . T h e oxidation of N A D P H is measured by the change in ex

tinction at 340 (334, 365) n m . T h e t w o forms of glutathione are measured in the same assay mixture.
T h e G S H is determined first (measurement of the extinction at 240 n m ) and then G S S G is estimated in the
same cuvette (measurement of the extinctions at 340, 334 or 365 n m ) .
T h e equilibria of b o t h reactions lie far to the right. U n d e r the conditions described below the reactions
proceed stoichiometrically.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r f o r a c c u r a t e m e a s u r e m e n t s at 2 4 0 n m a n d
3 4 0 , 3 3 4 o r 365 n m ; b e n c h c e n t r i f u g e .
Reagents
1. T r i p o t a s s i u m p h o s p h a t e , K P 0
3 4 •3 H 0 2 4. Methylglyoxal
2. A l b u m i n ( e g g ) freshly distilled: steam distill a commercial
3. G l y o x a l a s e I, G l - I methylglyoxal solution (e.g. 3 0 % a q u e o u s solut
from yeast, solution in 3 0 % glycerol; ^ 200 ion from F l u k a & C o . , Switzerland) in the
U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , see Parnas- Wagner micro-distillation a p p a r a t u s .
p. 469.
5. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo 6. G l u t a t h i o n e r e d u c t a s e , G R
tide phosphate, N A D P H from yeast, crystalline suspension in 3.2 M
sodium salt, N A D P H - N a . Commercial prep
4
a m m o n i u m s u l p h a t e solution; ^ 120 U / m g .
aration, see p. 547. (25 ° C ) ; commercial p r e p a r a t i o n , see p . 465.
7. P e r c h l o r i c a c i d , c a . 7 0 % ( w / w ) ; s p . gr. 1.67.
G l u t a t h i o n e reductase a n d glyoxalase I m u s t n o t c o n t a i n m o r e t h a n 0 . 1 % of G - 6 - P D H , 6 - P G D H a n d
N A D P H oxidase (relative to their respective specific activities). Glyoxalase I must be absolutely free from
glyoxalase II.
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y d i s t i l l e d w a t e r . T o p r e v e n t t h e g r o w t h o f m i c r o
o r g a n i s m s sterilize all c o n t a i n e r s .
I. P h o s p h a t e ( 1 . 7 5 M K P 0 ) : 3 4
D i s s o l v e 4 6 . 5 g. K P 0 - 3 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
3 4 2
II. A l b u m i n (ca. 1 % w / v ) :
D i s s o l v e 100 m g . a l b u m i n ( e g g ) in 10 m l . d i s t i l l e d w a t e r a n d filter o r c e n t r i f u g e off i n s o l u b l e
material.
III. G l y o x a l a s e I, G l - I (1 m g . p r o t e i n / m l . ) :
Dilute the stock suspension with 3 0 % glycerol.
IV. M e t h y l g l y o x a l ( c a . 0.1 M ) :
D i l u t e t h e a q u e o u s s o l u t i o n (distillate) ca. 5 - f o l d w i t h d i s t i l l e d w a t e r . C h e c k t h e c o n c e n
tration by e n z y m a t i c assay, see p. 1496.
V . R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( c a . 11 m M N A D P H ) :
D i s s o l v e 10 m g . N A D P H - N a 4 i n 1 m l . 1% N a H C 0 . 3
V I . G l u t a t h i o n e r e d u c t a s e , G R (1 m g . p r o t e i n / m l . ) :
D i l u t e t h e s t o c k s u s p e n s i o n w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V I I . P e r c h l o r i c a c i d (ca. 1.0 M ) :
D i l u t e 8.6 m l . 7 0 % p e r c h l o r i c a c i d w i t h d i s t i l l e d w a t e r t o 1 0 0 m l .
Store all solutions a n d suspensions, stoppered, in a refrigerator at 0 to 4 °C. P r e p a r e the dilute methylglyoxal
solution, the albumin a n d N A D P H solutions freshly each week. T h e enzyme suspensions are stable for at
least 6 m o n t h s and all other solutions are stable indefinitely.
Procedure
Collection:
C o l l e c t b l o o d w i t h o u t v e n e s t a s i s a n d i m m e d i a t e l y d e p r o t e i n i z e . A n t i c o a g u l a n t s in t h e u s u a l
c o n c e n t r a t i o n s d o n o t interfere.
Glutathione 1645
H o m o g e n i z e i m m e d i a t e l y t i s s u e o b t a i n e d b y f r e e z e - c l a m p i n g ( s e e p . 4 0 0 ) in a P o t t e r - E l v e h j e m
o r U l t r a Turrax h o m o g e n i z e r .
Deproteinization:
W h o l e b l o o d : Deproteinize immediately to avoid oxidation of the glutathione. Pipette

successively into a centrifuge tube 5.00 ml. ice-cold perchloric acid solution (VII) a n d 5.00 ml.
b l o o d . M i x w e l l w i t h a t h i n g l a s s r o d a n d c e n t r i f u g e for 10 m i n . at 3 0 0 0 g. A d d . 0 . 9 0 m l .
p h o s p h a t e s o l u t i o n (I) t o 5 . 0 0 m l . o f t h e s u p e r n a t a n t fluid, a l l o w t o s t a n d for 15 m i n . in a n
ice b a t h a n d filter off t h e p e r c h l o r a t e p r e c i p i t a t e . U s e 0 . 5 m l . o f t h e filtrate (buffered t o c a . p H 7)
after e q u i l i b r a t i o n t o 2 5 ° C for t h e a s s a y .
T i s s u e : C a r e m u s t b e t a k e n w i t h h o m o g e n a t e s t h a t t h e filtrate after n e u t r a l i z a t i o n is virtually
free f r o m p e r c h l o r a t e a n d is a t p H 7. A n y a l t e r a t i o n in t h e v o l u m e o f s o l u t i o n I a d d e d m u s t
b e c o r r e c t e d for in t h e c a l c u l a t i o n s .
Stability of sample:
G S H c o n t a i n e d in b l o o d is o x i d i z e d t o G S S G v e r y r a p i d l y . If b l o o d is a l l o w e d t o s t a n d for
c a . 2 hr. b e f o r e d e p r o t e i n i z a t i o n t h e n all t h e g l u t a t h i o n e is f o u n d a s G S S G .
E v e n in d e p r o t e i n i z e d s o l u t i o n s G S H is o x i d i z e d at a significant rate. It is t h e r e f o r e n e c e s s a r y
t o w o r k r a p i d l y t o o b t a i n p r e c i s e r e s u l t s . T h e s a m e is t r u e o f t i s s u e e x t r a c t s .
Assay System
W a v e l e n g t h : 2 4 0 n m for G S H ; 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m for G S S G ; light p a t h : 1 c m . , silica

c u v e t t e s ; final v o l u m e : 3 . 1 8 m l . for G S H a n d 3.31 m l . for G S S G ; r o o m t e m p e r a t u r e . G S H
m e a s u r e m e n t s a g a i n s t b l a n k , G S S G m e a s u r e m e n t s a g a i n s t air.
GSH Determination (240 nm)
C o n c e n t r a t i o n in a s s a y
Pipette into cuvettes Sample Blank
mixture (sample)
Sample (deproteinized,
neutralized) 0.50 ml. 0.40 ml. u p t o 120 fiM GSH
(GSSG)
Distilled water 2.50 ml. 2.50 ml.
Albumin solution (II) 0.15 ml. 0.15 ml. 0.47 m g . / m l .
Gl-I suspension (III) 0.01 m l . 3.1 / i g . / m l . ^ 0.6 U / m l .
M i x , read extinction E. l
Methylglyoxal solution (IV) 0.02 ml. — ca. 0 . 6 2 m M
M i x . R e a d t h e e x t i n c t i o n s at 8, 10, 1 2 , 14 a n d 16 m i n . a n d
determine extinction E 2 by extrapolation to the time of Gl-I
a d d i t i o n (refer t o p. 3 0 8 ) .
Methylglyoxal solution (IV) 0.02 ml. —

M i x . R e a d t h e e x t i n c t i o n s at 2 , 4 , 6 a n d 8 m i n . , a n d d e t e r m i n e E 3
by extrapolation to the time o f the second methylglyoxal

addition. E 3 - E 2 = A E m e t h y l g l y o x a l ; E 2 — Ej - A E m e t h y l g l y o x a l
— d E G S H .
GSSG Determination (340, Hg 334, Hg 365 nm)
Pipette the following into the sample

C o n c e n t r a t i o n in a s s a y m i x t u r e
cuvette only:
N A D P H solution (V) 0.10 ml. 0.33 m M
M i x a n d read e x t i n c t i o n E . 4
G R suspension (VI) 0.01 m l . 3.1 jug./ml. > 0.18 U / m l .
M i x . R e a d e x t i n c t i o n at 8, 10, 12, 14 a n d 16 m i n . a n d
determine extinction E 5 by extrapolation to the time
o f G R a d d i t i o n (refer t o p . 3 0 8 ) . E 4 — E 5 = Ê G S S G
is u s e d for the c a l c u l a t i o n s .
Calculations
Both reactions proceed stoichiometrically u n d e r the conditions described a b o v e . T h e calculation formula

(2) on p. 312 applies. The extinction coefficient of S-lactoyl-GSH at 240 n m is 3.37 c m . / ^ m o l e according 2
to Racker and that of N A D P H is 6.22 c m . / / / m o l e at 340 n m . T h e results are obtained in //mole G S H

5 2
Glutathione 1647
or G S S G / m l . sample. This value m u s t be multiplied by a factor if the sample has been deproteinized,
neutralized or diluted in any way. In the case of whole b l o o d the specific gravity (ca. 1.06) and the water
content (80%) m u s t be taken into account.
In this m e t h o d , where whole blood is diluted 1 + 1 for deproteinization a n d 5.0 + 0.9 on neutralization
the factor is 2.18. T h e following relationships h o l d :
Wavelength : 240 nm 334 nm 340 nm 365 nm
GSH c = AE x 4.11 frimole/ml.]

AE x 1263 [/ig./ml.]
GSSG AE x 2.37 AE x 2.32 AE x 4.18 [//mole/ml.]
AE x 1449 AE x 1421 AE x 2563 [pg./ml]
With a m e a n value of 400 jig. G S H / m l . whole b l o o d the s t a n d a r d deviation was 24 /ig. a n d the coefficient
of variation 6.0%.
Normal Values
Blood contains 28 to 52 mg. glutathione/100 ml. which is exclusively in the e r y t h r o c y t e s . Widely differing 8
values have been reported for o t h e r tissues. T h e glutathione content of cat o r g a n s has been studied by
H. H. Tallan et a l . . 9
S o u r c e s o f Error
Effects of drugs and other therapeutic measures: None known.
Interference in the assay: Impurities of the reagents, especially of the enzymes, results in high values.
If the glyoxalase I contains glyoxalase II t o o little G S H will be recovered, while the presence of N A D P H
oxidase in the glutathione reductase gives G S S G values which are t o o high. G l u t a t h i o n e a d d e d to tissue
h o m o g e n a t e s is not fully recovered; the reasons for this are n o t k n o w n .
Specificity
In t h e p r e s e n c e o f l a r g e a m o u n t s o f a s p a r t a t h i o n e o r i s o g l u t a t h i o n e , h i g h v a l u e s for G S H are
f o u n d . G R is a b s o l u t e l y specific for G S S G .
1 0
References
1 A. Fujita & I. Numata, Biochem. Z. 300, 246, 257 [1939].

2 G. L. Ellman, Arch. Biochem. Biophys. 82, 70 [1959].
3 K. Shinohara, J. biol. C h e m . 109, 665 [1935]; 110, 263 [1935].
4 / . W. Patterson & A. Lazarow i n : G l u t a t h i o n e , a S y m p o s i u m . A c a d e m i c Press, N e w York 1954, p . 63.
5 E. Racker, J. biol. C h e m . 190, 685 [1951].
6 Th. Wieland, K. Dose & G. Pfleiderer, Biochem. Z. 326, 442 [1955].
7 T. W. Rail & A. L. Lehninger, J. biol. C h e m . 194, 119 [1952].
8 Hoppe-Seyler-Thierfelder: H a n d b u c h der physiologisch- u n d pathologisch-chemischen Analyse, 10th
Edn., Springer-Verlag, Berlin, G o t t i n g e n , Heidelberg 1953, vol. V, p . 44.
9 H. H. Tallan, S. Moore & W. H. Stein, J. biol. C h e m . 211, 927 [1954].
10 O. K. Behrend, J. biol. C h e m . 141, 503 [1952].
D-Amino Acids
Paul Boulanger and Roger O s t e u x t
The oxidative d e a m i n a t i o n of a - a m i n o acids h a s been k n o w n since 1909. Neubauer 1

a n d Knoop2
showed
that this process c o n s u m e d oxygen a n d resulted in the formation of a m m o n i a a n d a-keto acids. Kidney
tissue contains an enzyme, D - a m i n o acid oxidase, D - A O D ( D - A m i n o a c i d : oxygen oxidoreductase, d e a m i n a -
ting; E C 1.4.3.3), which specifically d e a m i n a t e s D - a m i n o a c i d s , a n d which requires flavine-adenine dinuc-
3
leotide ( F A D ) as coenzyme. Greenstein

4 5
used this enzyme t o p r e p a r e the p u r e L-isomers from a m i n o
acid racemates.
Table 1 shows the activity of a D - a m i n o acid oxidase p r e p a r a t i o n from sheep kidney with various D - a m i n o
a c i d s . We found t h a t a n enzyme from pig kidney was also able t o d e a m i n a t e D-aspartic acid a n d D-gluta-
6
mic acid if the enzyme c o n c e n t r a t i o n was increased a n d the reaction time was p r o l o n g e d .
Table 1. T h e r a t e of oxidation of D - a m i n o acids by D - a m i n o acid oxidase from sheep kidney.
p\. 0 c o n s u m e d / h r . / m g .
2 fil. 0 c o n s u m e d / h r . / m g .
2
D - A m i n o acid dry weight of enzyme D - A m i n o acid d r y weight of enzyme

preparation preparation
Alanine 64 Tyrosine 190

a-Aminobutyric acid 31 Histidine 6.2
a-Aminovaleric acid 20 Tryptophan 37
a-Aminocaproic acid 36 Lysine 0.6
Valine 35 Ornithine 3.1
Leucine 14 Serine 42
Isoleucine 22 Threonine 2.1
Aspartic acid 1.4* Proline 148
G l u t a m i c acid 0* Pipecolinic acid 2.6
a-Aminoadipic acid 0 Methionine 80
Phenylalanine 26 Cystine 1.9
* see text
D - A m i n o acid oxidase is found in the kidney a n d liver of all m a m m a l s a n d some o t h e r vertebrates, the
kidney of sheep a n d pig being especially rich in the enzyme. T h e enzyme from the h e p a t o p a n c r e a s of
Octopus 28
is active with D-aspartic acid a n d D-glutamic acid. D - A m i n o acid oxidase p r e p a r a t i o n s from
Neurospora crassa , 8
Aspergillus niger ,
9
Proteus morganii 10
a n d o t h e r bacteria are significantly less active
t h a n those from pig or sheep kidney.
Application of Method: In biochemistry a n d in food chemistry.
Manometric Method
Principle
(1) R—CH—COOH + 0 2
D
" A O D
) R—C—COOH + H 0
2 2
I II
NH 2 NH
t Deceased
D - A m i n o Acids 1649
T h e imino acid decomposes s p o n t a n e o u s l y :
(2) R—C—COOH + H 0 - • R—C—COOH + 2 NH 3
II I I
NH O
If the hydrogen peroxide formed in reaction (1) is destroyed by catalase, the over-all reaction is:
(3) R — C H — C O O H + V 0 -> R—C—COOH + 2 2 NH 3
I I I
NH 2 O
One mole of D - a m i n o acid yields one mole of 2-oxo acid a n d one mole of a m m o n i a , a n d 0.5 mole of oxygen
is consumed. This oxygen c o n s u m p t i o n is determined and, except in the case of D - a m i n o a d i p i c acid (as
well as glutamic acid a n d lysine, which usually d o n o t react completely), is a m e a s u r e of the total a m o u n t
of D - a m i n o acids contained in the sample.
The o p t i m u m p H for the reaction is 8.3. T h e enzyme c o n c e n t r a t i o n should be high. F o r a reaction v o l u m e

of 3 ml., the quantity of crude enzyme should c o r r e s p o n d to 0.25 g. of fresh kidney tissue, a n d the quantity
of p u r e enzyme to 1 g. of fresh tissue. T h e a m i n o acid concentration in the assay should be 5 - 1 0 m M .
The reaction proceeds best in an a t m o s p h e r e of oxygen.
Equipment
Conventional W a r b u r g a p p a r a t u s . M a n o m e t e r vessels with side-arm a n d centre well, 14 m l . ; 5 ml. vessels

for the micro m e t h o d .
Reagents
1. S o d i u m p y r o p h o s p h a t e , A . R . , 5. C a t a l a s e
Na P O 10H O
4 2 7 2
p o w d e r ; commercial p r e p a r a t i o n , see p . 438.
2. P o t a s s i u m h y d r o x i d e , A . R . , 2 N 6. D - A m i n o a c i d o x i d a s e
3. H y d r o c h l o r i c a c i d , A . R . , 1 N isolation of the enzyme, see p . 1654.
4. D - A l a n i n e commercial p r e p a r a t i o n , see p . 4 3 1 .
c h r o m a t o g r a p h i c a l l y p u r e , free from L-alanine
The crude D - a m i n o acid oxidase p r e p a r a t i o n obtained from sheep or pig kidney (p. 1654) satisfies the
requirements, as d o the commercially available lyophilized catalase p r e p a r a t i o n s .
I. P y r o p h o s p h a t e buffer (0.1 M ; p H 8 . 3 ) :
D i s s o l v e 8 . 9 2 2 g. N a P 0
4 2 7 • 1 0 H O in 100 m l . d o u b l y d i s t i l l e d w a t e r , a d d 8 m l . 1 N H C 1
2
and dilute to 200 ml. with d o u b l y distilled water.

II. D - A l a n i n e ( 1 0 / i m o l e / m l . ) :
D i s s o l v e 89.1 m g . D - a l a n i n e in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
III. D - A m i n o a c i d o x i d a s e :
U s e t h e c r u d e e n z y m e p r e p a r e d a c c o r d i n g t o p. 1 6 5 4 d i r e c t l y .
K e e p the p y r o p h o s p h a t e buffer (I) in a well-stoppered container at -f 1 to + 4 °C. T h e enzyme solution

(III) keeps for several days at -f 4 °C.
Procedure
T h e p r e l i m i n a r y t r e a t m e n t o f t h e s a m p l e d e p e n d s o n its c o m p o s i t i o n . It is useful t o c o n f i r m t h e
e x p e r i m e n t a l r e sults b y d e t e r m i n i n g t h e a m o u n t s o f a m m o n i a a n d o f 2 - o x o a c i d f o r m e d in t h e
reaction mixture.
A m i n i m u m o f 10 /xmole D - a m i n o a c i d , in n o t m o r e t h a n 2 m l . s o l u t i o n ( p H 8.3), are r e q u i r e d
for t h e m a n o m e t r i c d e t e r m i n a t i o n . R e m o v e H C 1 f r o m a c i d p r o t e i n h y d r o l y s a t e s b y c o n t i n u o u s
a e r a t i o n a n d w a r m i n g , o r b e t t e r still, b y p a s s i n g t h e s o l u t i o n t h r o u g h a c a t i o n e x c h a n g e c o l u m n
a n d e l u t i n g w i t h d i l u t e a m m o n i a s o l u t i o n . C o n c e n t r a t e t h e e l u a t e at l o w
1 1
temperature,
preferably lyophilize to a v o i d racemization o f the a m i n o acids. All the a m m o n i a must be
r e m o v e d s i n c e it w o u l d interfere w i t h t h e d e t e r m i n a t i o n o f t h e a m m o n i a l i b e r a t e d o n d e a m i n a -
t i o n . S o l u t i o n s c o n t a i n i n g D - a m i n o a c i d s (e. g. b i o l o g i c a l fluids s u c h a s p l a s m a o r s e r u m , o r
e n z y m a t i c r e a c t i o n m i x t u r e s ) m a y b e u s e d w i t h o u t p r e t r e a t m e n t , if t h e y c o n t a i n sufficient
D - a m i n o acids, d o not have t o o high a blank oxygen c o n s u m p t i o n and d o not contain t o o m u c h
a m m o n i a o r 2 - o x o a c i d s . O t h e r w i s e d e p r o t e i n i z e a n d c o n c e n t r a t e t h e free a m i n o a c i d s b y
c h r o m a t o g r a p h y o n a c a t i o n e x c h a n g e resin.
Assay System
T h e d e t e r m i n a t i o n is c a r r i e d o u t w i t h a W a r b u r g a p p a r a t u s . V e s s e l s w i t h s i d e - a r m a n d c e n t r e
w e l l ; t e m p e r a t u r e 37 ° C ; g a s p h a s e : o x y g e n ; final v o l u m e 3.0 m l . F i v e flasks are r e q u i r e d for
each determination:
F l a s k 1. T h e r m o b a r o m e t e r .
F l a s k 2. E n z y m e b l a n k for t h e e s t i m a t i o n o f 0 2 uptake o f the crude D - a m i n o acid oxidase

s o l u t i o n ( o m i t t e d if t h e purified e n z y m e is u s e d ) .
F l a s k 3 . S t a n d a r d t o c h e c k t h a t t h e a s s a y is f u n c t i o n i n g ( 2 0 / / m o l e o f D - a l a n i n e s h o u l d b e
o x i d a t i v e l y d e a m i n a t e d in c a . 2 0 m i n . w i t h t h e c o n s u m p t i o n o f 2 2 4 p\. 0 ).
2
F l a s k 4. S a m p l e b l a n k for t h e e s t i m a t i o n o f t h e 0 2 uptake o f the s a m p l e (not always necessary;

b u t t h e s o l u t i o n s e r v e s for t h e d e t e r m i n a t i o n o f a m m o n i a i n t h e s a m p l e b e f o r e t h e
action of the e n z y m e ) .
F l a s k 5. S a m p l e for d e t e r m i n a t i o n .
Flask N o .
Pipette into manometer flasks:
1 2 3 4 5
Main compartment:
P y r o p h o s p h a t e buffer (I) 0.75 ml. 0.75 ml. 0.75 ml. 0.75 ml. 0.75 ml.
Catalase 0.5 m g . 0.5 m g . 0.5 m g . 0.5 m g .
D-Alanine solution (II) 2.00 ml.
Sample 2.00 ml. 2.00 ml.
Water 2.25 ml. 2.00 ml.
Side-arm:
Water — 0.25 ml.
D - A m i n o acid oxidase (III) 0.25 ml. 0.25 ml. 0.25 ml.
Centre well:
2 N K O H ( o n filter p a p e r ) 0.20 ml. 0.20 ml. 0.20 ml. 0.20 ml. 0.20 ml.
G a s t h e flasks for 2 m i n . w i t h o x y g e n , e q u i l i b r a t e for 5 t o 10 m i n . a n d t h e n c l o s e t h e m a n o m e t e r

t a p s . Take a n initial r e a d i n g o f t h e m a n o m e t e r s , tip t h e c o n t e n t s o f t h e s i d e - a r m s i n t o t h e m a i n
c o m p a r t m e n t a n d t a k e r e a d i n g at 5 m i n . i n t e r v a l s u n t i l t h e 0 2 u p t a k e in all flasks is p r a c t i c a l l y
zero.
T h e rate o f t h e r e a c t i o n v a r i e s c o n s i d e r a b l y d e p e n d i n g o n t h e n a t u r e o f t h e a m i n o a c i d a n d t h e
c o m p o s i t i o n o f t h e s a m p l e s o l u t i o n . T h e r e a c t i o n t i m e is u s u a l l y m u c h l o n g e r t h a n t h e 2 0 m i n .
r e q u i r e d for D - a l a n i n e .
U s e t h e c o n t e n t s o f t h e flasks for t h e d e t e r m i n a t i o n o f a m m o n i a a n d 2 - o x o a c i d s .
Calculations
Correct the m a n o m e t e r readings for changes in the t h e r m o b a r o m e t e r values (flask 1) a n d multiply by the
flask constants (refer to p . 249). This gives the p\. oxygen c o n s u m e d . Subtract the value obtained for flask 2
(enzyme blank) from the values for flasks 3 - 5 . T h e 0 2 u p t a k e in flask 3 should n o w be 224 pi. (correspon-
ding to 20 /imole D-alanine). T h e value for flask 4 gives the blank oxygen c o n s u m p t i o n of the sample.
Subtraction of this a m o u n t from the 0 2 u p t a k e for flask 5 gives the a m o u n t of oxygen required for the
oxidative d e a m i n a t i o n of the D - a m i n o acids in the sample.
As 1 //mole of D - a m i n o acid c o r r e s p o n d s to / /zmole of 0 1
2 2 = 11.2 jil., the D - a m i n o acid content per ml.
sample is
fi\. oxygen u p t a k e
[^mole/ml.]
11.2 x v o l u m e of sample t a k e n for assay
Determination of Ammonia Liberated
Pipette 1.5 ml. of solution from the main c o m p a r t m e n t of each Warburg vessel with a long, d r a w n - o u t
pipette into 10 ml. conical centrifuge tubes a n d mix with 1 ml. 50% (w/v) trichloroacetic acid solution a n d
2.5 ml. water. Centrifuge at 3000 r p m for 15 min. a n d use the s u p e r n a t a n t fluids for the determination of
a m m o n i a by the Conway m e t h o d 1 2
or by the enzymatic m e t h o d (p. 1802). T h e contents of flask 4 serve as a
control ( N H content before the enzymatic reaction) a n d the contents of flask 1 as a reagent blank.
3
Identification of the 2-Oxo Acids Formed
The 2-oxo acids formed can be identified by p a p e r c h r o m a t o g r a p h y of their 2,4-dinitrophenylhydrazones.

The hydrazones can be converted to the original a m i n o acids by hydrogenolysis a n d these can be identified
by paper c h r o m a t o g r a p h y . Both m e t h o d s have the same d i s a d v a n t a g e : they are n o t suitable for the di-
nitrophenylhydrazones of 2-oxo acids formed by the d e a m i n a t i o n of basic D - a m i n o acids (diaminobutyric
acid, ornithine, lysine, arginine a n d histidine), because these hydrazones are not extracted by ether from
acid solution.
Carefully r e m o v e t h e K O H p a p e r s f r o m t h e c e n t r e w e l l s o f t h e m a n o m e t e r flasks. Transfer t h e

c o n t e n t s o f t h e flasks t o 10 m l . g r a d u a t e d c e n t r i f u g e t u b e s , m i x w i t h 1 m l . 1 0 % ( w / v ) s o d i u m
tungstate solution and 1 ml. 0.66 N H S 0 , dilute to the mark with water and centrifuge. P o u r
2 4
t h e s u p e r n a t a n t fluids i n t o 5 0 m l . s e p a r a t i n g f u n n e l s a n d a d d 2 m l . o f a s a t u r a t e d s o l u t i o n o f
2 , 4 - d i n i t r o p h e n y l h y d r a z i n e in 2 N H C 1 . A l l o w t o s t a n d for 1 t o 2 h o u r s a n d t h e n extract t h e
h y d r a z o n e s several t i m e s w i t h p e r o x i d e - f r e e ether. W a s h t h e c o m b i n e d e t h e r e x t r a c t s w i t h a
little w a t e r a n d t h e n e x t r a c t w i t h s m a l l a m o u n t s o f 1 0 % ( w / v ) s o d i u m c a r b o n a t e s o l u t i o n u n t i l
t h e h y d r a z o n e s are c o m p l e t e l y r e m o v e d . C a r e f u l l y acidify t h e a q u e o u s c a r b o n a t e s o l u t i o n s
with 1 0 % (v/v) H S 0 2 4 a n d r e - e x t r a c t t h e h y d r a z o n e s w i t h a little ether. C o m b i n e t h e e t h e r
s o l u t i o n s . A l l o w t h e e t h e r t o e v a p o r a t e in s m a l l d i s h e s in t h e d a r k . T a k e u p t h e r e s i d u e s in 0.5 m l .
e t h a n o l a n d u s e t h e s e s o l u t i o n s for t h e i d e n t i f i c a t i o n o f 2 - o x o a c i d s b y p a p e r c h r o m a t o g r a p h y 1 3
o r for h y d r o g e n o l y s i s .
It is difficult to give the reproducibility of the m e t h o d . If the d e a m i n a t i o n of neutral D - a m i n o acids is taken

as 100, that of the a m i n o dicarboxylic acids a n d the basic a m i n o acids is less t h a n 5. The accuracy for neutral
a m i n o acids is 1 0 % , whereas for acidic a n d basic a m i n o acids the m e t h o d is a qualitative rather t h a n a
true determination.
Benzoic acid inhibits the e n z y m e ; this interference is easy to avoid. F o r the d e t e r m i n a t i o n of a m m o n i a ,

any c o n t a m i n a t i o n of the reagents or of the sample with a m m o n i a (even from the air) m u s t be avoided.
Spe c ific ity o f M e t h o d
See introduction. L - A m i n o acids d o n o t react.
Micro Methods
If the D - a m i n o acid content of the sample is t o o low for the m a n o m e t r i c m e t h o d then it can be m e a s u r e d
by one of the following enzymatic m e t h o d s .
1. A f t e r P a p e r C h r o m a t o g r a p h y
Principle
The a m i n o acids are separated by two dimensional p a p e r c h r o m a t o g r a p h y using well-known m e t h o d s 1 4
and the c h r o m a t o g r a m is sprayed with a solution of purified D - a m i n o acid oxidase a n d c a t a l a s e . T h e 15
2-oxo acids formed are located in UV light (yellow fluorescence) after spraying with Wielands reagent .16
R e a g e n t s and S o l u t i o n s
All s o l u t i o n s r e q u i r e d for p a p e r c h r o m a t o g r a p h y a n d
I. D - A m i n o a c i d o x i d a s e - c a t a l a s e :
D i s s o l v e 5 m g . c a t a l a s e p o w d e r in d o u b l y d i s t i l l e d w a t e r , a d d 2 m l . D - a m i n o a c i d o x i d a s e
solution and m a k e u p to 100 ml.
11. Wieland 's r e a g e n t :
D i s s o l v e 1 0 0 m g . o - p h e n y l e n e d i a m i n e in 1 0 0 m l . 5 % ( w / v ) t r i c h l o r o a c e t i c a c i d .
Procedure
Spray a t w o dimensional c h r o m a t o g r a m o f the sample with D - a m i n o acid oxidase/catalase

s o l u t i o n (I) a n d i n c u b a t e for 1 t o 2 h o u r s in a c l o s e d c o n t a i n e r w i t h a h i g h h u m i d i t y . S p r a y w i t h
Wieland's reagent. C o m p a r e the dirty-yellow fluorescence in U V light o r t h e p i n k colour
obtained on heating with a standard chromatogram.
S o u r c e s o f Er r or and S e n s i t i v i t y
The D - a m i n o acid oxidase solution also has considerable fluorescence; the reaction therefore loses greatly
in sensitivity.
2 . In C o m b i n a t i o n with I o n E x c h a n g e C h r o m a t o g r a p h y 1 7
Principle
Half the sample is incubated with highly active D - a m i n o acid oxidase (without addition of catalase) for ca.
3 h o u r s at 37.5 °C. T h e difference between the a m i n o acid values (obtained by ion exchange c h r o m a t o
graphy) before a n d after i n c u b a t i o n c o r r e s p o n d s to the D - a m i n o acid c o n t e n t of the s a m p l e . E x a m p l e ,
18
see .
19
Appendix
Isolation of D-Amino Acid Oxidase
C r u d e extract from pig kidney has a higher activity with D-aspartic acid a n d D-glutamic acid, but is less
stable t h a n the enzyme from sheep kidney. It dissociates readily into F A D a n d inactive protein. Pig kidney
should be used to pre p are the crude enzyme, a n d sheep kidney to obtain a purified p r e p a r a t i o n .
Solutions
l a ) P y r o p h o s p h a t e buffer (50 m M ; p H 8.3):

Dilute solution I (p. 1649) in the ratio 1 : 1 with distilled water,
l b ) P y r o p h o s p h a t e buffer (17 m M ; p H 8.3):
Dilute solution l a in the ratio 1 : 2 with distilled water.
Ic) P y r o p h o s p h a t e buffer (67 m M ; p H 8.3):
Dilute solution I (p. 1649) in the ratio 2 : 1 with distilled water.
Procedure
a) Acetone-dried powder. R e m o v e fat a n d decapsulate kidneys from freshly slaughtered pigs or sheep
(frozen kidney can be used if w o r k e d u p immediately after thawing), cut into small pieces and homogenize
with 3 volumes of acetone at + 4 °C. Quickly filter the suspension. Suspend the moist residue in the same
volume of cold acetone a n d filter. Wash again with acetone a n d then three times with the same v o l u m e
of ether at + 4 °C.
Allow the p o w d e r to dry as a thin layer in air a n d store, stoppered, at + 4 °C. The enzyme is stable for several
years.
b) Preparation of crude enzyme. Stir 1 g. acetone-dried p o w d e r with 4 ml. p y r o p h o s p h a t e buffer (solution

lb) for 20 min. Centrifuge at 3000 r p m for 30 t o 40 min., remove the thin fatty layer, decant the strongly
coloured s u p e r n a t a n t fluid a n d filter if necessary.
Use this crude extract for the m a n o m e t r i c d e t e r m i n a t i o n s .
c) Preparations of purified enzyme. Proceed according t o , but use s o d i u m sulphate instead of a m m o n i u m

2 0
sulphate for the fractional precipitation, so t h a t the a m m o n i a liberated u n d e r the assay conditions can be
determined.
Suspend 10 g. acetone-dried p o w d e r from sheep kidney in 250 ml. 17 m M p y r o p h o s p h a t e buffer (solution
lb), stir for 45 min. at 38 °C a n d then centrifuge at 3000 r p m for 30 min. D e c a n t the s u p e r n a t a n t fluid,
adjust to p H 5.1, quickly heat to 38 °C a n d then cool to 15 °C in ice water. Immediately centrifuge oft'
the precipitate a n d filter the s u p e r n a t a n t fluid. A d d 17 g. a n h y d r o u s s o d i u m sulphate to every 100 ml.
filtrate and stir for 2 h o u r s at r o o m t e m p e r a t u r e . Centrifuge off the precipitate a n d dissolve in 10 ml.
67 m M p y r o p h o s p h a t e buffer (solution Ic). Use this enzyme solution for the determination. The purified
enzyme exhibits no d e a m i n a t i n g activity with L-amino acids a n d glycine.
References
1 O. Neubauer, Dtsch. A r c h . klin. M e d . 95, 211 [1909].

2 F. Knoop, Hoppe-Seylers Z. physiol. C h e m . 67, 489 [1910].
3 H. A. Krebs, Biochem. J. 29, 1620 [1935].
4 O. Warburg & W. Christian. Biochem. Z. 296, 294 [1938]; 298, 150 [1938].
5 / . P. Greenstein, A d v a n c e s Protein C h e m . 9, 121 [1954]; J. biol. C h e m . 192, 535 [1951].
6 H. A. Krebs: T h e Enzymes. A c a d e m i c Press, N e w Y o r k 1951, Vol. II, p a r t I, p . 508.
7 H. Blaschko & J. Hawkins, Biochem. J. 52, 306 [1952].

8 N. H. Horowitz, J. biol. C h e m . 154, 141 [1944].
9 R. L. Emerson, M. Puziss & 5 . G. Knight, A r c h . Biochem. Biophysics 25, 299 [1950].
10 P. K. Stumpf& D. E. Green, F e d . P r o c . 5, 157 [1956].
11 P. Boulanger, G. Biserte & F. Courtot, Bull. Soc. chim. biol. F r a n c e 34, 366 [1952].
12 E. J. Conway: Microdiffusion Analysis a n d Volumetric Error. C r o s b y L o c k w o o d a n d Son Ltd.,
L o n d o n 1957.
13 D. Cavallini & N. Frontali, Biochim. biophys. A c t a 13, 439 [1954].
14 E. & M. Lederer: C h r o m a t o g r a p h y . Elsevier, A m s t e r d a m 1953, p . 197.
15 T. S. G. Jones, Biochem. J. 42, L I X [1949].
16 T. Wieland, Angew. C h e m . 60, 171 [1951].
17 S. Moore & D. H. Spackman & W. H. Stein, Analytic. C h e m . 30, 1185 [1958].
18 G. Biserte & M. Dautrevaux, Bull. Soc. chim. biol. F r a n c e 39, 795 [1957].
19 P. Boulanger & R. Osteux, Bull, d u C a n c e r 45, 350 [1958].
20 E. Negelein & H Bromel, Biochem. Z . 300, 225 [1939].
L-Amino Acids
Determination by Isotope Dilution Technique
in the tRNA Loading Test
K l a u s B e a u c a m p and H a n s Elmar Walter
The transfer ribonucleic acids ( t R N A ) transfer a m i n o acids to the peptide chain growing on the r i b o s o m e
in protein biosynthesis. T h e a m i n o acids are activated by enzymes k n o w n as a m i n o - a c y l - t R N A synthetases
or a m i n o - a c y l - t R N A ligases ( E C 6.1.1.-). F o r each of the 20 a m i n o acids t h a t take p a r t in protein bio
synthesis there is at least one specific t R N A (e. g. t R N A P h e
, tRNA V a l
, tRNA S e r
, etc.) a n d one synthetase.
Both the t R N A ' s a n d the synthetases are highly specific with respect to the assigned a m i n o acid. This
property can be used for the quantitative d e t e r m i n a t i o n of L-amino a c i d s , a n d has been exploited for 1
some time for the d e t e r m i n a t i o n of the acceptor activities of t R N A for a m i n o acids a n d the activity of
synthetases with the aid of radioactively labelled a m i n o acids (see p . 1894).
Application of Method: T h e m e t h o d can be r e c o m m e n d e d where one or a few a m i n o acids are to be deter

mined in several a m i n o acid mixtures or where one a m i n o acid is to be determined in m a n y samples in
a series of experiments.
Principle
(1) Amino a c i d + t R N A + A T P s y n t h e t a s e
> A m i n o - a c y l - t R N A + A M P + PP;
If additional radioactively labelled a m i n o acid of the same type is a d d e d to the assay mixture, the concen
trations of the other reactants being kept constant, the radioactively labelled a m i n o acid is diluted by
the a m i n o acid already present in the sample, a n d the radioactive loading of the t R N A is correspondingly
less p r o n o u n c e d . T h e content of the desired a m i n o acid in the sample can be determined from the radio
activity as measured against a s t a n d a r d . T h e d e t e r m i n a t i o n is t h u s based on the principle of isotope
dilution analysis as described in a n o t h e r connection by Hales a n d Randle 2
(see also p . 296).
T h e fact t h a t a single m e t h o d is used for m a n y a m i n o acids m e a n s t h a t truly o p t i m u m conditions with

respect to the t R N A c o n c e n t r a t i o n , the synthetase activity, the salt a n d buffer concentrations, etc. is
never achieved in any given case. However, the m e t h o d described here allows the d e t e r m i n a t i o n of a m i n o
acids within the indicated limits of error.
Equipment
1. L a b o r a t o r y c e n t r i f u g e
2. C o u n t e r for r a d i o a c t i v i t y , e . g . Tricarb s c i n t i l l a t i o n c o u n t e r ( P a c k a r d C o r p . o r N u c l e a r
Chicago)
3. F i l t r a t i o n a p p a r a t u s , e. g. H . H o e l z e l T e c h n i k , D - 8 2 5 D o r f e n
4. M i c r o p i p e t t e s f r o m E p p e n d o r f G e r a t e b a u , N e t h e l e r & H i n z G m b H D - 2 H a m b u r g 6 3 ,
West G e r m a n y
5. C e n t r i f u g e t u b e s o r p l a s t i c v e s s e l s ( d i s p o s a b l e t u b e s ) 1 c m . x 5 c m . o r 1 c m . x 7.5 c m . ;
e . g . " P o l y r o h r c h e n g l a s k l a r " 1 4 . 5 / 5 0 f r o m G r e i n e r , D - 7 4 4 0 N u r t i n g e n , West G e r m a n y
L - A m i n o Acids 1657
6. Ice b a t h , w a t e r b a t h 37 ° C , d r y i n g o v e n 8 0 ° C o r infrared l a m p
7. G l a s s - f i b r e filter, 2.5 c m . d i a m e t e r , e . g . t y p e G F / C f r o m W h a t m a n , M a i d s t o n e , Kent
(England)
8. S p e c i a l f o r c e p s for filter, e. g. M i l l i p o r e N o . 6 2 0 0 0 0 6
9. G l a s s o r p l a s t i c s a m p l e b o t t l e s for s c i n t i l l a t i o n c o u n t e r , e. g. m e a s u r i n g b o t t l e s a n d s c r e w
c a p s f r o m H o r m u t h a n d Vetter, D - 6 9 H e i d e l b e r g , W e s t G e r m a n y
10. R e f r i g e r a t o r a n d d e e p - f r e e z e ( — 2 0 ° C )
11. S t o p clock
12. Water-jet p u m p
13. R o t a r y e v a p o r a t o r
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 9. A m i n o - a c y l - t R N A s y n t h e t a s e s
2. A d e n o s i n e t r i p h o s p h a t e , A T P Use p r e p a r a t i o n described in A p p e n d i x p . 1662.
d i s o d i u m salt A T P - N a H - 3 H 0 , commercial
2 2 2 10. Transfer r i b o n u c l e i c a c i d s ( t R N A ) from
p r e p a r a t i o n s , see p . 527. E. coli
3. M a g n e s i u m chloride, M g C l 2 6 H 0 , A . R.
2
commercial p r e p a r a t i o n s , see p . 557.
4. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 11. Trichloroacetic acid A . R .
KH P0 H 0, 2 4 2 A.R. 12. E t h a n o l 9 8 % ( v / v )
5. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , m a y be d e n a t u r a t e d
K HP0 ,2 4 A.R. 13. S c i n t i l l a t o r r e a g e n t s
6. P o t a s s i u m c h l o r i d e , K C 1 , A . R . e. g. " P P O " a n d " P O P O P " (2,5-diphenyloxazole
7. G l u t a t h i o n e , G S H a n d 2,2 '-p-phenylenebis-(5)-phenyloxazole) from
commercial p r e p a r a t i o n s , see p . 538. Merck, D a r m s t a d t , or Omnifluor from New
8. [ C ] - a n d [ C ] - L - a m i n o a c i d s
1 2 1 4
E n g l a n d N u c l e a r C o r p . , Boston, M a s s .
[ C]-Labelled
14
amino acids from Schwarz/ 14. Toluene
M a n n , U S A ( u n d e r the n a m e Stan STAR), 15. D i e t h y l e t h e r
N e w E n g l a n d N u c l e a r C o r p . , or R a d i o c h e m .
Centre, A m e r s h a m (England). A m i n o acids
with activities of a b o u t 50 Ci/mole are suitable.
Purity of Reagents
F o r radioactively labelled L - a m i n o acids, see m a n u f a c t u r e r s ' literature. t R N A : the p r e p a r a t i o n should

contain at least 9 0 % of t R N A , based on the extinction at 260 n m , 19 O D 2 6 0 units being assumed for 1 m g .
of t R N A .
P r e p a r e all a q u e o u s s o l u t i o n s w i t h fresh d i s t i l l e d w a t e r .
I. Tris b u f f e r * ( 0 . 2 5 M t r i s ; 25 m M M g C l 2 ; 25 m M K C 1 ; 5 m M G S H ; p H 7 . 4 ) :
D i s s o l v e 3 . 0 2 5 g. t r i s - H C l + 0 . 5 g. M g C l - 6 H O + 0 . 1 8 7
2 2 g. K C 1 + 0 . 1 8 3 g. G S H in
8 0 m l . w a t e r , a d j u s t t o p H 7.4 o n t h e p H m e t e r w i t h 1 M H C 1 , a n d m a k e u p t o 1 0 0 m l .
with water.
* 0.25 M cacodylate buffer, p H 7.4, can in principle be used instead of tris buffer, p H 7.4; for example,
as solution I, b u t use 5.35 g. s o d i u m cacodylate (hydroxydimethylarsine oxide, s o d i u m salt) instead
of tris.
II. P h o s p h a t e buffer ( 0 . 2 5 M p h o s p h a t e ; 2 5 m M M g C l ; 25 m M
2 KC1; 5 m M GSH;
p H 7.0):
a) D i s s o l v e 5.7 g. K H P 0 - 3 H 0 in w a t e r a n d m a k e u p t o 1 0 0 m l .
2 4 2
b) D i s s o l v e 3 . 4 g. K H P 0 2 4 in w a t e r a n d m a k e u p t o 100 m l .
A d d s o l u t i o n 4> ( a p p r o x . 6 4 m l . ) t o s o l u t i o n a until a p H o f 7.0 is r e a c h e d o n t h e p H
m e t e r . D i s s o l v e 0.5 g. M g C l - 6 H O + 0 . 1 8 7 g. K C 1 + 0 . 1 8 3 g. G S H in 100 m l . buffer.
2 2
III. A d e n o s i n e - 5 ' - t r i p h o s p h a t e ( 1 0 m M ) :
D i s s o l v e 6 0 . 5 m g . A T P - N a H - 3 H 0 in 10 m l . w a t e r .
2 2 2
I V . t R N A s o l u t i o n 1 (2 m g . t R N A / m l . ) :
D i s s o l v e 10 m g . t R N A in 5 m l . w a t e r (for M e t , Ser, Tyr, Val).
V. t R N A solution 2 (4 m g . t R N A / m l . ) :
D i s s o l v e 2 0 m g . t R N A in 5 m l . w a t e r (for A r g , He, L e u , L y s , P r o , T h r ) .
VI. t R N A solution 3 (10 mg. t R N A / m l . ) :
D i s s o l v e 10 m g . t R N A in 1 m l . w a t e r (for H i s , P h e ) .
VII. L - A m i n o acid [ C ] calibration solutions (0.4 m M ) :
1 2
D i s s o l v e 4 0 / / m o l e o f t h e c o r r e s p o n d i n g L - a m i n o a c i d in 1 0 0 m l . w a t e r a n d d i l u t e a s
r e q u i r e d (see u n d e r " P r o c e d u r e " )
VIII. L - A m i n o acid [ C ] standard solution (40 /xM):
1 4
D i l u t e t h e c o m m e r c i a l p r e p a r a t i o n (1 m M ; 5 0 C i / m o l e ) , e . g . 0 . 0 5 m l . o f a " S t a n -
S T A R " a m i n o a c i d + 1 . 2 m l . w a t e r ; u s e 0 . 0 5 m l . o f this d i l u t i o n .
IX. Synthetase solution (approx. 3 - 4 mg. protein/ml.):
I m m e d i a t e l y b e f o r e u s e , c e n t r i f u g e a p o r t i o n o f t h e s u s p e n s i o n d e s c r i b e d in t h e A p p e n d i x
p. 1 6 6 2 a n d t a k e u p t h e p r e c i p i t a t e in buffer ( s o l u t i o n I). If n e c e s s a r y , the s o l u t i o n
c o n t a i n i n g 3 - 4 m g . p r o t e i n / m l . s h o u l d b e d i l u t e d w i t h t h e s a m e buffer s h o r t l y b e f o r e
the determination.
X. Trichloroacetic acid 5 % ( w / v ) :
D i s s o l v e 5 0 g. C C l C O O H in w a t e r a n d m a k e u p t o 1 litre.
3
X L Trichloroacetic acid 1 0 % ( w / v ) :
D i s s o l v e 1 0 0 g. C C l C O O H in w a t e r a n d m a k e u p t o 1 litre.
3
XII. Scintillation fluid:

D i s s o l v e 4 g. o f P P O a n d 1 0 0 m g . P O P O P in t o l u e n e a n d m a k e u p t o 1 litre.
Solution I a n d II can be kept for 3 weeks. Solutions III to VIII can be stored for only a few days at 0 °C
(attack by bacteria), but will keep for several weeks at — 20 °C. However, the safest course is to p r e p a r e n o t
m o r e t h a n one week's supply of these solutions. Solution IX see " P r e p a r a t i o n of the S o l u t i o n s " . Solution X I I
is stable for several weeks in the d a r k .
Procedure
Preliminary Treatment of Sample
Samples containing protein (serum, urine, etc.) m u s t be deproteinized. D i s s o l v e the sample

in w a t e r if n e c e s s a r y , a d d t w o v o l u m e s o f s o l u t i o n X I in t h e c o l d , a n d c e n t r i f u g e . Extract
t h e s u p e r n a t a n t 3 - 4 t i m e s w i t h t h r e e t i m e s its v o l u m e o f ether. E v a p o r a t e t h e a q u e o u s p h a s e
t o d r y n e s s (e. g. in a B u e c h i r o t a r y e v a p o r a t o r ) a n d t a k e u p in buffer s o l u t i o n ( s o l u t i o n I o r
s o l u t i o n II) ( c o n c e n t r a t i o n a c c o r d i n g t o " D e t e r m i n a t i o n o f t h e M e a s u r i n g R a n g e " , see b e l o w ) .
Quantities of Synthetases and t R N A to Use and Choice of Buffer
Synthetases m g . of t R N A
A m i n o acid Solution Buffer
(Solution IX) test
Arg Diluted 1 :10 V 0.2

His Diluted 1 :10 VI 0.5
He Diluted 1 :10 V 0.2
Leu Undiluted V 0.2 Tris p H 7.4
Lys Diluted 1 :10 V 0.2 (Solution I)
Met Diluted 1 :10 IV 0.1
Phe Diluted 1 :10 VI 0.5
Pro Undiluted V 0.2 P h o s p h a t e ; p H 7.0
(Solution II)
Ser Diluted 1 :10 IV 0.1
Thr Undiluted V 0.2 Tris p H 7.4
Tyr Undiluted IV 0.1 (Solution I)
Val Diluted 1 :10 IV 0.1
Calibration Curve and Determination of the Measuring Range
F o r each a m i n o acid to be determined, use 0.05 ml. of the c o r r e s p o n d i n g calibration solution III undiluted,
or diluted with 0.05 ml. of water (dilution 1 + 1) or with 0.15 ml. of water (dilution 1 + 3). Use 0.05 ml.
of each of these mixtures for c a l i b r a t i o n ; this c o r r e s p o n d s to 2 0 , 1 0 , a n d 5 n m o l e of a m i n o acid respectively.
The calibration curve is then linear in the range from 2 to 25 nmole/0.05 ml.
It is advisable to establish three calibration points (5, 10, 20 nmole) first with " c o l d " [ C ] a m i n o acid to 12
check t h a t the course of the calibration curve is satisfactory. L a t e r it is usually sufficient to determine only
one calibration p o i n t (10 nmole). T h e calibration line is fixed with sufficient accuracy by the intersection
with the ordinate at a value of 1.
It is i m p o r t a n t t h a t the t R N A c o n c e n t r a t i o n used should be limiting with respect to the a m i n o acid. If
the t R N A c onc e nt ratio n is t o o high, the calibration curve is not linear a n d does n o t cut the ordinate at a
value of 1. In this case, the t R N A c o n c e n t r a t i o n to be chosen m u s t be d e t e r m i n e d from a saturation curve.
Various quantities of t R N A m u s t be used here with a c o n s t a n t a m i n o acid c o n c e n t r a t i o n .
The quantity of t R N A used should preferably be t h a t c o r r e s p o n d i n g to roughly the middle of the linear
portion of the curve obtained.
If the a m i n o acid content in the test material is u n k n o w n , a dilution series m u s t be p r e p a r e d in o r d e r to
determine the measuring range ( 2 - 2 5 nmole/0.05 ml.). Use 0.05 ml. p o r t i o n s of the prepared sample
without dilution a n d at dilutions of 1 :10, 1 :100, a n d 1 : 1 0 0 0 with water, a n d determine the c / c values Q x
(see p . 1661, Calculations). O n e of the dilutions should give an c / c value t h a t lies in the range of the
D x
calibration curve. T h e dilution for the sample can thus be chosen accordingly. (With some experience, the
a m i n o acid content can be d e t e r m i n e d with sufficient accuracy ( + 25 %) from the value lying in the measur
ing range.)
Assay System
I n c u b a t i o n v o l u m e : 0.5 m l . ; 3 7 ° C .
P r e p a r e m i x t u r e s in d u p l i c a t e for t h e s a m p l e s ( a s w e l l a s o n e d i l u t i o n , e . g . 1 + 1). A n y " i n t e r
n a l " m e a s u r i n g error t h u s b e c o m e s c l e a r l y v i s i b l e .
In p r i n c i p l e , t h e d e t e r m i n a t i o n c a n b e c a r r i e d o u t in s m a l l e r v o l u m e s , e . g . w i t h 1/5 o f t h e
q u a n t i t i e s i n d i c a t e d in t h e p i p e t t i n g s c h e m e . In this c a s e , h o w e v e r , t h e c o m p l e t e mixture
s h o u l d b e " c o l l e c t e d " in a b e n c h c e n t r i f u g e ( s h o r t c e n t r i f u g a t i o n t i m e ) i m m e d i a t e l y b e f o r e
i n c u b a t i o n . If t h e r e a c t i o n s are s t a r t e d w i t h s o l u t i o n I X a t 1 0 s e c . i n t e r v a l s a n d stopped
w i t h s o l u t i o n X after i n c u b a t i o n , u p t o 6 0 a s s a y s c a n b e c a r r i e d o u t in a s i n g l e o p e r a t i o n . T h e
s a m p l e t u b e s c a n r e m a i n in a n ice b a t h for u p t o 2 hr. after a d d i t i o n o f s o l u t i o n X . D u r i n g
this time, filtration, w a s h i n g , a n d t r a n s f e r i n t o c o u n t i n g v i a l s c a n be c a r r i e d o u t w i t h o u t
difficulty.
Pipette into 4 ml. tubes Standard Calibration Sample C o n c e n t r a t i o n in

(in a n ice b a t h ) : (ml.) (ml.) (ml.) assay mixture
Buffer s o l u t i o n (I o r II) 0.20 0.20 0.20 0.1 M tris o r

phosphate
10 m M M g C l 2
10 m M KC1
2 mM GSH
A T P solution (III) 0.10 0.10 0.10 2 mM ATP
t R N A solution (IV, V, or VI) 0.05 0.05 0.05 0.2 o r 0 . 4 o r
1.0 m g . / m l .
Water 0.05 — —
L - A m i n o acid — 0.05 — 4 0 , 2 0 , 10 juM
calibration solution (VII)
(or 1 + 1 o r 1 + 3 d i l u t i o n )
L - A m i n o acid [ C ] solution (VIII)
1 4
0.05 0.05 0.05
Sample solution — — 0.05
Synthetase solution (IX) 0.05 0.05 0.05 c a . 0.7 o r
7 mg./ml.
M i x carefully a n d p l a c e in a 37 ° C w a t e r b a t h for 10 m i n .
Trichloroacetic acid (X) ca. ca. ca.

(cold) 3.0 3.0 3.0
A d d trichloroacetic acid ( T C A ) from wash bottle to about 1 c m b e l o w

t h e r i m o f e a c h t u b e , a n d r e c o o l in t h e ice b a t h . M o i s t e n filter in filtration
a p p a r a t u s w i t h T C A a n d filter c o n t e n t s o f t u b e s t h r o u g h s e p a r a t e filters
u n d e r v a c u u m (waterjet p u m p ) . W a s h o u t e a c h t u b e 3 t i m e s w i t h a p p r o x .
3 m l . o f T C A a n d p o u r w a s h i n g s t h r o u g h filter. W a s h t w i c e w i t h a p p r o x .
3 ml. of ethanol from w a s h bottle and (without releasing v a c u u m ) trans
fer filter i n t o c o u n t i n g vial b y m e a n s o f t o n g s . D r y for 3 0 m i n . at 8 0 ° C
and allow to cool.
Scintillation fluid (XII) 5.0 5.0 5.0
M e a s u r e r a d i o a c t i v i t y in c o u n t e r .
Calculations
Divide the c p m values o b t a i n e d in the c o u n t e r for the s t a n d a r d (c ) by the c p m values of the calibration
c
mixtures a n d of t h e samples (c ). Plot the values c / c from the s t a n d a r d a n d the calibration mixtures
x D x
(ordinate) against the calibration values (abscissa) (Fig. 1). T h e result is a straight line cutting the o r d i n a t e
at a value of 1. This line is valid for the range from a b o u t 2 t o a b o u t 25 n m o l e of a m i n o acid in the test.
T h e values in nmole/test c o r r e s p o n d i n g t o the c / c values of the samples c a n be found from this calibration
D x
line.
c /c i
0 x
5 10 20 Tn'test
Fig. 1
• •: C a l i b r a t i o n line gives g o o d experimental values
o o. £ j j b
a r a t j o n p o i n t s d o n o t give a straight line passing t h r o u g h a value of 1 on the o r d i n a t e ;
curve b e n d s ; experimental values incorrect.
Accuracy and Precision
T h e values are reproducible with an accuracy of between + 5 % a n d ± 1 5 % , d e p e n d i n g on the a m i n o

acid a n d the position of the experimental p o i n t s o n the calibration l i n e . W i t h a c c u r a t e w o r k , the position
2
of the calibration points on the calibration line provides a g o o d indication. If deviations of m o r e t h a n

± 1 0 % are found here, the accuracy of the values for the sample will h a r d l y be any better (see e.g. o p e n
circles in Fig. 1). We have so far o b t a i n e d g o o d results with the following a m i n o a c i d s : A r g , His, He, Leu, Lys,
M e t , Phe, Ser, T h r , Tyr, a n d Val; here again, values between 5 a n d 15 n m o l e give the best agreement with
reference values. Values below 2 n m o l e a n d a b o v e 25 n m o l e should be avoided (choose a different dilution).
The results o b t a i n e d for P r o a n d Trp were p o o r e r ; the reasons for this are n o t yet k n o w n .
N o d e t e r m i n a t i o n s were carried o u t for the a m i n o acids Ala, A s p , Asn, G l u , a n d G i n , since these can be
determined by o t h e r simpler m e t h o d s (see, e.g. p . 1679). D e t e r m i n a t i o n s were n o t carried o u t for Gly a n d
Cys, in the latter case because the a m i n o acid is partly present in an oxidized form a n d c a n n o t be determined
by this m e t h o d .
Excessively low values m a y be d u e to excessively high salt c o n c e n t r a t i o n s ( > 0.2 M ) .

If the synthetase m i x t u r e used h a s been stored for t o o long, e n d o g e n o u s a m i n o acids released by proteolysis
can lead to incorrect values. In cases of d o u b t , it is advisable to dialyse the synthetase mixture against
solution I before use.
Synthetases m a y lose activity d u r i n g storage. To check for this, e.g. the 10 n m o l e calibration sample is
incubated for 10,15, a n d 20 min. If the longer incubation times d o n o t cause higher levels of incorporation,
the synthetases are active. If the i n c o r p o r a t i o n level is increased, it is necessary either to increase the
incubation time or to increase the q u a n t i t y of synthetase a d d e d .
E r r o r s such as those described in the literature as a result of defective loading of t R N A are of n o i m p o r t a n c e
in this m e t h o d , since they fall within the limits of error.
Appendix
Preparation of the Amino-acyl-tRNA Synthetases
A mixture of the synthetases from E. coli can be p r e p a r e d as described i n or by the following p r o c e d u r e .

3
Disintegrate E. coli with glass b e a d s or in a M a n t o n - G a u l i n high-pressure dispersion press. Centrifuge

off the cell c o m p o n e n t s , a d d a b o u t 1.5 ml. of 5 % streptomycin sulphate solution per 10 ml. of extract
to the s u p e r n a t a n t , a n d centrifuge off the precipitate. F r a c t i o n a t e the s u p e r n a t a n t with a m m o n i u m
sulphate a n d take u p the fraction between 3 0 % a n d 5 0 % saturation (1.2 M to 2.1 M ) with a little buffer
solution (0.01 M tris, p H 7.9; 1 M KC1, 0.01 M M g C l ; 0.1 m M d i t h i o t h r e i t o l ; 5 % glycerol). Introduce
2
the very concentrated, viscous, brown-yellow solution into an " A g a r o s e A 1.5 m " c o l u m n equilibrated
with the same buffer, a n d c h r o m a t o g r a p h with the same buffer. M e a s u r e the U V extinction of the fractions
at 280 n m a n d at 260 n m . T h r e e m a i n p e a k s appear, the second of these c o n t a i n i n g the synthetases. Collect
the fractions of this second p e a k a n d a d d a m m o n i u m sulphate to give a p p r o x . 8 0 % saturation (approx.
3.2% M ) .
In this form, the synthetase mixture is stable for m a n y m o n t h s at 0 to 4 °C. A lyophilizate of the a m
m o n i u m sulphate precipitate (take u p precipitate in solution I to a c o n c e n t r a t i o n of 10 m g . protein/ml.)
can also be used for several m o n t h s if stored at 0 to 4 °C.
References
1 / . B. Rubin & G. Goldstein, A n a l . Biochem. 33, 244 [1970].

2 C. N. Hales & P. J. Randle, Biochem. 88, 137 [1963].
3 K. H. Munch & P. Berg in G. L. Cantoni & D. R. Davies: Proc. in Nucleic Acid Res., vol. I, p . 375, H a r p e r
a n d Row, N e w York, E v a n s o n , L o n d o n [1966].
L-Lysine, L-Arginine, L-Ornithine, L-Tyrosine, L-Histidine,

L-Glutamic Acid, L-Aspartic Acid
Manometric Method
Ernest F. Gale
Certain bacteria, grown u n d e r suitable conditions, p r o d u c e specific L-amino acid d e c a r b o x y l a s e s 1 - 3

.
In most cases, the p H o p t i m a of these decarboxylases are in the acid range, so that the C 0 2 p r o d u c e d can
be measured manometrically.
Application of Method: In biochemistry, in the analysis of proteins a n d in microbiology.

Principle
A m i n o acid decarboxylases catalyse reactions of the t y p e :
(1) R—CH—C0 H 2 > R—CH —NH 2 2 + C0 2
NH 2
The C 0 2 p r o d u c e d is determined in a W a r b u r g m a n o m e t e r a n d is a m e a s u r e of the a m i n o acid c o n t e n t

of the sample. Specific decarboxylases are available for the following a m i n o a c i d s :
a) L-Lysine —• C a d a v e r i n e (L-Lysine carboxy-lyase; E C 4.1.1.18)

p H o p t i m u m : 6.0
b) L-Arginine —• A g m a t i n e (L-Arginine carboxy-lyase; E C 4.1.1.19)
p H o p t i m u m : 5.2
c) L-Ornithine —• Putrescine (L-Ornithine carboxy-lyase; E C 4.1.1.17)
p H o p t i m u m : 5.5
d) L-Tyrosine —• T y r a m i n e (L-Tyrosine carboxy-lyase; E C 4.1.1.25)
p H o p t i m u m : 5.5
e) L-Histidine —> H i s t a m i n e (L-Histidine carboxy-lyase; E C 4.1.1.22)
p H o p t i m u m : 4.5
f) L - G l u t a m i c acid —• y-Aminobutyric acid ( L - G l u t a m a t e 1-carboxy-lyase; E C 4.1.1.15)
p H o p t i m u m : 4.5
g) L-Aspartic acid —• a-Alanine ( L - A s p a r t a t e 4-carboxy-lyase; E C 4.1.1.12)
p H o p t i m u m : 5.5
T h e a m i n o acid decarboxylases have very s h a r p p H o p t i m a . Consequently the r e c o m m e n d e d buffer

solutions m u s t be strictly a d h e r e d t o a n d the p H of the sample m u s t be adjusted accordingly. A d d sufficient
enzyme so t h a t 2 0 0 - 3 0 0 u\. C 0 2 is liberated in 15 min.
Equipment
Warburg b a t h a n d m a n o m e t e r s ; flasks w i t h o n e s i d e - a r m (for r e a c t i o n s b , d, e, f) a n d w i t h

t w o s i d e - a r m s (for r e a c t i o n s a, c, g ) .
Reagents
T h e letters a) t o g) c o r r e s p o n d t o t h e s e v e n b - d ) Citric acid

a m i n o acids listed a b o v e a n d indicate the e - g ) S o d i u m acetate, a n h y d r o u s
r e a g e n t s w h i c h are r e q u i r e d for t h e d e t e r e - g ) Acetic acid
2 . a, c, g) S u l p h u r i c a c i d , c a . 2 N
mination of the respective amino acids.
3. A m i n o acid d e c a r b o x y l a s e s
1. R e a g e n t s for buffers a) L-Lysine d e c a r b o x y l a s e
a) P o t a s s i u m d i h y d r o g e n p h o s p h a t e , acetone-dried p o w d e r of Bacterium cadaveris
KH P0 2 4
( N C I B * N o . 6578). F o r c o n d i t i o n s of g r o w t h
a - d ) Disodium hydrogen phosphate, a n d p r e p a r a t i o n of t h e a c e t o n e powder , 2
Na HP0 -2H 0
2 4 2
see A p p e n d i x , p . 1668.
* N a t i o n a l Collection of Industrial Bacteria, A d d r e s s : Torry R e s e a r c h Station, A b e r d e e n , Scotland.

b) L-Arginine decarboxylase e) L-Histidine decarboxylase

acetone-dried p o w d e r from Escherichia coli acetone-dried p o w d e r from Clostridium wel-
( N C I B N o . 7020). F o r conditions of g r o w t h chii B W 21 ( N C I B N o . 6785). F o r conditions
a n d p r e p a r a t i o n of the acetone p o w d e r , see 2
of g r o w t h a n d p r e p a r a t i o n of the acetone-
A p p e n d i x , p . 1668. dried p o w d e r 2 , 4
, see Appendix, p . 1668.
c) L-Ornithine decarboxylase f) L - G l u t a m i c acid decarboxylase
washed cells of Clostridium septicum Pasteur washed cells of Clostridium welchii S R 12*
( N C I B N o . 547). F o r conditions of g r o w t h , 2
( N C I B N o . 6784). F o r conditions of growth
see A p p e n d i x , p . 1668. a n d p r e p a r a t i o n of acetone-dried p o w d e r , 2
d) L-Tyrosine decarboxylase see Appendix, p . 1668.

acetone-dried p o w d e r of Streptococcusfaeca- g) L-Aspartic acid decarboxylase
lis ( N C I B N o . 6782). F o r conditions of acetone-dried p o w d e r from Nocardia glo-
growth a n d p r e p a r a t i o n of the acetone-dried berula ( N C I B N o . 8852). F o r conditions
p o w d e r , see A p p e n d i x , p . 1668.
2
of g r o w t h a n d p r e p a r a t i o n of the acetone-
dried p o w d e r , see A p p e n d i x , p . 1668.
3
Specificity o f E n z y m e P r e p a r a t i o n s
To obtain p r e p a r a t i o n s of the required specificity, the correct strain of o r g a n i s m m u s t be used, a n d the

conditions of g r o w t h a n d m e t h o d of p r e p a r a t i o n of the acetone-dried p o w d e r s m u s t be strictly adhered to.
T h e lysine decarboxylase p r e p a r a t i o n m a y contain traces of arginine decarboxylase; however, the activity
of the latter disappears if the acetone-dried p r e p a r a t i o n is kept for 2 - 3 days at 0 to 4 °C. Likewise, the
histidine decarboxylase p r e p a r a t i o n s from CI. welchii occasionally have weak glutamic acid decarboxylase
activity. In this case, suspend the acetone-dried p o w d e r in 50 m M b o r a t e buffer ( p H 8.5) (40 mg./ml.) a n d
incubate overnight at 37 °C. Centrifuge for 30 min. at 4000 g a n d use the clear, yellow s u p e r n a t a n t as
the histidine decarboxylase p r e p a r a t i o n . 4
T h e letters a) t o g ) c o r r e s p o n d t o t h e s e v e n a m i n o a c i d s l i s t e d in t h e o r d e r g i v e n o n p . 1 6 6 3 a n d
i n d i c a t e t h e s o l u t i o n s r e q u i r e d for t h e d e t e r m i n a t i o n o f t h e r e s p e c t i v e a m i n o a c i d s . P r e p a r e
all s o l u t i o n s w i t h freshly d i s t i l l e d w a t e r .
I. Buffer s o l u t i o n s
a) P h o s p h a t e buffer ( 0 . 2 M ; p H 6 . 0 ) :
M i x 13.0 m l . 0 .2 M N a H P 0 2 4 s o l u t i o n ( 3 5 . 6 g. N a H P 0 - 2 H 0 / l 0 0 0 m l . ) w i t h
2 4 2
8 7 . 0 m l . 0.2 M K H P 0 2 4 s o l u t i o n ( 2 7 . 2 g. K H P 0 / 1 0 0 0 m l . ) .
2 4
b) P h o s p h a t e - c i t r a t e buffer ( p H 5 . 2 ) :
M i x 4 6 . 4 m l . 0.1 M citric a c i d ( 1 9 . 2 g./lOOO m l . ) w i t h 5 3 . 6 m l . 0 . 2 M Na HP0
2 4
s o l u t i o n ( 3 5 . 6 g. N a H P O / 1 0 0 0 m l . ) ,
2 4
c, d ) P h o s p h a t e - c i t r a t e buffer ( p H 5 . 5 ) :
M i x 4 3 . 1 m l . 0.1 M citric a c i d ( 1 9 . 2 g./lOOO m l . ) w i t h 6 5 . 9 m l . 0 . 2 M Na HP0
2 4
s o l u t i o n ( 3 5 . 6 g. N a H P 0 - 2 H 0 / l 0 0 0 m l . ) .
2 4 2
* It is very i m p o r t a n t t h a t this particular strain is used.

e, 0 A c e t a t e buffer ( 0 . 2 M ; p H 4 . 5 ) :
M i x 42.5 ml. 0.2 M N a acetate solution (16.4 g./l 0 0 0 ml.) with 57.5 ml. 0.2 N acetic
a c i d ( 1 2 . 0 g. a c e t i c a c i d / 1 0 0 0 m l . ) ,
g ) A c e t a t e buffer (0.1 M ; p H 5 . 5 ) :
M i x 8 8 . 0 m l . 0.1 M N a a c e t a t e s o l u t i o n ( 8 . 2 g . / l 0 0 0 m l . ) w i t h 1 2 . 0 m l . 0.1 N a c e t i c
a c i d ( 6 . 0 g. a c e t i c a c i d / 1 0 0 0 m l . ) .
II. E n z y m e s u s p e n s i o n s
a) L-Lysine d e c a r b o x y l a s e
S u s p e n d 1 0 0 m g . a c e t o n e - d r i e d p o w d e r in 5 m l . buffer ( s o l u t i o n l a ) .
b) L-Arginine decarboxylase
S u s p e n d 1 0 0 m g . a c e t o n e - d r i e d p o w d e r in 5 m l . buffer ( s o l u t i o n l b ) .
c) L - O r n i t h i n e d e c a r b o x y l a s e
S u s p e n d 2 5 0 m g . w a s h e d cells ( d r y w e i g h t ) in 5 m l . buffer ( s o l u t i o n I c , d ) .
d) L-Tyrosine decarboxylase
S u s p e n d 1 0 0 m g . a c e t o n e - d r i e d p o w d e r in 5 m l . buffer ( s o l u t i o n I c , d ) .
e) L - H i s t i d i n e d e c a r b o x y l a s e
S u s p e n d 3 0 0 m g . a c e t o n e - d r i e d p o w d e r in 5 m l . buffer ( s o l u t i o n I e , f ) .
0 L-Glutamic acid decarboxylase
S u s p e n d 2 0 0 m g . w a s h e d cells ( d r y w e i g h t ) in 5 m l . buffer ( s o l u t i o n I e , f )
g) L-Aspartic acid decarboxylase
S u s p e n d 5 0 m g . a c e t o n e - d r i e d p o w d e r in 5 m l . buffer ( s o l u t i o n I g ) .
T h e buffer solutions keep indefinitely in stoppered bottles at 0 to 4 °C. T h e stability of the acetone-dried
p o w d e r s varies from p r e p a r a t i o n to p r e p a r a t i o n . N o r m a l l y , they retain their activity for 2 - 3 m o n t h s
(sometimes years) when stored in a desiccator. Occasionally the p r e p a r a t i o n s lose their activity within a
few days. Suspensions of CI. welchiiSR 12 keep for several weeks at 4 °C. In c o n t r a s t , the o r n i t h i n e decarboxy
lase activity of suspensions of CI. septicum is m u c h less stable a n d m a y be lost within 2 - 3 days a t 4 °C.
It is best to use a freshly p r e p a r e d suspension for each estimation.
Procedure
T h e a m i n o acid solution to be analysed must n o t contain any inhibitors o f the respective

a m i n o a c i d d e c a r b o x y l a s e p r e p a r a t i o n s . T h e p H o f t h e s a m p l e m u s t b e sufficiently n e a r t o
p H o p t i m u m o f t h e e n z y m e , s o t h a t w h e n buffer is a d d e d t h e o p t i m u m p H is a t t a i n e d . D e
c a r b o x y l a s e p r e p a r a t i o n s d o n o t u s u a l l y a t t a c k c a r b o h y d r a t e s . H o w e v e r , if t h e s a m p l e c o n t a i n s
f e r m e n t a b l e s u g a r s , for e x a m p l e , g l u c o s e , it is a d v i s a b l e t o i n c l u d e a c o n t r o l c o n t a i n i n g t h e
s a m e c o n c e n t r a t i o n o f s u g a r (this is e s p e c i a l l y i m p o r t a n t w h e n w a s h e d c e l l s are u s e d ) .
Manometric Measurements
W a r b u r g m a n o m e t e r s ; v e s s e l s w i t h s i d e - a r m s ; t e m p e r a t u r e : 37 ° C ; g a s p h a s e : air.
F o r e a c h e s t i m a t i o n 3 - 4 v e s s e l s are n e c e s s a r y : 1 - 2 e x p e r i m e n t a l v e s s e l s , 1 c o n t r o l v e s s e l
(without substrate) and 1 thermobarometer. Prepare the vessels as f o l l o w s :
Experimental Control Thermo

Pipette into vessels:
vessel vessel barometer
Main compartment sample 0 . 5 - 1 . 0 ml. — —

distilled water
— 0 . 5 - 1 . 0 ml. 2.5 m l .
buffer 1 . 5 - 1 . 0 ml. 1 . 5 - 1 . 0 ml.
—
Side-arm enzyme suspension 0.5 m l . 0.5 m l .
—
E q u i l i b r a t e t h e v e s s e l s for 5 - 1 0 m i n . C l o s e t h e t a p s a n d r e a d t h e m a n o m e t e r s . T i p t h e e n z y m e
s u s p e n s i o n i n t o t h e m a i n c o m p a r t m e n t a n d r e c o r d t h e i n c r e a s e in p r e s s u r e until t h e r e a c t i o n
ceases ( 1 0 - 3 0 min.).
In t h e d e t e r m i n a t i o n o f l y s i n e , o r n i t h i n e a n d a s p a r t i c a c i d t h e p H at t h e e n d o f t h e r e a c t i o n
is > 5 . 8 a n d t h e r e f o r e s o m e C 0 2 is r e t a i n e d . To d e t e r m i n e this r e t e n t i o n u s e m a n o m e t e r
vessels with d o u b l e side-arms. Prepare the second side-arm with
0.4 ml. 2 N H S 0 . 2 4
A t t h e e n d o f the e n z y m a t i c r e a c t i o n tip t h e a c i d i n t o t h e m a i n c o m p a r t m e n t a n d read t h e

i n c r e a s e in p r e s s u r e .
Calculations*
T h e volume of C 0 2 p r o d u c e d is calculated from the m a n o m e t e r readings ( m m . m a n o m e t e r fluid) (after

correction for the t h e r m o b a r o m e t e r changes) by multiplication by the m a n o m e t e r c o n s t a n t k : 5
where
V g = volume of the gas p h a s e in the m a n o m e t e r [pi.]
V f = volume of fluid in the m a n o m e t e r [pi.]
a = solubility [ c m / c m ] of C 0
3 3
2 in water at 760 m m . a n d t e m p e r a t u r e T
T = absolute t e m p e r a t u r e of the reaction [°K]
P D = 760 m m . H g pressure expressed in terms of m a n o m e t r i c fluid (usually P = 10000 m m . ) . G
* Refer to p . 249.
The C 0 2 p r o d u c t i o n is usually s o m e w h a t less t h a n 1 0 0 % theory. Assays o n s t a n d a r d solutions gave

the following values (last c o l u m n : factor with which v o l u m e C 0 2 p r o d u c e d in the experimental vessel
m u s t be multiplied to o b t a i n the m g . a m i n o acid in the reaction m i x t u r e ) :
Yield
100 fj\. CO are p r o d u c e d by Factor
%
2
0.652
a) 0.652 m g . lysine 98
98
0.775
b) 0.775 m g . arginine 95
95
0.590
c) 0.590 m g . ornithine 98
98
0.810
d) 0.810 m g . tyrosine 96
96
0.692
e) 0.692 m g . histidine 96
96
0.656
f) 0.656 m g . glutamic acid 98
98
0.596
g) 0.596 m g . aspartic acid 97
97
Example
D e t e r m i n a t i o n of L-lysine. Experimental p r o t o c o l ( m a n o m e t e r readings corrected for the t h e r m o b a r o

meter c h a n g e s ) :
C o n t r o l vessel Experimental vessel
Increase in pressure d u r i n g the enzymatic reaction 2 mm. 154 m m .

Increase in pressure after tipping acid 13 m m . 25 m m .
Total increase in pressure 15 m m . 179 m m .
Manometer constant 1.73 1.89
Volume of C 0 2 evolved 15 x 1.73 = 26 u\. 179 x 1.89 - 338 fil
Volume of C 0 2 liberated from lysine 338 - 26 = 312 ul.
312 x Q-^^ = 2.08 m g . lysine/reaction mixture.

98
T h e error of this m e t h o d is t h a t usual for m a n o m e t r i c m e t h o d s , namely 4 - 5 % . T h e o u t p u t of C 0 2 should

n o t be less t h a n 20 ul. a n d n o t m o r e t h a n 400 p\.
T h e m o r e complex the sample the less the accuracy of the d e t e r m i n a t i o n ; the e r r o r is higher if there is
retention of C 0 , incorrect adjustment of p H o r insufficient buffering capacity. P r o d u c t i o n of C 0
2 2 from
other c o m p o u n d s (see Specificity of M e t h o d ) results in high values.
S p ec ific ity o f M e t h o d
E a c h enzyme p r e p a r a t i o n is specific for its respective L - a m i n o acid substrate. T h e carboxyl a n d a-amino

g r o u p of the a m i n o acid m u s t n o t be s u b s t i t u t e d . Occasionally a n a m i n o acid derivative with an O H
1
g r o u p in the rest of the molecule is a t t a c k e d : lysine decarboxylase reacts slowly with hydroxylysine;
tyrosine decarboxylase a t t a c k s p h e n y l a l a n i n e at 5 - 1 0 % of the rate at which tyrosine is decarboxylated
6
a n d it also reacts with L-3.4-dihydroxyphenylalanine . G l u t a m i c acid decarboxylase p r e p a r a t i o n s m a y

7
liberate C 0 2 from certain isomers of 3-hydroxyglutamic a c i d , a n d also from L-aspartic acid if traces of
8
pyruvate, 2-oxoglutarate o r o t h e r oxo acids are present in the s a m p l e . T h e reaction with L-aspartic acid is
9
prevented by the addition of c e t y l t r i m e t h y l a m m o n i u m b r o m i d e (0.25% w/v).
Other Methods of Determination
Decarboxylases can also be used for the d e t e r m i n a t i o n of a m i n o acids labelled in the carboxyl g r o u p
with 1 4
C . T h e reagents a n d p r o c e d u r e are as described above, b u t the 1 4
C0 2 m u s t be absorbed in N a O H
a n d the radioactivity measured.
Appendix
Preparation of the Enzyme Preparations
It is essential to keep exactly t o the culturing conditions given in the original p u b l i c a t i o n s " . T h e follow 2 4
ing figures are intended only as an initial guide. T h e letters a) to g) c o r r e s p o n d to those on page 1663.
Culturing conditions
a, b) 30 hr. at 25 °C in a m e d i u m c o n t a i n i n g 3 % casein hydrolysate a n d 2 % glucose,

c, e, 0 16 hr. at 37 °C in a m e d i u m containing 3 % casein hydrolysate, 2 % glucose, 0.1 % yeast extract a n d
cardiac muscle particles. A n a e r o b i c conditions,
d) 16 hr. at 37 °C in a m e d i u m containing 3 % casein hydrolysate, 2 % glucose a n d 0.1 % yeast extract,
g) 60 hr. at 30 °C in 2 % p e p t o n e . A e r o b i c conditions.
In a), b) a n d d) the bacteria are allowed to grow in a bottle filled u p to the neck b u t unsealed. T h e g r o w t h
conditions are then partially a n a e r o b i c .
Preparation of the acetone dry powder
Prepare a thick suspension o r c r e a m with the cells in distilled water. P o u r the suspension quickly whilst
stirring into five times its v o l u m e of cold acetone a n d c o n t i n u e stirring until the cells coagulate. Suck off
precipitate (Buchner funnel), wash on the funnel once with acetone a n d once with ether a n d dry in air.
References
1 E. F. Gale, Advances in Enzymology 6, 1 [1946].

2 E. F. Gale in D. Glick: M e t h o d s of Biochemical Analysis, vol. IV, p . 285. Interscience, N e w Y o r k 1957.
3 L. V. Crawford, Biochem. J. 68, 221 [1958].
4 H. M. R. Epps, Biochem. J. 39, 42 [1945].
5 W. W. Umbreit, R. H. Burris & /. F. Stauffer, M a n o m e t r i c Techniques. Burgess Publ. C o . , Minneapolis,
M i n n . , 1949.
6 R. W. McGilvery & P. P.Cohen, J. biol. C h e m . 174, 813 [1948].
7 H. M. R. Epps, Biochem. J. 47, 605 [1944].
8 W. W. Umbreit & P. Heneage, J. biol. C h e m . 221, 15 [1953].
9 A. Meister, H. A. Sober & S. V. Tice, J. biol. C h e m . 189, 577, 591 [1951].
L-Lysine, L-Arginine, L-Histidine, L-Ornithine and L-Tyrosine

Colorimetric Method with Fluorodinitrobenzene
Joel Hutzler*
Progress in the chemistry of a m i n o acids has always been d e p e n d e n t o n i m p r o v e d analytical m e t h o d s . M a n y

new m e t h o d s based o n c o l u m n c h r o m a t o g r a p h y have been widely used for a m i n o acid d e t e r m i n a t i o n s .
Nevertheless, there is a need for simple, accurate, a n d fast microanalytical m e t h o d s t h a t d o n o t require
expensive i n s t r u m e n t s a n d t h a t allow a series of d e t e r m i n a t i o n s of specific a m i n o acids.
Various a m i n o acids can be determined by m e a s u r e m e n t of c a r b o n dioxide liberated o n t r e a t m e n t with
specific L-amino acid decarboxylases ( L - A m i n o a c i d carboxy-lyase, E C 4 . 1 . 1 - ) 1 , 2
. T h e amine that r e m a i n s
m a y also be converted into the c o r r e s p o n d i n g d i n i t r o p h e n o l derivative by reaction with fluorodinitro
benzene ( F D N B ) , with subsequent s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of this derivative. This m e t h o d is
m o r e sensitive, a n d will be described here.
Application of Method: T h e m e t h o d is used for the d e t e r m i n a t i o n of the a b o v e - m e n t i o n e d a m i n o acids in

plasma a n d urine, a n d particularly for a series of d e t e r m i n a t i o n s of a single a m i n o acid or for confirmation
of the identity of a n a m i n o acid tentatively identified by other m e t h o d s . T h e process can be easily extended
to additional a m i n o acids if c o r r e s p o n d i n g enzymes are available. In the case of labelled a m i n o acids in
biological solutions, the c a r b o n skeleton can be isolated by this m e t h o d .
Principle
(1) Lysine l y s i n e d e c a r b o x y l a s e
» Cadaverine + C0 2
(pyrodoxal phosphate) i
I
(2) Cadaverine + 2 F D N B • Cadaverine • 2 D N B + 2HF
pH 7 - 1 0
Lysine is used here as an example. W h e n the reaction is complete, the m i x t u r e is m a d e strongly alkaline to
destroy unreacted F D N B . T h e D N B derivative of the a m i n e is extracted with c h l o r o f o r m , a n d the extinct
ion is m e a s u r e d in the chloroform p h a s e at 400 n m .
A g m a t i n e , the reaction p r o d u c t of arginine, only reacts with o n e molecule of F D N B .
The measurement presents n o difficulties in n o r m a l biological material. Excessively low results are to be
expected in pathological specimens containing unusually high contents of a m i n o acids, p h o s p h a t e s , o r heavy
metals, since these are k n o w n inhibitors of the enzyme o r of the F D N B reactions. H o w e v e r , we h a v e never
encountered difficulties of this n a t u r e . A large excess of buffer a n d enzyme is used in the m e t h o d described
here to minimize the possibility of interference. T h e decarboxylation usually proceeds to an extent of
8 0 - 9 0 % in the first 10 min.
T h e n o r m a l contents of lysine a n d tyrosine in p l a s m a can be readily d e t e r m i n e d . T h e c o n t e n t s of arginine
a n d histidine in p l a s m a after starvation ( a p p r o x . 0.08 ^ m o l e / m l . ) lie a r o u n d the sensitivity limit of the m e t h o d ;
elevated values can be easily m e a s u r e d if plasma is deproteinized by ultrafiltration. This m e t h o d is better
t h a n protein precipitation by Z n S 0 a n d N a O H .
4
F o r o p t i m u m experimental conditions, see Table 1.
* S u p p o r t e d by the N a t i o n a l Science F o u n d a t i o n .
Table 1. Experimental c o n d i t i o n s .
Decarboxylation F D N B reaction Extraction

Enzyme* pH Reaction Temp. Time pH KOH Solvent
product (° Q (min.) (N)
(amine)
Lysine decarboxylase, 6.0 Cadaverine 60 30 8.7 1.0 Chloroform
B. cadaveris ( N C I B * * 6578),
0.1 U / m g . (37 °C)
Arginine decarboxylase, 5.2 Agmatine 45 60 10.0 5.0 Chloroform
E. coli ( N C I B * * 7020),
0.2 U / m g . (37 °C)
Histidine decarboxylase, 4.5 Histamine 60 60 8.7 1.0 Chloroform
CI welchii ( N C I B * * 6785),
0.07 U / m g . (37 °C)
Tyrosine decarboxylase 5.5 Tyramine 60 30 8.7 1.0 Chloroform
S.faecalis (NCTC 6783),
0.1 U / m g . (37 °C)
Ornithine decarboxylase, 5.5 Putrescine*** 60 30 8.7 1.0 Methylene
CI septicum ( N C I B * * 547) chloride
0.1 U / m g . (37 °C)
* Typical p r e p a r a t i o n s that have been successfully used in o u r l a b o r a t o r y .

** N C I B = N a t i o n a l Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland.
These n u m b e r s are identical with the N C T C n u m b e r s ( N a t i o n a l Collection of Type Cultures, L o n d o n ,
England), except in the case of S. faecalis.
** Only putrescine has been determined in o u r l a b o r a t o r y , n o t ornithine.
Equipment
A n a r r o w b a n d w i d t h s p e c t r o p h o t o m e t e r ( e . g . B e c k m a n D U o r D B ; t h e e x t i n c t i o n s will
t h e n b e linear). If a w i d e b a n d - w i d t h i n s t r u m e n t m u s t b e u s e d , it will b e n e c e s s a r y t o c o n s t r u c t
a c a l i b r a t i o n c u r v e , s i n c e t h e e x t i n c t i o n s will b e n o n - l i n e a r .
S h a k i n g i n c u b a t o r , 37 ° C a n d 6 0 ° C ; s t o p p e r e d c u v e t t e s 9 - 1 2 m l . , light p a t h a p p r o x . 1 c m . . +
Dialysis tubing, diameter / inch 1

4
+ +
.
Reagents
1. P o t a s s i u m h y d r o x i d e , A . R . 9. A m i n o a c i d s
2. H y d r o c h l o r i c a c i d , A . R . lysine, arginine, histidine, ornithine, tyrosine
3. B o r i c acid, A . R., H B 0 3 3
10. Decarboxylases
4. Ethylenediaminetetra-acetate, EDTA acetone-dried p o w d e r of various bacteria, see
disodium salt, E D T A - N a H - 2 H 0 , A . R .
2 2 2
Table 1. Lysine, arginine, histidine, a n d tyrosine
5. l - F l u o r o - 2 , 4 - d i n i t r o b e n z e n e , FDNB decarboxylases are commercial p r o d u c t s m a n u
6. C h l o r o f o r m , A . R . ( w i t h 0 . 7 5 % e t h a n o l ) factured by Worthington. Ornithine decarboxy
7. E t h a n o l , a n h y d r o u s , A . R . lase is u n s t a b l e ; for p r e p a r a t i o n see Gale, p . 1668;
8. P y r i d o x a l p h o s p h a t e solutions keep for only a few d a y s + + +
.
+
Culture tubes with Teflon-coated screw caps (Kimble N o . 45066-A, 13 x 100 m m . ) with ± 0 . 5 %
deviation at 50% transmission m a y be used.
+ +
F o r the p r e p a r a t i o n of ultrafiltrates, e.g. from A. H. T h o m a s Co., P. O. Box 779, Philadelphia, Pa.
19105.
+ + +
Recently Worthington has m a r k e t e d partially purified decarboxylase p r e p a r a t i o n s which should
be of a d v a n t a g e in the assay.
11. Methylene chloride, A . R. 14. P r o p i o n i c a c i d , A . R .

for the d e t e r m i n a t i o n of o r n i t h i n e for the d e t e r m i n a t i o n of arginine, tyrosine, a n d
12. M a l e i c a c i d , A . R . ornithine
for the d e t e r m i n a t i o n of lysine 15. Z i n c s u l p h a t e , Z n S 0 - 7 H 0 , A . R.
4 2
13. Acetic acid, A . R. 16. S o d i u m h y d r o x i d e , A . R .

for the d e t e r m i n a t i o n of histidine
Purity of Reagents
T h e reagents o b t a i n a b l e in the U S A u n d e r the designation "highest p u r i t y " m a y be used as supplied.
U s e o n l y freshly d i s t i l l e d w a t e r .
I. P o t a s s i u m h y d r o x i d e ( 1 . 0 N ) :
D i s s o l v e 198 g. K O H p e l l e t s ( a p p r o x . 8 5 % K O H ) in d i s t i l l e d w a t e r a n d m a k e u p t o
3 0 0 0 ml. Titrate with standard acid.
II. P o t a s s i u m h y d r o x i d e ( a p p r o x . 5 N ) :
D i s s o l v e 3 3 2 g. o f K O H p e l l e t s in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
III. H y d r o c h l o r i c a c i d ( a p p r o x . 0 . 4 5 N ) :
D i l u t e 37 m l . c o n e . H C 1 ( 3 6 - 3 8 % ) t o 9 6 0 m l . w i t h d i s t i l l e d w a t e r .
I V . Buffer s o l u t i o n
F o r the determination o f lysine, ornithine, histidine, and tyrosine.
a ) B o r a t e buffer ( 1 . 0 M ; p H 8 . 7 ) :
D i s s o l v e 6 1 . 8 g. b o r i c a c i d i n 2 6 0 m l . 1 N K O H a n d a p p r o x . 6 5 0 m l . d i s t i l l e d w a t e r
w i t h stirring a n d h e a t i n g ; filter a n d m a k e u p t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r .
For the determination o f arginine
b ) B o r a t e buffer ( 1 . 0 M ; p H 1 0 ) :
D i s s o l v e 6 1 . 8 g. b o r i c a c i d in 6 0 0 m l . 1 N K O H a n d 3 0 0 m l . d i s t i l l e d w a t e r w i t h
stirring a n d h e a t i n g ; filter a n d m a k e u p t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r .
V. l-Fluoro-2,4-dinitrobenzene, F D N B ( 0 . 4 % w / v ) :
D i s s o l v e 2 0 0 m g . F D N B in 5 0 m l . o f a n h y d r o u s e t h a n o l ( c a u t i o n : v e s i c a n t ) .
V I . Buffer f o r d e c a r b o x y l a t i o n
F o r the determination o f lysine
a ) M a l e a t e buffer ( 0 . 2 M ; p H 6 . 0 ) :
D i s s o l v e 2 3 . 2 g. m a l e i c a c i d a n d 0 . 9 g. E D T A i n d i s t i l l e d w a t e r , a d d 2 7 0 m l . 1 N K O H ,
a n d dilute t o 1 0 0 0 ml. with distilled water.
F o r the determination of arginine
b) P r o p i o n a t e buffer ( 0 . 2 M ; p H 5 . 2 ) :
D i s s o l v e 15 m l . p r o p i o n i c a c i d a n d 0 . 9 g. E D T A i n w a t e r , a d d 1 1 5 m l . o f 1 N K O H ,
and dilute to 1 0 0 0 ml. with water.
For the determination of tyrosine and ornithine
c) P r o p i o n a t e buffer ( 0 . 2 M ; p H 5 . 5 ) :
D i s s o l v e 15 m l . p r o p i o n i c a c i d a n d 0 . 9 g. E D T A in d i s t i l l e d w a t e r , a d d 1 4 6 m l .
1 N K O H , a n d dilute t o 1 0 0 0 ml. with distilled water.
F o r the determination o f histidine

d ) A c e t a t e buffer ( 0 . 2 M ; p H 4 . 5 ) :
D i s s o l v e 11.5 m l . a c e t i c a c i d a n d 0 . 9 g. E D T A in d i s t i l l e d w a t e r , a d d 5 6 m l . 1 N K O H ,
a n d dilute t o 1 0 0 0 ml. with distilled water.
VII. Zinc sulphate (approx. 0.6 N ) :
D i s s o l v e 9 0 g. Z n S 0 - 7 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
4 2
V I I I . S o d i u m h y d r o x i d e ( a p p r o x . 0.5 N ) :
D i s s o l v e 2 0 g. N a O H i n d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
Note: Titrate the solutions for the deproteinization in the presence of the decarboxylation buffer as
follows. Mix 5 ml. decarboxylation buffer, 20 ml. zinc sulphate (solution VII), a n d 150 ml. distilled
water, a n d while stirring, titrate with s o d i u m hydroxide (solution V I I I ) t o p H 8.1. Adjust the sodium
hydroxide solution (solution V I I I ) so t h a t 20 + 0.1 ml. are r e q u i r e d ; with p h e n o l p h t h a l e i n , 20.4 +
0.1 ml. N a O H should give a lasting pink colour.
I X . S t a n d a r d a m i n o a c i d s o l u t i o n s (1 ^ m o l e / m l . ) :
D i s s o l v e 0.5 m m o l e ( e . g . 9 1 . 3 5 m g . o f l y s i n e • HC1) in w a t e r t o 5 0 0 m l .
MW MW MW
Lys 146.2 Lys HC1 182.7 Lys 2HC1 219.1
Arg 174.2 Arg HC1 210.7 — —
His 155.2 His HC1H 0 2 209.6 His •2HC1 228.1
Orn 132.2 Orn H Q 168.6 Orn •2HC1 205.1
Tyr 181.2 — — —
X. Enzyme suspension:
P r e p a r e i m m e d i a t e l y b e f o r e u s e . D i l u t e 5 m l . o f t h e a p p r o p r i a t e d e c a r b o x y l a t i o n buffer
( V I ) w i t h 4 5 m l . w a t e r , a d d 1 0 0 m g . e n z y m e p o w d e r , stir b y h a n d w i t h a g l a s s h o m o g e n i z e r
t o f o r m a u n i f o r m s u s p e n s i o n , a n d c e n t r i f u g e for 10 m i n . at 8 0 0 g. D i s c a r d t h e s u p e r
n a t a n t fluid ( w h i c h c o n t a i n s u n w a n t e d fine p a r t i c l e s a n d a n u m b e r o f a m i n e - l i k e s u b
s t a n c e s ) . R e s u s p e n d t h e p r e c i p i t a t e u n i f o r m l y in 2 5 m l . d e c a r b o x y l a t i o n buffer a n d a d d
1 mg. pyridoxal phosphate.
T h e decarboxylation buffers will keep for m a n y m o n t h s at a b o u t 0 °C. T h e stabilities of the acetone-dried

enzyme powders vary in an unpredictable m a n n e r . They are best kept in a desiccator at — 20 °C. Ornithine
decarboxylase is less stable, a n d should always be used when fresh. Store the alkali solutions in tightly c a p p e d
plastic bottles to prevent a b s o r p t i o n of C 0 . These solutions, as well as the b o r a t e buffer, can be kept at
2
r o o m t e m p e r a t u r e . Store the F D N B solution in a cool place, and p r e p a r e fresh solution every week. T h e
standard a m i n o acid solutions are best kept below 0 °C. If crystallization occurs (particularly in the case of
tyrosine), carefully redissolve the crystals before use.
Procedure
T h e m e t h o d h a s b e e n u s e d b y u s o n l y f o r p l a s m a a n d u r i n e . H o w e v e r , it s h o u l d a l s o b e s u i t a b l e
for u s e , either d i r e c t l y o r after s l i g h t m o d i f i c a t i o n , for t i s s u e h o m o g e n a t e s , s e r u m , c e r e b r o s p i n a l
fluid, and other biological material. T h e determination o f amine in accordance with equation
(2) c a n b e applied t o the c o r r e s p o n d i n g a m i n e s from norvaline, serine, phenylalanine, a n d
t r y p t o p h a n ( m e t h o d f o r l y s i n e ) . It is w e l l k n o w n t h a t t h e s e a r e r e p r e s e n t a t i v e o f v a r i o u s
classes o f a m i n o acids.
Collection and treatment of sample: C o l l e c t b l o o d w i t h s y r i n g e s c o n t a i n i n g 0 . 2 m g . o f h e p a r i n /

ml. or 1 m g . o f E D T A / m l . O b t a i n p l a s m a i m m e d i a t e l y a n d k e e p at — 2 0 ° C until analysis.
K e e p urine frozen at — 2 0 °C.
Stability of sample: T o o b t a i n u n i f o r m r e s u l t s , a s t a n d a r d i z e d m e t h o d m u s t b e u s e d t o d e t e r m i n e
t h e a m i n o a c i d c o n t e n t o f b l o o d p l a s m a . I f p l a s m a is i m m e d i a t e l y s e p a r a t e d f r o m e r y t h r o c y t e s ,
t h e a m i n o a c i d level is c o n s t a n t f o r 2 4 hr. at 0 t o -f 4 ° C , a n d f o r s e v e r a l w e e k s a t — 2 0 ° C . I f u r i n e
is k e p t at r o o m t e m p e r a t u r e , t h e a m i n o a c i d c o n t e n t r e m a i n s c o n s t a n t if t h e g r o w t h o f b a c t e r i a is
a v o i d e d , b u t s o m u c h a m m o n i a is f o r m e d f r o m urine that t h e c o n t r o l v a l u e b e c o m e s very
h i g h . T h i s r e d u c e s t h e s e n s i t i v i t y o f t h e m e t h o d , b u t d o e s n o t affect t h e r e s u l t .
Fig. 1. Ultrafiltration a p p a r a t u s .
Two types (A a n d B) used in o u r l a b o r a t o r y ; the dimensions m a y be c h a n g e d as required. C shows a v a c u u m
distribution vessel for several samples, a = glass t u b e with tapered e n d ; outside d i a m e t e r 8 m m . , length
125 m m . b = r u b b e r stopper, c = filter tube, 22 m m . x 175 m m . d = dialysis tube, diameter a p p r o x .
6 m m . e = 1 m l . plastic syringe (disposable), c u t d o w n , f = stainless h y p o d e r m i c needle, N o . 20, IV2 inch,
as v a c u u m connector, g = test tube, 22 m m . x 175 m m . h = r u b b e r b u l b for sealing u n u s e d tubes,
j = wide-necked flask, diameter 55 m m . k = glass tube, outside diameter 8 m m . , length 60 m m . (may also
be as e,f)- T h e ends v of t h e tubes a r e connected by v a c u u m t u b i n g t o t h e p o i n t s v of A a n d B , a n d o n e
end v t o t h e v a c u u m p u m p .
Deproteinization: T h e only suitable deproteinization m e t h o d s are the precipitation o f protein

w i t h z i n c s u l p h a t e / s o d i u m h y d r o x i d e s o l u t i o n a n d ultrafiltration. T h e z i n c m e t h o d is faster,
b u t m u s t b e carried o u t w i t h g r e a t a c c u r a c y , g i v e s l o w e r c o l o u r v a l u e s , a n d is u n s u i t a b l e f o r
t h e d e t e r m i n a t i o n o f a r g i n i n e o r h i s t i d i n e . U l t r a f i l t r a t i o n is s i m p l e a n d u n i v e r s a l l y a p p l i c a b l e .
Plasma, deproteinization with ZnSO^/NaOH: T h e p l a s m a p r o t e i n s a r e r e m o v e d o n l y after t h e

decarboxylation reaction, see pipetting scheme. Samples treated with zinc sulphate have
s o m e w h a t r e d u c e d c o l o u r v a l u e s , a n d t h e s t a n d a r d s m u s t t h e r e f o r e a l s o b e t r e a t e d w i t h this
reagent. Standards can be prepared by the addition o f k n o w n quantities o f a m i n o acids to plasma.
U s e p l a s m a f r o m f a s t i n g y o u n g a d u l t s ; t h i s c o n t a i n s s o little a m i n o a c i d t h a t t h e r e s u l t i n g
i n c r e a s e in e x t i n c t i o n c a n b e e a s i l y c a l c u l a t e d . W e prefer t h e s e s t a n d a r d s t o a q u e o u s s t a n d a r d
s o l u t i o n s , s i n c e l o w v a l u e s a r e o b t a i n e d if p r o t e i n s are n o t p r e c i p i t a t e d c o r r e c t l y .
Ultrafiltration: M o i s t e n a piece o f dialysis tubing 1 3 - 1 8 cm. long, and k n o t o n e end. Push the tube
o v e r t h e t a p e r e d e n d o f a g l a s s t u b e , p a s s it t h r o u g h t h e b o r i n g in a r u b b e r s t o p p e r , a n d fix it in
t h e b o r i n g w i t h t h e g l a s s t u b e ( s e e F i g . 1). F i l l t h e d i a l y s i s t u b e w i t h p l a s m a u n t i l t h e s u r f a c e o f
t h e l i q u i d p r o j e c t s i n t o t h e g l a s s t u b e ( t h e v o l u m e o f t h e d i a l y s i s t u b e is d o u b l e d w h e n v a c u u m is
a p p l i e d , F i g . 1 A ) . T h e ultrafiltration is c a r r i e d o u t a t 0 t o + 4 ° C u n d e r a n e g a t i v e p r e s s u r e o f
a b o u t 5 0 0 - 5 5 0 m m . H g . G r e a t e r p r e s s u r e d r o p s c a n l e a d t o l o s s o f m a t e r i a l a s a result o f
e v a p o r a t i o n . 5 0 t o 6 0 % o f o r i g i n a l v o l u m e is filtered o n ultrafiltration o v e r n i g h t .
T h e a p p a r a t u s c a n b e s i m p l i f i e d a s s h o w n in F i g . 1 B . T h e s i m u l t a n e o u s ultrafiltration o f several
s a m p l e s c a n b e carried o u t b y t h e u s e o f a v a c u u m d i s t r i b u t o r v e s s e l m a d e o f t h i c k g l a s s ( F i g . 1 C ) .
T h e v a c u u m t u b e s are a t t a c h e d t o b o r e h o l e s i n t h e v e s s e l s t o p p e r . S e e a l s o Smith . 4
Assay System with Deproteinization (for e. g. Plasma)
After the decarboxylation reaction, the plasma proteins are removed with Z n S 0 / N a O H . This 4
slightly r e d u c e s t h e c o l o u r . S t a n d a r d m i x t u r e s s h o u l d b e d e p r o t e i n i z e d in t h e s a m e w a y a s t h e
s a m p l e s . A s i n g l e b l a n k is c a r r i e d t h r o u g h i n e a c h a n a l y t i c a l series. T h e e x t i n c t i o n o f t h e b l a n k
s h o u l d b e n e a r z e r o w h e n t h e F D N B ( s o l u t i o n V ) is fresh.
S t a n d a r d s : 0.5 m l . p l a s m a h a v i n g a l o w a m i n o a c i d c o n t e n t w i t h 0, 0 . 0 5 , 0 . 1 0 , 0 . 2 0 , 0 . 3 0 , a n d
0.40 ml. (//mole) standard solution ( I X ) + 0 . 5 ml. e n z y m e suspension ( X ) .
C o n t r o l : 0.5 m l . p l a s m a + 0 . 5 m l . d e c a r b o x y l a t i o n buffer ( V I ) , b u t d o n o t a d d e n z y m e . P l a s m a
c o n t r o l v a l u e s a r e c o n s t a n t a n d l o w ( t h i s is n o t t h e c a s e w i t h u r i n e , w h e r e t h e y fluctuate
considerably and m a k e a control value necessary for each measurement).
B l a n k : 0.5 m l . d i s t i l l e d w a t e r + 0.5 m l . d e c a r b o x y l a t i o n buffer ( V I ) .

I n c u b a t i o n t e m p e r a t u r e : 37 ° C ; i n c u b a t i o n t i m e : 4 5 m i n . ; i n c u b a t i o n v o l u m e : 1.5 m l . C o l o u r
r e a c t i o n in 2 . 6 m l . ; w a v e l e n g t h : 4 0 0 n m ; light p a t h ; 1 c m . M e a s u r e a g a i n s t c h l o r o f o r m .
P i p e t t e i n t o 16 m m . x 125 m m . c e n t r i f u g e t u b e s . C o n c e n t r a t i o n in assay mixture
S a m p l e ( e . g . 0.5 m l . p l a s m a ) + water 1.0 m l . U p to 0.3 m M a m i n o acid

D e c a r b o x y l a t i o n buffer (VI) 0.5 m l . 67 m M maleate, acetate, or
( o n l y in b l a n k a n d c o n t r o l ) p r o p i o n a t e ; 0.8 m M EDTA
Enzyme suspension (X) 0.5 m l . 1.3 m g . o f e n z y m e / m l .
(only in s a m p l e and standards)
Pipette rapidly, keeping the e n z y m e suspension uni

form by shaking. Incubate for 45 min. with shaking.
ZnS0 4 solution (VII) 2.00 ml.
M i x ; d e c a r b o x y l a t i o n r e a c t i o n is s t o p p e d .
N a O H solution (VIII) 2.00 ml.
A l l o w N a O H solution to run slowly out o f the pipette

and d o w n the inner wall o f the tube (cover with a layer
o f N a O H ) . S t o p p e r the tube a n d m i x rapidly by tilting
(do not shake!). A h o m o g e n e o u s milky solution should
b e f o r m e d . C e n t r i f u g e f o r 2 0 m i n . a t 1 5 0 0 g. D o n o t
d e c a n t s u p e r n a t a n t fluid, b u t r e m o v e w i t h a p i p e t t e .
Pipette into centrifuge tubes: C o n c e n t r a t i o n in a s s a y m i x t u r e
Supernatant 2.0 ml.

B o r a t e buffer (IV) 0.4 ml. 154 m M borate
F D N B solution (V) 0 .2 m l . 1.7 m M FDNB
(Warning: Poison. D o not pipette by mouth). Pipette

i n t o o n l y 2 c u v e t t e s at a t i m e . C l o s e c u v e t t e s i m m e d i a
tely, a n d m i x w e l l b y s h a k i n g v i g o r o u s l y . I n c u b a t e ( f o r
t i m e a n d t e m p e r a t u r e s e e T a b l e 1). A l l o w t o c o o l t o
r o o m temperature.
K O H s o l u t i o n (I o r II a c c o r d i n g t o T a b l e 1) 1.0 m l .
Organic solvent according to Table 1 4.0 ml.
C l o s e t u b e s a n d s h a k e w e l l . C e n t r i f u g e briefly to
separate phases. R e a d extinction.
Assay System Without Deproteinization (Protein-Free solution)
U s e 0 . 0 5 m l . o r 0 . 1 0 m l . o f u r i n e s a m p l e s ; u p t o 0 . 6 0 m l . in t h e c a s e o f v e r y d i l u t e u r i n e ( d u p l i c a t e
determinations). A control m u s t b e carried t h r o u g h for e a c h urine s a m p l e .
U s e 0.3 m l . o f p l a s m a ultrafiTtrates ( d u p l i c a t e d e t e r m i n a t i o n s ) .
Standards, control, blank, and other incubation and assay c o n d i t i o n s as described a b o v e .
I n c u b a t i o n t e m p e r a t u r e : 3 7 ° C ; i n c u b a t i o n t i m e : 4 5 m i n . ; i n c u b a t i o n v o l u m e : 1.1 m l . ; c o l o u r
r e a c t i o n in 1.7 m l . ; w a v e l e n g t h : 4 0 0 n m ; m e a s u r e a g a i n s t c h l o r o f o r m .
P i p e t t e i n t o test t u b e s : C o n c e n t r a t i o n in a s s a y m i x t u r e
Sample + water 0.6 m l . up to 0.4 m M a m i n o acid

D e c a r b o x y l a t i o n buffer (VI) 0.5 m l . 91 m M m a l e a t e , a c e t a t e o r
(only in blank and control) p r o p i o n a t e ; 1.1 m M E D T A
Enzyme suspension (X) 0.5 m l . 1.8 m g . o f e n z y m e / m l .
( o n l y in s a m p l e a n d s t a n d a r d s )
Pipette rapidly, keeping the e n z y m e suspension uni

f o r m b y s h a k i n g . I n c u b a t e for 4 5 m i n . w i t h v i g o r o u s
shaking.
HC1 (III) 0.2 ml. 69 m M HC1
M i x ; d e c a r b o x y l a t i o n r e a c t i o n is s t o p p e d . C e n t r i f u g e
for 2 0 m i n . at 1 5 0 0 g. U s e s u p e r n a t a n t fluid.
Pipette into cuvettes: C o n c e n t r a t i o n in a s s a y m i x t u r e
Supernatant 1.0 m l . u p t o 0.7 m M a m i n o a c i d

B o r a t e buffer (IV) 0.4 ml. 235 m M borate;
( a c c o r d i n g t o T a b l e 1) 0.5 m l . E D T A
1 N KOH (I) 0.1 m l .
M i x carefully t o r e m o v e undiluted K O H from the wall

o f the vessel. C o n t i n u e with addition o f F D N B as des
c r i b e d in t h e a b o v e p i p e t t i n g s c h e m e . 2.5 m M FDNB
Calculations
Since the s t a n d a r d mixtures are treated as samples, the experimental values are c o m p a r e d to the s t a n d a r d
values. Subtract the extinction of the control from the extinction of the sample to o b t a i n A E S a m p l e . Proceed
similarly for the s t a n d a r d s (procedure " w i t h deproteinization"). In the p r o c e d u r e " w i t h o u t deproteiniza
tion", subtrakt the extinction of the t u b e with 0 ml. of s t a n d a r d solution from the extinction of the
Fig. 2. S t a n d a r d curves.
A - L-Lysine
0 0.05 0.10 0.20 0.30 0.45 B = L-Tyrosine
ûmole amino acid C = L-Arginine a n d L-histidine.
s t a n d a r d s . This gives A E . If c
S t s t a n d a r d is the a m i n o acid c o n c e n t r a t i o n (/imole/ml.) of the s t a n d a r d a n d v
the v o l u m e of the sample, then
c S a m p ie=^ S a m p l e
- C s t a n d a r d
[/miole/ml.]
Plot A E s t (ordinate) against c (abscissa); this should give a straight line (Fig. 2). A s t a n d a r d curve of this
type is preferably constructed for each series of d e t e r m i n a t i o n s ; the average J E S t found in this way for
1 /xmole of a m i n o acid is used in the calculation.
The best results are o b t a i n e d if the assay mixture contains a b o u t 0.20 /imole of the a m i n o acid to be determin
ed. Lysine, ornithine, a n d tyrosine give better values t h a n histidine a n d arginine. In the d e t e r m i n a t i o n of
lysine at several c o n c e n t r a t i o n s , we found the following values:
\imole of lysine s N CV
0 7.35 8 13.4
0.04 4.92 8' 11.3
0.08 7.16 7 9.3
0.16 12.6 7 8.6
0.32 20.7 8 6.2
0.64 19.0 8 3.1
N o r m a l Values
T h e values given in the literature for a m i n o acids in p l a s m a vary widely a n d d e p e n d on the assay m e t h o d
a n d on the deproteinization m e t h o d . F o r lysine, we found 3.3 mg./lOO ml. of p l a s m a after ultrafiltration
5
a n d 2.6 mg/100 ml. after deproteinization with Z n S 0 / N a O H . Stein 4

6
found 2.72 mg./lOO ml. by c o l u m n
c h r o m a t o g r a p h y , a n d Hier 1
found 2.95 mg./lOO ml. by a microbiological m e t h o d . A c c o r d i n g t o Stein,
the values for arginine, histidine, ornithine, a n d tyrosine are 1.45, 1.29, 0.91, a n d 1.04 m g . respectively per
100 ml. of fasting plasma.
M a n y c o m m o n buffers a n d deproteinizing agents interfere with the F D N B reaction. F o r the analysis of

new test material, b o t h factors m u s t first be checked.
T h e principal source of e r r o r is an insufficiency of F D N B as a result of a n excess of substrate or o t h e r
F D N B - c o n s u m i n g substances, the presence of which is shown by high c o n t r o l values. T h e sample m a y be
correspondingly diluted. T h e extinction of the c o n t r o l should be less t h a n 0.200.
Specificity o f M e t h o d
T h e specificity of the m e t h o d d e p e n d s mainly o n the enzyme. Gale et a l . 1 , 2

have s h o w n t h a t a) the enzymes
are specific for L - a m i n o acids, b) substituents on the a m i n o or carboxyl g r o u p h i n d e r decarboxylation,
c) additional hydroxyl g r o u p s elsewhere in the a m i n o acid molecule greatly reduce the decarboxylation
activity, a n d d) other decarboxylases in the enzyme p r e p a r a t i o n s are the m a i n cause of non-specificity. Gale
et a l . ' , Dickerman
1 2
et a l . , a n d Hutzler
8
et a l . have found t h a t traces of lysine, arginine, and tyrosine
3
decarboxylases in other enzyme p r e p a r a t i o n s are the principal sources of error. E q u i m o l a r quantities of the
corresponding substrates give a m a x i m u m of 4 % of the A E value of the a m i n o acids to be determined. Of
m a n y substances tested, m o s t react with < 0.5 % of the rate of the a m i n o acid u n d e r investigation . A m m o n i a
3
a n d amines increase the control value, but d o n o t affect the result of the d e t e r m i n a t i o n . F u r t h e r purification
of the dry enzyme p o w d e r s improves the specificity . 1,2
References
1 E. F. Gale, A d v a n . Enzymol. 6, 1 [1946].

2 E. F. Gale in D. Glick: M e t h o d s of Biochemical Analysis. Interscience, N e w Y o r k 1957, Vol. IV, p . 285.
3 / . Hutzler, M. Odievre & J. Dancis, Anal. Biochem. 79, 529 [1967].
4 /. Smith: C h r o m a t o g r a p h i c a n d Electrophoretic Techniques (/. Smith, Editor), Vol. 1, p . 62, Interscience,
N e w Y o r k 1960.
5 W. D. Block, M. E. Markovs & B. F. Steele, Proc. Soc. Expl. Biol., M e d . 122, 1089 [19661.
6 W. Stein & S. Moore, J. biol. C h e m . 211, 915 [1954].
7 S. W. Hier & O. Bergeim, J. biol. C h e m . 163, 129 [1946].
8 H. W. Dickerman & M. L. Carter, Anal. Biochem. 3, 195 [1962].
L-Alanine
Determination with Alanine Dehydrogenase
Dermot H. Williamson
L-Alanine d e h y d r o g e n a s e (L-Alanine: N A D oxidoreductase, d e a m i n a t i n g , E C 1.4.1.1) was first described

a n d partially purified from B. subtilis by Wiame a n d Pierard . 1,2
A similar enzyme h a s been isolated from
B. cereus . T h e p r e p a r a t i o n of the crystalline enzyme from B. subtilis* h a s led t o its use for the d e t e r m i n a t i o n
3
of L - a l a n i n e .
5,6
Application of Method: In biochemistry, clinical biochemistry a n d microbiology.
Principle
(1) L-Alanine + N A D +
+ H Q ,2
a l a n i n e
^ Pyruvate + N A D H + N H +
dehydrogenase
T h e increase in extinction at 340 (334 or 365) n m d u e t o the formation of N A D H is a m e a s u r e of the

reaction.
T h e equilibrium c o n s t a n t for reaction (1) is 8.06 x 1 0 ~ 1 4

( p H 8.98) a n d the o p t i m u m p H is 1 0 - 1 0 . 5 . 7
T h e reaction proceeds quantitatively from left t o right at low H +

ion c o n c e n t r a t i o n ( p H 10), a n d with the
inclusion of hydrazine t o t r a p the p y r u v a t e formed in the reaction as the h y d r a z o n e . If possible exclude 6
NH4 ions from the assay system.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r f o r a c c u r a t e m e a s u r e m e n t s at 3 4 0 , 3 3 4 o r
365 n m .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 6. L - A l a n i n e
2. Hydrazine hydrate ( 9 9 - 1 0 0 % ) 7. L - A l a n i n e d e h y d r o g e n a s e
3. H y d r o c h l o r i c a c i d , 1 N from Bacillus subtilis 5
ca. 130 U / m g . protein
4. Ethylenediaminetetra-acetic acid, E D T A (oxidative d e a m i n a t i o n r e a c t i o n ) . F o r c o m
7
d i s o d i u m salt, E D T A - N a H • 2 H 0
2 2 2 mercial p r e p a r a t i o n , see p . 427.
5. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , N A D
free acid; commercial p r e p a r a t i o n , see p . 545.
Purity of Reagents
Alanine dehydrogenase should n o t c o n t a i n m o r e t h a n 0 . 1 % L D H or M D H (relative t o the alanine de

hydrogenase activity). Less p u r e p r e p a r a t i o n s can be used if the a m i n o acid fraction is first separated from
6
interfering substances (see DL-Serine a n d T h r e o n i n e , p . 1727).

P r e p a r e all s o l u t i o n s w i t h d i s t i l l e d o r d e i o n i z e d w a t e r .
I. Tris s o l u t i o n ( 0 . 2 M ) :
D i s s o l v e 4 . 8 4 g. tris in 2 0 0 m l . d i s t i l l e d w a t e r .
II. H y d r a z i n e - t r i s buffer ( 4 0 m M tris, 1 M h y d r a z i n e , 1.4 m M E D T A ; p H 1 0 . 0 ) * :
M i x 5.0 m l . h y d r a z i n e h y d r a t e , 2 0 m l . tris s o l u t i o n (I), 5 0 m g . E D T A - N a H 2 ' 2 H 0 a n d
2 2
4 0 ml. distilled water. Adjust to p H 10.0 with N HC1 (glass electrode) a n d dilute to 100 ml.
with distilled water.
III. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( c a . 2 4 m M / ? - N A D ) :
D i s s o l v e 4 0 m g . N A D in 2 m l . d i s t i l l e d w a t e r .
IV. L-Alanine dehydrogenase:
E n z y m e p r e p a r a t i o n d i a l y s e d a g a i n s t 2 0 m M p h o s p h a t e , p H 7 . 4 , t o r e m o v e NH4 ions,
a n d d i l u t e d w i t h t h i s buffer t o g i v e a n a c t i v i t y o f 15 U / m l .
P r e p a r e the hydrazine-tris buffer (solution II) freshly each day. Store the N A D solution (HI) at —15 °C
a n d the tris solution (I) at 0 - 4 °C. Store the dilute alanine dehydrogenase solution (IV) at - 1 5 °C.
Procedure
Collection:
T h i s m e t h o d h a s o n l y b e e n t e s t e d o n p u r e s o l u t i o n s a n d o n e x t r a c t s o f liver t i s s u e p r e p a r e d
as described. DL-Serine and D L - T h r e o n i n e , p. 1727. Obtain tissues f r o m experimental animals
w i t h freeze c l a m p s (refer t o p . 4 0 0 ) . T h e a l a n i n e c o n t e n t o f rat liver i n c r e a s e s r a p i d l y p o s t
mortem . 6
Deproteinization:
Refer to p. 1729.
Isolation of the amino acid fraction :
T h i s is o n l y n e c e s s a r y if t h e a l a n i n e d e h y d r o g e n a s e p r e p a r a t i o n is c o n t a m i n a t e d w i t h l a r g e
a m o u n t s o f L D H o r M D H . F o r p r o c e d u r e refer t o p . 1 7 2 9 .
Stability of sample :
L - A l a n i n e is s t a b l e p r o v i d i n g b a c t e r i a l c o n t a m i n a t i o n is a v o i d e d .
L-Alanine also reacts quantitatively at p H 9.0 a n d this m e a n s t h a t L-alanine a n d L-glutamate can be

determined on the same sample by the successive addition of the respective dehydrogenases.
L-Alanine 1681
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 3.1 m l . ; m e a s u r e a g a i n s t
a c u v e t t e c o n t a i n i n g h y d r a z i n e - t r i s buffer ( s o l u t i o n II).
Sample 2.0 ml. 1 0 - 1 0 0 / i M L-alanine

H y d r a z i n e - t r i s buffer (II) 1.0 m l .
N A D solution (III) 0.1 m l . 0.8 m M
M i x and read extinction E . t
Alanine dehydrogenase (IV) 0.01 m l . 40 m U / m l .
M i x a n d r e a d t h e e x t i n c t i o n at 4 0 , 5 0 a n d 6 0 m i n . ;
by extrapolation determine extinction E . 2
E 2 — E x = A E is u s e d for t h e c a l c u l a t i o n s .
Calculations
U n d e r the conditions described a b o v e the reaction proceeds quantitatively a n d therefore the calculation
formula ( 2 ) o n p . 312 is used.
T h e results are o b t a i n e d in jumole L-alanine/ml. sample.
Wavelength: 334 nm " 340 nm 365 nm
c = 0.254 x AE 0.249 x AE 0.456 x AE [/imole/ml.]
T h e precision of the m e t h o d over the range 0 . 0 5 0 - 0 . 3 0 0 jumole is 99 ± 5.4% (S.D.).
N o r m a l Values
T h e L-alanine content of livers from fed rats is 1.23 ± 0.64 /zmole/g. fresh wt. a n d for starved rats (48 hr.)
is 0.46 ± 0.36 /zmole/g. fresh w t . . 6
Interference in the assay: C o n t a m i n a t i o n of the alanine dehydrogenase with excessive a m o u n t s of L D H or

M D H leads to high values unless the a m i n o acid fraction is first isolated (refer to p . 1729).
Specificity
L-Alanine de hydrogenase also reacts with the following a m i n o acids in decreasing o r d e r of activity:
L-y-aminobutyrate, L-valine, L-isoleucine, L-serine a n d L - n o r v a l i n e . However, because of their low affinity
7
for the enzyme a n d slow reaction rates relative to L-alanine they cause little interference in the assay.
References
1 J. M. Wiame & A. Pierard, N a t u r e 176, 1073 [1955].

2 A. Pierard & J. M. Wiame, Biochim. biophys. A c t a 37, 490 [I960].
3 N. G. McCormick & H. O. Halvorson, J. Bacteriol. 87, 68 [1964].
4 A. Yoshida & E. Freese, Biochim. biophys. A c t a 92, 33 [1964].
5 A. Yoshida, A n a l . Biochem. 11, 383 [1965].
6 D. H. Williamson, O. Lopes-Vieira & B. Walker, Biochem. J. 104, 497 [1967].
7 A. Yoshida & E. Freese, Biochim. biophys. A c t a 96, 248 [1965].
Determination with GPT and LDH

Marianne Grassl
L-Alanine is widely distributed in N a t u r e in the free state as well as being a constituent of proteins, so
that a n u m b e r of m e t h o d s for its d e t e r m i n a t i o n ( e . g . ) after preliminary c h r o m a t o g r a p h i c or electro
1,2 3
p h o r e t i c s e p a r a t i o n a r e available. These c o l o u r tests, like t h e gas c h r o m a t o g r a p h i c m e t h o d a r e however

4 5
only suitable w h e n a m i n o acid mixtures from peptide o r protein hydrolysates are t o be analysed. In the
following a specific enzymatic m e t h o d * is described, which d e p e n d s o n t h e reaction of L-alanine with
2-oxoglutarate ( O x o G ) in t h e presence of g l u t a m a t e - p y r u v a t e t r a n s a m i n a s e , G P T ( L - A l a n i n e : 2-oxo-
glutarate aminotransferase, E C 2.6.1.2), where the p y r u v a t e formed is d e t e r m i n e d with the aid of lactate
dehydrogenase, L D H ( L - L a c t a t e : N A D oxidoreductase, E C 1.1.1.27).
Application of Method: In biochemistry, possibly in foodstuff chemistry.
Principle
(1) L-Alanine + 2-Oxoglutarate 7 ^ Pyruvate + L - G l u t a m a t e
(2) Pyruvate + N A D H + H +
L-Lactate + N A D +
T h e decrease of N A D H , as m e a s u r e d by t h e c h a n g e of extinction at 340 (334, 365) n m , is p r o p o r t i o n a l t o

the a m o u n t of L-alanine present.
T h e equilibrium of reaction (2) in the n e u t r a l range is quantitatively in favour of N A D . Even with relatively
large a m o u n t s of G P T reaction (1) proceeds relatively slowly so t h a t the d e t e r m i n a t i o n takes 30 t o 60 min.
A l t h o u g h the Michaelis c o n s t a n t of G P T for alanine is very high this s u b s t r a t e is virtually quantitatively
converted in the system.
* End-point method
L-Alanine 1683
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e for p r e c i s e m e a s u r e m e n t s at 3 4 0 ,
3 3 4 or 365 n m .
Reagents
1. H y d r o c h l o r i c a c i d , A . R., 1 N 6. L a c t a t e d e h y d r o g e n a s e , LDH
2. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris from rabbit or pig muscle crystallized, solution
3. S o d i u m h y d r o x i d e , A . R., 1 N in 5 0 % glycerol (v/v); ^ 5 0 0 U/mg. (25 °C);
4. 2-Oxoglutarate, OxoG commercial preparation, see p. 4 8 1 .
free a c i d ; c o m m e r c i a l p r e p a r a t i o n , see p . 548. 7. G l u t a m a t e - p y r u v a t e t r a n s a m i n a s e , GPT
5. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo from pig heart, suspension in 3.2 M ammonium
tide, N A D H sulphate solution; ^ 8 0 U / m g . (25 °C); com
d i s o d i u m salt, N A D H - N a 2 ; commercial p r e p mercial preparation, see p. 463.
a r a t i o n , see p . 545.
Purity of Reagents
T h e enzymes m u s t be free from other N A D - d e p e n d e n t dehydrogenases (especially from glutamate

dehydrogenase, < 0.001 % ) , D - a m i n o acid oxidase a n d aminotransferases.
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y d i s t i l l e d w a t e r . T o p r e v e n t t h e g r o w t h o f m i c r o
o r g a n i s m s sterilize t h e c o n t a i n e r s .
I. Tris buffer (0.1 M ; p H 7 . 6 ) :

D i s s o l v e 1.2 g. tris in c a . 8 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7.6 w i t h 1 N H C 1 a n d d i l u t e
t o 100 m l . w i t h d i s t i l l e d w a t e r .
II. 2 - O x o g l u t a r a t e ( 0 . 2 M ) :
D i s s o l v e 3 0 m g . 2 - o x o g l u t a r i c a c i d in c a . 0.5 m l . d i s t i l l e d w a t e r , n e u t r a l i z e w i t h 1 N
N a O H a n d d i l u t e t o 1.0 m l . w i t h d i s t i l l e d w a t e r .
III. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 1 2 m M ) :
D i s s o l v e 10 m g . N A D H - N a 2 in 1 m l . d i s t i l l e d w a t e r .
I V . L a c t a t e d e h y d r o g e n a s e , L D H (1 m g . p r o t e i n / m l . ) :
If n e c e s s a r y , d i l u t e t h e s t o c k s o l u t i o n w i t h 5 0 % g l y c e r o l s o l u t i o n a c c o r d i n g l y .
V. Glutamate-pyruvate transaminase, G P T (10 mg. protein/ml.):

U s e the stock suspension undiluted.
Store all solutions a n d enzyme suspension at 4 °C. T h e tris buffer a n d 2-oxoglutarate solution are stable
for ca. 2 weeks a n d L D H a n d G P T for ca. 6 - 1 2 m o n t h s .
Procedure
N o assays h a v e been carried o u t by us o n biological material. This m e t h o d s h o u l d h a v e the

s a m e r a n g e o f a p p l i c a b i l i t y a s t h e k i n e t i c m e t h o d o f G. Pfleiderer et a l . .
6
S o l u t i o n s c o n t a i n i n g p r o t e i n c a n b e d e p r o t e i n i z e d b y h e a t i n g f o r 3 m i n . in a b o i l i n g w a t e r
b a t h f o l l o w e d b y c e n t r i f u g a t i o n . T h e s a m p l e is s t a b l e for at l e a s t a w e e k at 4 ° C i n n e u t r a l
solution.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 3 . 0 0 m l . ; r o o m t e m p e r
ature. R e a d a g a i n s t air.
Concentration in
Pipette into cuvettes:
assay mixture
Tris buffer (I) 2.75 ml. 92 m M

Sample 0.05 ml. 0 . 0 3 - 0 . 1 5 m M L-alanine
2-Oxoglutarate solution (II) 0.10 ml. 6.7 m M
N A D H solution (III) 0.05 ml. 0.2 m M
L D H suspension (IV) 0.01 m l . 6 U/ml.
M i x a n d r e a d e x t i n c t i o n E^.
G P T suspension (V) 0.04 ml. 10 U / m l .
Mix. After ca. 60 min. read the extinction every

5 min.; by extrapolation of these values to the time o f
G P T addition (see p. 308) determine extinction E . 2
Ex — E 2 = A E is u s e d for t h e c a l c u l a t i o n s .
T h e i n c r e a s e i n e x t i n c t i o n d u e t o t h e a d d i t i o n o f G P T ( V ) a l o n e is d e t e r m i n e d b y t h e f u r t h e r
a d d i t i o n o f s u s p e n s i o n ( V ) . T h e o b s e r v e d e x t i n c t i o n c h a n g e is u s e d t o c o r r e c t E .
2
Calculations
U n d e r the a b o v e conditions the reaction proceeds stoichiometrically a n d therefore the calculation formula
(2) on p . 312 applies. T h e results are o b t a i n e d in /imole L-alanine/ml. sample. T h e following relationships
hold:
Wavelength: 334 nm 340 nm 365 nm

c = AE x 9.84 AE x 9.65 AE x 17.65 [/imole/ml.]
c = AE x 0.876 AE x 0.859 AE x 1.57 [mg./ml.]
Insufficient purity of the enzymes used (see p . 1683) can result in false values. G P T can be inhibited by a
large excess of other a m i n o acids.
L-Alanine 1685
Specificity of M e t h o d
G P T from heart is strictly stereospecific a n d only reacts with L-alanine. O t h e r a m i n o acids are n o t determin
ed in this assay . 2-Aminobutyric acid also reacts with G P T , b u t the resulting 2 - o x o b u t y r a t e hardly reacts
7
under these conditions with L D H from muscle. W i t h L D H from heart, which is less specific with regard
to substrate, 2 - a m i n o b u t y r i c acid is also determined.
Other Enzymatic Methods
A kinetic assay based o n the s a m e principle has been described by G. Pfleiderer . 6

T h e m e t h o d described here
can also be carried o u t fluorimetrically .
8
T h e m e t h o d r e p o r t e d by A. Yoshida9
using L-alanine d e h y d r o g e n
ase from Bacillus subtilis is also suitable (see p . 1679).
References
1 W. Troll &R. K. Cannan, J. biol. C h e m . 200, 803 [1953].

2 Roussel-UclafD. Verdier & Fr. Romainville, A n n . P h a r m . F r a n c . 25, N o . 6, 497 [1967].
3 S.Jacobs in D. Glick: M e t h o d s of Biochemical Analysis, Vol. XIV, p . 177, Interscience Publishers
N e w York 1966.
4 S. Blackburn in D. Glick- M e t h o d s of Biochemical Analysis, Vol. XIII, p . 1, Interscience Publishers
N e w York 1965.
5 B. Weinstein in D. Glick: M e t h o d s of Biochemical Analysis, Vol. XIV, p . 203, Interscience Publishers
N e w York 1966.
6 G. Pfleiderer, L. Grein & Th. Wieland, A n n . A c a d . Sci. fennicae, Ser. A II, 60, 381 [1955].
7 P. P. Cohen in J. B. Sumner & K. Myrback: T h e Enzymes, Vol. / , p . 1040, A c a d e m i c Press, New York
1951.
8 / . M. Ghysen, D. J. Tipper & J. L. Strominger in C. P. Colowick & N. O. Kaplan: M e t h o d s of E n z y m -
ology, Vol. VIII, p . 698, A c a d e m i c Press N e w Y o r k 1966.
9 A. Yoshida, A n a l . Biochem. 11, 383 [1965].
D -Alanine
Marianne Grassl
Free D-alanine is extremely u n c o m m o n in N a t u r e , b u t it h a s been detected in peptides from the cell walls
of Bacillus subtilis as a c o m p o n e n t of the teichoic acid a n d from L. plantarum
1 2
a n d m u t a n t s of S. albus .
3
B o t h the D - a n d L-isomers are formed in the chemical synthesis of a l a n i n e a n d in various reactions. A n

elegant specific d e t e r m i n a t i o n of D-alanine is possible with the aid of the reaction catalysed by D - a m i n o
acid oxidase ( D - A m i n o acid:oxygen oxidoreductase, d e a m i n a t i n g , E C 1.4.3.3) which is coupled with
the lactate d e h y d r o g e n a s e r e a c t i o n . 4
Application of Method: In biochemistry a n d possibly in food chemistry.
Principle
(1) D-Alanine + 0 2 + H 0
2 ""l^Zr" > Pyruvate + NH 3 + H 0
2 2
^ g e L e > L-Lactate + N A D +
The decrease of N A D H , as m e a s u r e d by the change in extinction at 340 (334, 365) n m , is p r o p o r t i o n a l

to the a m o u n t of D-alanine present.
T h e equilibrium of reaction (2) d e p e n d s on the p H . In the neutral region it lies o n the side of L-lactate.
However, since reaction (1) is m u c h faster in weakly alkaline media, a c o m p r o m i s e is necessary. T h e deter
m i n a t i o n is carried o u t at p H 8.5; the pyruvate formed in reaction (1) is still converted a l m o s t quantitatively
into L-lactate, while reaction (1) is still sufficiently fast. T h e h y d r o g e n peroxide formed at the same time must
be eliminated by catalase, since oxidation of pyruvate to acetic acid a n d c a r b o n dioxide m a y otherwise
occur.
Apparatus
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r c a p a b l e o f a c c u r a t e m e a s u r e m e n t s at 3 4 0 , 3 3 4 ,
o r 365 n m .
Reagents
1. H y d r o c h l o r i c a c i d , A . R . 5. L a c t a t e d e h y d r o g e n a s e , L D H
2. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris from rabbit o r pig skeletal muscle, crystalline
3. Oxygen suspension in 3.2 M ammonium sulphate
4. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo s o l u t i o n ; ^ 5 0 0 U / m g . (25 °C). F o r commercial
tide, N A D H p r e p a r a t i o n , see p . 4 8 1 .
disodium salt, N A D H - N a . F o r 2 commercial
p r e p a r a t i o n s , see p . 545.
D-Alanine 1687
6. Catalase 7. D - A m i n o a c i d o x i d a s e , D - A O D
from bovine liver, crystalline suspension in a q u e from pig kidneys, crystalline suspension in 3.2 M
ous s o l u t i o n ; ^ 50 000 U / m g . (25 °C). F o r c o m a m m o n i u m sulphate s o l u t i o n ; ^ 15 U / m g .
mercial p r e p a r a t i o n , see p . 438. (25 °C). F o r c o m m e r c i a l p r e p a r a t i o n , s e e p . 4 3 1 .
Purity of Reagents
T h e enzymes m u s t be free from o t h e r N A D - d e p e n d e n t dehydrogenases.
M a k e u p all s o l u t i o n s w i t h f r e s h l y p r e p a r e d , d o u b l y d i s t i l l e d w a t e r . S t e r i l i z e t h e v e s s e l s t o p r e
vent the growth o f micro-organisms.
I. H y d r o c h l o r i c a c i d ( I N ) :
D i l u t e 2 0 m l . c o n e , h y d r o c h l o r i c a c i d ( a p p r o x . 12 M ) w i t h 2 0 0 m l . d i s t i l l e d w a t e r .
II. Tris b u f f e r (0.1 N ; p H 8 . 5 ) :
D i s s o l v e 1.2 g. tris in a p p r o x . 8 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 8.5 w i t h 1 N H C 1 ( I ) , a n d
m a k e u p t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r . T h e n g a s w i t h o x y g e n f o r 10 m i n .
III. R e d u c e d nicotinamide-adenine dinucleotide (12 m M ) :
I V . L a c t a t e d e h y d r o g e n a s e , L D H (5 m g . p r o t e i n / m l . ) :
D i l u t e stock s u s p e n s i o n as necessary with 3.2 M a m m o n i u m sulphate s o l u t i o n .
V. Catalase (0.2 mg. protein/ml.):
D i l u t e stock suspension as necessary with distilled water.
VI. D - A m i n o a c i d o x i d a s e , D - A O D (5 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n as n e c e s s a r y w i t h 3 . 2 M a m m o n i u m s u l p h a t e s o l u t i o n .
K e e p all solutions a n d suspensions, stoppered in a refrigerator at 0° to 4 °C. F r e s h tris buffer should be

p r e p a r e d after 2 weeks, fresh N A D H solution after 1 week, a n d fresh catalase solution every day. L D H a n d
D - A O D suspensions can be kept a b o u t 6 t o 12 m o n t h s .
Procedure
W e h a v e n o t y e t c a r r i e d o u t a n y d e t e r m i n a t i o n s in b i o l o g i c a l m a t e r i a l .
Stability of sample: D - A l a n i n e k e e p s f o r s e v e r a l d a y s in a q u e o u s s o l u t i o n a s l o n g a s n o g r o w t h o f
micro-organisms occurs.
Assay System
ature. M e a s u r e a g a i n s t air.
Tris buffer (II) 2.80 ml. 9 4 m M tris

Sample 0.05 ml. 0.03-0.15 m M D-alanine
NADH solution (HI) 0.05 ml. 0.2 m M NADH
L D H suspension (IV) 0.01 m l . ca. 8 U / m l .
Catalase solution (V) 0.01 m l . ca.33 U/ml.
Mix, read extinction E j .
D - A O D suspension (VI) 0.10 ml. ca. 2.5 U / m l .
Mix. After the reaction (approx. 60 min.), read extinct

i o n E . E -E
2 2 t = A E is u s e d in t h e c a l c u l a t i o n s .
T h e i n c r e a s e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f D - A O D ( V I ) is d e t e r m i n e d at t h e e n d o f t h e
r e a c t i o n b y a further a d d i t i o n o f s u s p e n s i o n ( V I ) . T h e e x t i n c t i o n d i f f e r e n c e t h a t o c c u r s m u s t b e
used to correct E . 2
Calculations
T h e reaction proceeds stoichiometrically u n d e r the conditions indicated. T h e calculation formula (2) o n

p. 312 can therefore be used. T h e result is obtained in /rniole of D-alanine per ml. of sample. T h e following
relationships are t h u s valid.
c= Êx9.90 Êx9.71 JExl7.8 [/miole/ml.]

c= A E x 0.882 JEx0.865 A E x 1.58 [mg./ml.]
Interference in the assay: I n a d e q u a t e purity of the enzymes (see p . 1687). can lead to incorrect values.
Fatty acids, L-leucine, L-phenylalanine, a n d adenine nucleotides m a y inhibit the o x i d a s e . 4
D - A m i n o acid oxidase from pig kidneys is strictly stereospecific, a n d reacts only with D - a m i n o a c i d s . A s a 5
results of the coupling with the reaction catalysed by L D H (from muscle), the d e t e r m i n a t i o n is practically
specific for D-alanine.
D-Alanine can also be determined by m a n o m e t r i c m e a s u r e m e n t of the oxygen c o n s u m e d o n reaction with

D - a m i n o acid oxidase (in the presence of c a t a l a s e ) ' o r by determination of the resulting p y r u v a t e via the
6 7
D-Alanine 1689
2,4-dinitrophenylhydrazone . T h e p y r u v a t e formed can also be d e t e r m i n e d

8
fluorimetrically by reaction
with o-phe nyle ne diamine . F u r t h e r m o r e a m e t h o d with a specific electrode, c o a t e d with carrier-fixed
9
D - a m i n o acid oxidase, is d e s c r i b e d .
10
However, these m e t h o d s are inferior to t h a t described here in specificity a n d convenience. Only 2-oxo-
butyrate, which is formed u n d e r these reaction conditions from D - 2 - a m i n o b u t y r i c acid, reacts slowly
with L D H . If L D H from heart is used, this reaction proceeds so rapidly t h a t it is difficult to differentiate
between D - 2 - a m i n o b u t y r i c acid a n d D-alanine.
References
1 J. Baddiley, J. R o y a l Inst. C h e m . 86, 366 [1962].

2 T. J. Steenson, M. E. Jennings, Biochem. J. 107, 18 [1968].
3 J.Haupt & M. Bocker, Z. Physiol. C h e m . 342, 132 [1965].
4 J.-M. Ghysen, D. J. Tipper, J. L. Strominger in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology,
A c a d e m i c Press, N e w Y o r k , 1966, Vol. V I I I , p . 685.
5 K. Burton in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c Press, N e w Y o r k 1955,
vol. II, p . 199.
6 A. E. Bender &H. A. Krebs, Biochem. J. 46, 210 [1950].
7 P. Boulanger & R. Osteux inH. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag C h e m i e ,
Weinheim 1962, 1st. edn., p . 367.
8 K Hayashi, M. Ishida, T. Hino & M. Ohara, N i p p o n N o g e i k a g a k u Kaishi 36, 269 [1962].
9 / . P . Greenstein, S. M. Birnbaum & L. Levinkow in E. E. Snell: Biochemical P r e p a r a t i o n s , J o h n Wiley
a n d Sons, N e w York 1953, vol. 3, p . 92.
10 G. G. Guilbault & E. Hrabankowa, A n a l . C h i m . A c t a 56, 285 [1971].
y-Aminobutyric Acid
L. T . G r a h a m jr. a n d M . H . A p r i s o n
Endogenously formed y-aminobutyric acid, y-ABA, in animals, is virtually limited to nervous tissue.
Significant a m o u n t s a r e found in certain bacteria a n d in plant material. A specific enzymatic m e t h o d
for the d e t e r m i n a t i o n of y-aminobutyric acid h a s been k n o w n for several y e a r s . F o r the analysis of smaller
1
tissue samples (a few mg. or ^g.) however, t h e high sensitivity of fluorimetric m e a s u r e m e n t s is r e q u i r e d . 2
Jakoby a n d Scott 3
have described a system with t w o enzymes from Pseudomonas fluoresceins: the t r a n s
a m i n a s e reaction, y-ABA-T ( 4 - A m i n o b u t y r a t e : 2-oxoglutarate aminotransferase, E C 2.6.1.19) is coupled
with the N A D P - l i n k e d d e h y d r o g e n a s e reaction, S S A - D H (Succinate semialdehyde: N A D ( P ) oxido-
reductase, E C 1.2.1.16).
Application of Method: In quantitative histochemical analysis and with small identifiable tissue samples
e.g. parts of the spinal cord of m a m m a l s , layers of the retina of a m p h i b i a n s , layers of the cerebellum
4 5
of m a m m a l s 6 , 7
, isolated nerve fibres a n d individual cells of crustaceans.
8 9
Principle
(1) y-Aminobutyrate + 2-Oxoglutarate y ~ A B A

- » G l u t a m a t e + Succinate semialdehyde
T
i 1
(2) Succinate semialdehyde + N A D P + S S A D H

> Succinate + N A D P H + H +
On completion of the reaction, the excess N A D P is destroyed with weak alkali; N A D P H is stable in weak
alkali a n d is converted to a highly fluorescent N A D P p r o d u c t with s t r o n g alkali a n d H 0 2 2
1 0
. T h e fluor
escence is a m e a s u r e of the y - a m i n o b u t y r a t e content.
T h e p H o p t i m u m for the coupled reaction is n a r r o w ; it is between p H 8 . 3 - 8 . 6 . T h e 2-oxoglutarate

c o n c e n t r a t i o n should n o t exceed 6.7 m M because excess inhibits the t r a n s a m i n a s e a n d also can interfere
with the formation of the fluorescent p r o d u c t in alkaline peroxide s o l u t i o n . 10
Equipment
Cahn e l e c t r o - b a l a n c e for w e i g h i n g s in a f r e e z e r 1 0 a
at — 2 0 ° C a n d a l s o for t h e l a b o r a t o r y b e n c h ;
refrigerated c e n t r i f u g e ; u l t r a c e n t r i f u g e , v a c u u m c e n t r i f u g e e v a p o r a t o r ; b u z z e r ; glass m i c r o
1 1 1 2
test t u b e s , 4 m m . e x t e r n a l d i a m e t e r ; s t o p p e r s for t h e s e t u b e s c o n s i s t i n g o f a s h o r t p i e c e o f r u b b e r
1 2
t u b i n g w i t h o n e e n d s e a l e d w i t h a g l a s s r o d ; Lang-Levy constriction micro-pipettes, prepared

in t h e l a b o r a t o r y o r o b t a i n a b l e f r o m H. E. Pedersen, D e n m a r k ; 3 m l . p y r e x test t u b e s ( 1 0 m m . x
x 75 m m . , C o r n i n g N o . 9 8 2 0 ) a s f l u o r i m e t e r t u b e s ; s p e c t r o p h o t o f l u o r i m e t e r o r filter fluori-
m e t e r w i t h e x c i t a t i o n w a v e l e n g t h 3 6 5 n m ( C o r n i n g filter N o . 7 - 3 7 ) a n d e m i s s i o n w a v e l e n g t h
at 4 7 0 n m ( C o r n i n g filter N o . 4 - 7 0 , 5 - 6 1 , 3 - 7 2 ) .
y-Aminobutyric Acid 1691
Reagents
1. S o d i u m p y r o p h o s p h a t e , 6. S o d i u m d i h y d r o g e n p h o s p h a t e ,
Na P O 10H O
4 2 7 2 Na HP0 -7H 0
2 4 2
2. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e 7. H y d r o g e n p e r o x i d e , H 0 , 3 0 % ( w / w )
2 2
phosphate, N A D P 8. S o d i u m h y d r o x i d e , 2 N a n d 10 N
sodium salt, N A D P - N a H , commercial p r e p
2 9. H y d r o c h l o r i c a c i d , 0.1 N
aration, see p . 547. 10. Ethanol
3. 2-Mercaptoethanol 11. E n z y m e preparation,
4. 2 - O x o g l u t a r i c a c i d see Appendix, p. 1694.
5. T r i s o d i u m p h o s p h a t e , Na P0 12H 0
3 4 2 12. y - A m i n o b u t y r i c a c i d
Purity of Reagents
All reagents should be A. R. quality, to exclude enzyme inhibitors and to keep non-specific fluorescence
to a m i n i m u m . The enzyme p r e p a r a t i o n should have little b a c k g r o u n d fluorescence a n d should be free
from N A D P H oxidases.
U s e d o u b l y distilled water.
D i s s o l v e 4 . 4 6 g. N a P O 1 0 H O i n 7 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 8 . 4 w i t h 0.1
4 2 7 2 N
H C 1 a n d d i l u t e t o 100 m l . w i t h d i s t i l l e d w a t e r .
II. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e (1.1 m M N A D P ) :
D i s s o l v e 4.7 m g . N A D P - N a H in 5 m l . d i s t i l l e d w a t e r .
2
III. 2 - M e r c a p t o e t h a n o l ( 6 0 m M ) :
D i l u t e 2 0 fil. 2 - m e r c a p t o e t h a n o l t o 5 m l . w i t h s o l u t i o n I.
IV. 2-Oxoglutaric acid (60 m M ) :
D i s s o l v e 4 4 m g . 2 - o x o g l u t a r i c a c i d i n 4 m l . s o l u t i o n I, n e u t r a l i z e w i t h 2 N N a O H a n d
d i l u t e w i t h distilled w a t e r t o 5 m l .
V. P h o s p h a t e solution (0.4 M P O j " ; 0.2 M H P O ^ " ) :
D i s s o l v e 1 5 . 2 g. N a P 0 1 2 H 0 a n d 5.3 g. N a H P 0 - 7 H 0 in d i s t i l l e d w a t e r a n d m a k e
3 4 2 2 4 2
u p t o 100 m l .
VI. Hydrogen peroxide (3%):
Dilute 1 ml. 30% H 0 2 2 t o 10 m l . w i t h d i s t i l l e d w a t e r .
VII. Alkaline peroxide solution:
D i l u t e 0.01 m l . 3 % H 0 2 2 s o l u t i o n ( V I ) t o 10 m l . w i t h 10 N N a O H .
VIII. Reagent mixture:
M i x 1.0 ml. s o l u t i o n I a n d 0.2 m l . o f s o l u t i o n s II, III a n d I V .
IX. Enzyme solution:
U s e the solution o b t a i n e d according to p. 1694.
X. Reaction mixture:
A d d 0 . 2 m l . e n z y m e s o l u t i o n I X t o 1.6 m l . s o l u t i o n V I I I .
X I . y - A m i n o b u t y r i c acid standard solution (4 yM):
D i s s o l v e 10.3 m g . y - a m i n o b u t y r i c a c i d in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l . ; d i l u t e
4 ml. o f this s o l u t i o n t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r .
Solutions I and V are stable at r o o m t e m p e r a t u r e , solutions II, III a n d VI are stable at 0 - 4 °C for a b o u t
two weeks, solution VIII for one week at — 20 °C a n d I X for several m o n t h s at - 70 °C. P r e p a r e solutions
IV, VII, X and X I just before use.
Procedure
Collection of sample:
F r e e z e t i s s u e s a m p l e s a s r a p i d l y a s p o s s i b l e ; w i t h s m a l l l a b o r a t o r y a n i m a l s if p o s s i b l e freeze
the w h o l e animal in dry i c e / p e t r o l e u m ether.
Treatment of sample:
W e i g h f r o z e n t i s s u e , a d d 1 0 - 3 0 m g . t o 2 m l . 7 5 % c o l d e t h a n o l in a p r e - c o o l e d g l a s s h o m o -
g e n i z e r (less t h a n 10 m g . t i s s u e in 1 m l . e t h a n o l ) . H o m o g e n i z e ; t r a n s f e r t h e h o m o g e n a t e
q u a n t i t a t i v e l y t o a s m a l l c o n i c a l g l a s s c e n t r i f u g e t u b e w i t h a s t o p p e r a n d c e n t r i f u g e for 10 m i n .
in a refrigerated c e n t r i f u g e at 2 5 0 0 0 r p m . D e c a n t t h e s u p e r n a t a n t fluid i n t o a s t a i n l e s s steel
c e n t r i f u g e t u b e ; r e s u s p e n d t h e p r e c i p i t a t e in 0.5 m l . c o l d 7 5 % e t h a n o l , c e n t r i f u g e a n d c o m b i n e
w i t h t h e first s u p e r n a t a n t fluid. T a k e t o d r y n e s s in a v a c u u m c e n t r i f u g e a n d r e s u s p e n d t h e
r e s i d u e in w a t e r (1 m l . p e r 4 m g . fresh w t . ) . T h e s u s p e n s i o n is u s u a l l y s l i g h t l y t u r b i d ; it is
c l e a r e d b y c e n t r i f u g a t i o n f o r 3 0 m i n . at 3 0 0 0 0 r p m in a n u l t r a c e n t r i f u g e .
D e c a n t t h e c l e a r s u p e r n a t a n t fluid i n t o a g l a s s t u b e ; it c a n b e u s e d f o r t h e a n a l y s i s o f v a r i o u s
a m i n o a c i d s ' . P i p e t t e a m e a s u r e d v o l u m e ( c o r r e s p o n d i n g t o 2 0 - 4 0 0 pg. fresh w t . o f t i s s u e )
2 4
i n t o r e a c t i o n t u b e s : ( 4 m m . x 5 0 m m . ) a n d t a k e t o d r y n e s s in t h e v a c u u m c e n t r i f u g e . T h e s e
t u b e s c o n t a i n i n g t h e d r i e d e x t r a c t are u s e d for t h e a s s a y .
Extraction with trichloroacetic acid:
Trichloroacetic acid c a n be used for the deproteinization and extraction o f frozen samples or
lyophilized thin tissue slices. Trichloroacetic acid inhibits the e n z y m e reaction, but small
s a m p l e s are sufficiently v o l a t i l e t o e v a p o r a t e in t h e v a c u u m c e n t r i f u g e d u r i n g t h e d r y i n g
p r o c e s s * . T r i c h l o r o a c e t i c a c i d h a s b e e n u s e d for t h e e x t r a c t i o n o f l y o p h i l i z e d layers o f r e t i n a ;
5
the m e a s u r e m e n t s w e r e c a r r i e d o u t in a final v o l u m e o f 120 pi. ( r e d u c e all v o l u m e s a c c o r d i n g l y ) .
It is p o s s i b l e t o s t o r e t h e a q u e o u s e x t r a c t f r o z e n for 2 4 hr., b u t t h e s t a b i l i t y is greatest in t h e

d r y s t a t e in a v a c u u m d e s i c c a t o r ( e i t h e r at t h e s t a g e o f t h e steel c e n t r i f u g e t u b e s o r t h e m i c r o
t u b e s ) . It is c o n v e n i e n t t o dry d o w n s e v e r a l y - a m i n o b u t y r i c a c i d s t a n d a r d s o l u t i o n s a n d t o
s t o r e in a d e s i c c a t o r .
* We t h a n k R. P. Shank for this information.

Assay System
I n c u b a t i o n t e m p e r a t u r e : 3 8 ° C ; i n c u b a t i o n v o l u m e : 6 5 |il. - fluorescence m e a s u r e m e n t s at
r o o m t e m p e r a t u r e ; p r i m a r y w a v e l e n g t h : 3 6 5 n m , s e c o n d a r y w a v e l e n g t h : 4 7 0 n m ; final v o l u m e
1.15 m l . P r e p a r e t h e f o l l o w i n g s t a n d a r d a n d b l a n k t u b e s f o r e a c h s e r i e s o f m e a s u r e m e n t s :
Blank containing sample ( B l k + S a m p ) : A s f o r s a m p l e b u t p i p e t t e p h o s p h a t e s o l u t i o n ( V ) b e f o r e

the reaction mixture ( X ) . (Determines the fluorescence of the sample).
Reagent blank ( w i t h o u t s a m p l e ) :
a) 15 |il. r e a c t i o n m i x t u r e ( X ) + 5 0 p\. p h o s p h a t e s o l u t i o n ( V ) , t r e a t a s f o r s a m p l e . T h i s b l a n k
( B l k ) is f o r t h e s t a n d a r d s .
t
b ) A s t h e fluorescence i n c r e a s e s s l i g h t l y at 3 8 ° C d u r i n g t h e i n c u b a t i o n i n t h e a b s e n c e o f y - a m i n o
b u t y r i c a c i d , a s e c o n d b l a n k ( B l k ) is r e q u i r e d w h i c h c o r r e c t s f o r t h i s d i f f e r e n c e b e t w e e n t e s t
2
a n d b l a n k c o n t a i n i n g s a m p l e : p i p e t t e 5 0 p\. p h o s p h a t e s o l u t i o n ( V ) b e f o r e 15 p\. r e a c t i o n
mixture ( X ) .
Standards: D r y d o w n 5 t o 5 0 p\. s t a n d a r d s o l u t i o n ( X L ) i n m i c r o t u b e s a s f o r t h e s a m p l e s
(2 x 1 0 " 1 1
to 20 x 1 0 " 1 1
m o l e y - a m i n o b u t y r i c a c i d ) a n d treat a s f o r s a m p l e s .
Pipette into tubes* c o n t a i n i n g the dried s a m p l e s : Concentration in assay mixture
Reaction mixture (X) 15 | d . 55 m M p y r o p h o s p h a t e

6.7 m M 2 - o x o g l u t a r a t e
6.7 m M 2 - m e r c a p t o e t h a n o l
0.12 m M N A D P
1 0 0 - 2 0 0 |ig. protein/ml.
M i x gently, stopper tubes a n d incubate for 30 m i n .

at 3 8 ° C . R e t u r n t o i c e w a t e r .
Phosphate solution (V) 50 pi 0.46 M phosphate
M i x v i g o r o u s l y , r e c a p t u b e s a n d p l a c e f o r 15 m i n . i n
a water bath at 6 0 ° C .
Pipette into 3 ml. fluorimeter tubes:
Alkaline H 0 2 2 solution (VII) 100 pi 2% H 0 2 2
Incubation mixture 50 pi 6.7 N N a O H
M i x a n d h e a t f o r 10 m i n . i n a w a t e r b a t h a t 6 0 ° C .
Distilled water 1000 |il.
Mix and measure fluorescence F.
* In ice water
Calculations
T h e tissue fluorescence F B I k + S a m p - F B l k 2 must be subtracted from the values for F S a m p ; AF S a m p = F S a m p -

- (F ik+samp +
B (pRiki —F B I k 2 ) ) . C o n v e r t t h e difference in fluorescence / 4 F S a m p to 1 0 " 1 1
mole y-amino
butyric acid by m e a n s of the s t a n d a r d curve. T h e s t a n d a r d curve is l i n e a r ( o r d i n a t e : A F
2
S t d = F S t d — F B l k i ;
abscissa: 2 x 1 0 " 1 1
t o 20 x 1 0 ~ m o l e y-aminobutyric acid).
u
T h e coefficient of variation for the m e t h o d is smaller t h a n 4 % .
N o r m a l Values
y-Aminobutyric acid is unevenly distributed in various areas of the nervous system. T h e concentration
extends from 60 nmole/g. fresh wt. in the spinal r o o t s of cats u p to 49 /miole/g. fresh wt. in the isolated
inhibitor nerves of lobster. M a m m a l i a n brain contains regions with 1 to 6 ^ m o l e y-aminobutyric acid/g.
fresh wt.
Interference in the assay technique: Care should be taken in the collection of the samples that the tissue is
rapidly frozen; it is possible that y - a m i n o b u t y r a t e concentration can increase post mortem and so give
falsely raised values.
All precautions should be taken to ensure t h a t the glassware is absolutely clean and the reagents are of the
highest quality.
T h e only other c o m p o u n d k n o w n to give an 2-oxoglutarate-dependent p r o d u c t i o n of N A D P H in the

enzyme system used here is /Miydroxy-y-aminobutyric acid. /?-Hydroxy-y-aminobutyric acid reacts con
siderably m o r e slowly t h a n y-aminobutyric acid. In addition, ^-hydroxy-y-aminobutyric acid does n o t
normally occur in nervous t i s s u e . 13
Appendix
Isolation of Enzyme System
G r o w Pseudomonasfluorescens ( A T C C 13430) with pyrrolidine or y-aminobutyric acid as s u b s t r a t e ; har

vest during the log-phase a n d extract a n d purify the enzyme system from the bacteria according to the
m e t h o d of Scott a n d Jakoby *' . 1 15
Modify this m e t h o d according to Baxter (unpublished) as follows:
after centrifugation of the thawed, suspended bacteria between 50 and 70% of the enzyme system is found
in the s u p e r n a t a n t fluid. D o not discard this s u p e r n a t a n t fluid, but treat with p r o t a m i n e sulphate, a m m o n i u m
sulphate and acetone as for the s u p e r n a t a n t obtained after ultrasonic t r e a t m e n t of the resuspended cell
f r a c t i o n ' . In this way two parallel fractions are obtained. In some cases the fraction from the first
14 15
s u p e r n a t a n t fluid is better. At this stage (first dialysed acetone fraction) dilute 5-10-fold, according to the
activity, with 1 m M 2-aminoethylisothiouronium b r o m i d e ( A E T ) ; sufficient activity is obtained to complete
the assay according to the above m e t h o d in 30 min. A E T increases the stability of the enzyme solution;
freeze the p r e p a r a t i o n in small p o r t i o n s at — 70 °C.
T h e commercially available p r o d u c t (lyophilized p o w d e r from PL Biochemicals Inc., Milwaukee) is n o t
as good as that described above. T h e fluorescence b l a n k is higher and the enzyme activity is lower. Suspend
5 mg. of this p r o d u c t in 1 ml. 1 m M A E T a n d freeze in small p o r t i o n s at — 70 °C. T h e conditions for the
measurements are the same as those described above except that the time required for the reaction is
longer.
References
1 C. F. Baxter in / . H. Quastel: M e t h o d s in Medical Research. Year B o o k Medical Publishers, C h i c a g o

1961, Vol. 9, p . 192.
2 L. T. Graham jr. & M. H. Aprison, Analytic. Biochem. 15, 487 [1966].
3 W. B. Jakoby & E. M. Scott, J. biol. C h e m . 234, 937 [1959].
4 L. T. Graham jr., R. P. Shank, R. Werman & M. H. Aprison, J. N e u r o c h e m . 14, 465 [1967].
5 L. T. Graham jr., R. N. Lolley & C. F. Baxter, F e d e r . P r o c . 27, 463 [1968].
6 H. E. Hirsch & E. Robins, J. N e u r o c h e m . 9, 63 [1962].
7 K. Kuriyama, B. Haber, B. Sisken & E. Roberts, P r o c . N a t l . A c a d . Sci. (Wash.) 55, 846 [1966].
8 E. A. Kravitz, S. W. Kuffler & D. D. Potter, J. N e u r o p h y s i o l . 26, 739 [1963].
9 M. Otsuka, E. A. Kravitz & D. D. Potter, J. N e u r o p h y s i o l . 30, 725 [1967].
10 O.H. Lowry, N. R. Roberts & /. /. Kapphahn, J. biol. C h e m . 224, 1047 [1957].
lOaM. H. Aprison & C. J. Hancock in G. A. Kerkut: Experiments in Physiology a n d Biochemistry. A c a d e m i c
Press, L o n d o n 1970, Vol. 3, p . 39.
11 R.W. Albers & O. H. Lowry, Analytic. C h e m . 27, 1829 [1955].
12 O.H. Lowry, N. R. Roberts, K. Y. Leiner, M. L. Wu & A. L. Farr, J. biol. C h e m . 207, 1 [1954]. .
13 K. A. C. Elliot, Brit. M e d . Bull. 21, 70 [1965].
14 E. M. Scott & W. B. Jakoby, J. biol. C h e m . 234, 932 [1959].
15 W. B. Jakoby in S. P. Colowick & N. O. Kaplan: M e t h o d s in E n z y m o l o g y . A c a d e m i c Press, N e w
Y o r k 1962, Vol. V, p . 765.
L-Aspartate and L-Asparagine
H . U . B e r g m e y e r , E . B e r n t , H . M o l l e r i n g a n d G . Pfleiderer
T h e successive e n z y m a t i c d e t e r m i n a t i o n of L - a s p a r t a t e a n d L-asparagine in o n e cuvette, p r o p o s e d by

Williamson 1
in 1962, b e c a m e possible with the availability of crystalline asparaginase (L-Asparagine
a m i n o h y d r o l a s e , E C 3.5.1.1). Previously the d e t e r m i n a t i o n of the a s p a r a g i n e c o n t e n t of proteins using
ion exchange resins h a d been difficult because asparagine moves with serine a n d glutamine on the ion
exchange c o l u m n .
T h e d e t e r m i n a t i o n m e t h o d e m p l o y i n g the enzymes G O T a n d M D H w a s described in 1969 by Kojima
and Wacker . 2
A m e t h o d of m e a s u r i n g a s p a r a g i n e via N H 3 formation with asparaginase a n d g l u t a m a t e dehydrogenase*

was p u b l i s h e d 3
in 1972. W h e n the sample c o n t a i n s relatively large quantities of glutamine additional
a m m o n i a m a y be formed d u e t o the side activity of the asparaginase of L-glutaminase ( a b o u t 2 % ) . We
therefore prefer o u r o w n m e t h o d .
Application of Method: In biochemistry, microbiology, in clinical chemistry a n d in foodstuff chemistry.
Principle
(1) L-Asparagine + H 0 2
A S P A R A G I N A S E
> L-Aspartate + NH 3
(2) L-Aspartate + 2-Oxoglutarate — • Oxaloacetate + L - G l u t a m a t e
(3) Oxaloacetate + N A D H + H + M P H
* * * > L-Malate + N A D +
A s p a r t a t e is determined first according t o e q u a t i o n s (2) a n d (3); the decrease of the extinction due t o
N A D H at 340, 334 or 365 n m is p r o p o r t i o n a l t o the a s p a r t a t e c o n c e n t r a t i o n . A d d i t i o n of asparaginase
4
to the assay system leads t o a further decrease of the N A D H c o n c e n t r a t i o n according to equation (1)
and this gives the asparagine content.
The equilibrium of reaction (3) K = 4.3 x 1 0 l./mole at p H 7.2 a n d 22 °C is strongly in favour of m a l a t e

4
f o r m a t i o n . T h e Michaelis c o n s t a n t of the enzyme for L-aspartate is very small, L - A s p a r t a t e is quantitative

5
ly converted t o L - ( + ) - m a l a t e . M e a s u r e m e n t s are m a d e at 25 °C. T h e c o n c e n t r a t i o n s of reactants stated

below give o p t i m u m c o n d i t i o n s .
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r f o r m e a s u r e m e n t s at 3 4 0 ( 3 3 4 o r 3 6 5 ) n m ,
bench centrifuge.
* L - G l u t a m a t e : N A D ( P ) o x i d o r e d u c t a s e (deaminating), E C 1.4.1.3.
'* G l u t a m a t e - o x a l o a c e t a t e t r a n s a m i n a s e ( L - A s p a r t a t e : 2-oxoglutarate aminotransferase, E C 2.6.1.1).
* M a l a t e d e h y d r o g e n a s e ( L - M a l a t e : N A D oxidoreductase, E C 1.1.1.37).
L - A s p a r t a t e a n d L-Asparagine 1697
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 7. G l u t a m a t e - o x a l o a c e t a t e t r a n s a m i n a s e ,
KH P0 2 4 GOT
2. D i s o d i u m h y d r o g e n p h o s p h a t e , from pig heart, suspension in 3.2 M a m m o n i u m
Na HP0 -2H 0
2 4 2 sulphate s o l u t i o n ; ^ 2 0 0 U / m g . (25 °C); c o m
3. R e d u c e d n i c o t i n a m i d e - a d e n i n e - mercial p r e p a r a t i o n , see p . 462.
dinucleotide, NADH 8. Asparaginase
d i s o d i u m salt, N A D H - N a ; c o m m e r c i a l p r e p
2 from E. coli, crystallized, solution in 5 0 % (v/v)
a r a t i o n , see p . 545. glycerol; ^ 80 U / m g . (25 °C); commercial p r e p
4. 2-Oxoglutarate a r a t i o n , see p . 435.
commercial p r e p a r a t i o n , see p . 548. 9. P e r c h l o r i c a c i d , A . R. 7 0 % ( w / w ) , s p . gr.
5. S o d i u m h y d r o x i d e , A . R., 2 N 1.67
6. M a l a t e d e h y d r o g e n a s e , MDH 10. P o t a s s i u m p h o s p h a t e , K P0 -3H 0
3 4 2
from pig heart, suspension in 3.2 M a m m o n i u m

sulphate s o l u t i o n ; ^ 1 1 0 0 U / m g . (25 °C); c o m
mercial p r e p a r a t i o n , see p . 485.
Purity of Reagents
M D H , G O T a n d asparaginase m u s t be as free as possible from g l u t a m a t e d e h y d r o g e n a s e activity. As

the enzymes are usually stored in a m m o n i u m sulphate-containing solutions, presence of g l u t a m a t e
dehydrogenase with excess 2-oxoglutarate a n d N A D H would result in slow reductive a m i n a t i o n of
oxoglutarate to glutamic acid with the oxidation of N A D H . However, G O T a n d M D H are easy to p r e
pare in a virtually p u r e s t a t e 6 - 8
, a n d are, together with asparaginase, available commercially in sufficient
purity; they can be stored at 0 - 4 °C for weeks without noticeable loss of activity.
U s e o n l y fresh d o u b l y d i s t i l l e d w a t e r .
I. P h o s p h a t e buffer ( 6 7 m M ; p H 7 . 2 ) :
a ) D i s s o l v e 1 1 . 8 7 6 g. N a H P 0 - 2 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2 4 2
b ) D i s s o l v e 9 . 0 7 8 g. K H P 0 2 4 in distilled water a n d m a k e u p t o 1000 ml. M i x a) a n d b) in

the ratio 72 : 28 v o l u m e s . Check the p H .
II. R e d u c e d n i c o t i n a m i d e - a d e n i n e - d i n u c l e o t i d e (ca. 12 m M j f t - N A D H ) :
Dissolve 50 mg. N A D H - N a 2 in 5 m l . d i s t i l l e d w a t e r .
III. 2 - O x o g l u t a r a t e (0.1 M ) :
D i s s o l v e 1.46 g. 2 - o x o g l u t a r i c a c i d in c a . 5 0 m l . d i s t i l l e d w a t e r w i t h g e n t l e w a r m i n g ,
neutralize with 2 N N a O H and dilute to 100 ml. with distilled water
I V . M a l a t e d e h y d r o g e n a s e , M D H (5 m g . p r o t e i n / m l . ) :
D i l u t e t h e s t o c k s u s p e n s i o n a c c o r d i n g l y w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V. Glutamate-oxaloacetate transaminase, G O T (2.0 mg. protein/ml.):
V I . A s p a r a g i n a s e (5 m g . p r o t e i n / m l . ) :
Dilute the stock suspension accordingly with 5 0 % glycerol.
V I I . P e r c h l o r i c a c i d (1 M ) :
D i l u t e 8.6 m l . 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h d i s t i l l e d w a t e r .
VIII. Potassium phosphate (1.75 M ) :
D i s s o l v e 4 6 . 5 g. K P 0 - 3 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
3 4 2
Procedure
Collection and treatment of sample:
F r e e p r o t e i n h y d r o l y s a t e s ( o b t a i n e d b y h e a t i n g 15 t o 7 2 hr. w i t h 5 0 % c o n e . H C 1 at 110 ° C ) f r o m
e x c e s s HC1 in vacuo o n a w a t e r b a t h o r in a v a c u u m d e s i c c a t o r o v e r c o n e . H S 0 2 4 and caustic
s o d a . T a k e u p t h e r e s i d u e in a little w a t e r , n e u t r a l i z e t h e s o l u t i o n w i t h 2 N N a O H a n d d i l u t e t o a
k n o w n v o l u m e . T h i s p r o c e d u r e , h o w e v e r , results in t h e c o n v e r s i o n o f a s p a r a g i n e t o a s p a r t a t e .
T o p r e v e n t a c i d h y d r o l y s i s h y d r o l y s e t h e p r o t e i n s e n z y m a t i c a l l y at n e u t r a l p H , e . g . with
a m i n o p e p t i d a s e . P r e c i s e i n c u b a t i o n t i m e s c a n n o t b e g i v e n b e c a u s e t h e rate o f h y d r o l y s i s
depends o n the a m i n o acid c o m p o s i t i o n .
F o r the d e t e r m i n a t i o n in b l o o d m i x 5 m l . w h o l e b l o o d w i t h 5 m l . i c e - c o l d p e r c h l o r i c a c i d
( s o l u t i o n V I I ) , a l l o w t o s t a n d for 10 m i n . in a n ice b a t h a n d t h e n c e n t r i f u g e for 10 m i n . at c a .
6 0 0 g. A d d 0.9 ml. p o t a s s i u m p h o s p h a t e s o l u t i o n ( V I I I ) t o 5 m l . s u p e r n a t a n t fluid, a l l o w t o
s t a n d for 15 m i n . in a n ice b a t h a n d t h e n filter. A d d 3 . 0 0 m l . o f this s o l u t i o n buffered at c a .
p H 7.0 ( c h e c k w i t h p H p a p e r ) t o the a s s a y s y s t e m . Treat s e r u m as for w h o l e b l o o d .
D e p r o t e i n i z e b l o o d o r s e r u m i m m e d i a t e l y after c o l l e c t i o n . A s p a r a g i n e is s t a b l e for at least 3 hr.

at 4 ° C in t h e a c i d p e r c h l o r i c a c i d e x t r a c t , b u t b e c a u s e o f t h e d a n g e r o f h y d r o l y s i s the e x t r a c t
should be kept ice-cold and s h o u l d be neutralized rapidly.
L - A s p a r t a t e a n d L-Asparagine 1699
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 o r H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 3 . 2 m l . ; r o o m
t e m p e r a t u r e . R e a d a g a i n s t air.
Pipette into cuvettes: C o n c e n t r a t i o n in assay mixture
Sample* (deproteinized,
neutralized) 3.00 ml. u p t o c a . 1 5 0 fiM asparagine +
aspartate
NADH solution (II) 0.05 ml. 0.19 m M NADH
2-Oxoglutarate (HI) 0.10 ml. 3.1 m M
MDH suspension (IV) 0.01 m l . c a . 17 U / m l .
M i x , follow the extinction c h a n g e until c o n s t a n t (ca.

2 min.). Read extinction E ^
G O T suspension (V) 0.02 ml. c a . 2.5 U / m l .
M i x , w a i t for t h e e n d o f t h e r e a c t i o n (ca. 1 5 - 2 0 m i n . )
a n d read E . If t h e e x t i n c t i o n c o n t i n u e s t o d e c r e a s e ,
2
extrapolate E to the time o f G O T addition according

2
to p. 308. E 1 - E 2 = Ê a s p a r t a t e .
Asparaginase (VI) 0.02 ml. c a . 2.5 U / m l .
M i x a n d after c o m p l e t i o n ( c a . 1 5 - 2 0 m i n . ) o f t h e r e a c t
i o n r e a d E . If a c o n s t a n t e n d - p o i n t is n o t a t t a i n e d
3
extrapolate to the point o f asparaginase addition.

^2 —
E 3 = A E a s p a r a g i n e .
* A d d only 0.10 ml. of c o n c e n t r a t e d protein hydrolysates (neutralized) a n d m a k e u p to 3.0 ml. with

buffer solution I.
Calculations
U n d e r the a b o v e conditions the reactions proceed stoichiometrically a n d therefore the calculation formula
(2) o n p . 312 applies. T h e results are o b t a i n e d in jumole a s p a r t a t e o r /imole asparagine per ml. sample.
The results m u s t be multiplied by a factor if the sample has been deproteinized, neutralized or diluted
in any way. In the assay of whole b l o o d the specific gravity (ca. 1.06) a n d the water c o n t e n t (ca. 8 0 % )
must be taken into account.
F o r this p r o c e d u r e with whole b l o o d a factor of 2.18 is obtained from these d a t a a n d the dilution of
1 + 1 on deproteinization a n d 2 + 0.32 on precipitation of perchlorate. T h e following relationships
therefore a p p l y :
c = AE x 0.381 AE x 0.372 AE x 0.648 [/imole/ml.]

c = JEx 50.73 AE x 49.76 AE x 91.02 [/ig./ml] (aspartate)
T h e same factors apply for the calculation of jimole or /zg. asparagine (the higher molecular weight is
compensated for by the larger assay volume).
With a mean value of 20.4 ug. a s p a r t a t e a n d 12 ug. asparagine per ml. blood, s t a n d a r d deviations of
0.85 pg. aspartate a n d 0.19 fig. asparagine/ml. b l o o d were found. T h e coefficients of variation are 4 . 2 %
for a s p a r t a t e a n d 1.6% for asparagine.
N o r m a l Values
We found in n o r m a l b l o o d ca. 20 ug. a s p a r t a t e a n d ca. 12 ug. a s p a r a g i n e / m l . T h e values for serum are

considerably lower; virtually n o a s p a r t a t e was found, whereas asparagine was ca. 5 ug./ml.
F o r the content of asparagine a n d glutamine in p l a s m a , brain a n d liver s e e 9 1 0
.
High concentrations of oxaloacetate ( > 0 . 1 m M ) interfere due to the a d d i t i o n a l oxidation of N A D H .

This does not normally occur in tissues or b l o o d because the oxaloacetate c o n c e n t r a t i o n is usually very
low. Addition of m o r e N A D H to the assay system will c o m p e n s a t e , but the change in volume must then
be taken into account in the calculations. In studies o n protein hydrolysates the analytical values o b t a i n e d
by this m e t h o d are in good agreement with those found with the chemical m e t h o d by ion exchange
chromatography . 4
S p ec ific ity o f M e t h o d
T h e t r a n s a m i n a t i o n reaction is specific. Only L-aspartate a n d the s u l p h o a n a l o g u e , cysteic acid, react;

but the latter is m u c h slower. T h e cysteic acid reaction is linear for a long time a n d therefore can be
eliminated by extrapolation (p. 308). Cysteic acid can be formed from cysteine- a n d cystine-containing
proteins a n d peptides in the presence of oxygen.
D - A s p a r t a t e can be determined if the enzymatically determined value for L-aspartate is subtracted from
the total concentration of a s p a r t a t e obtained with ninhydrin (after c h r o m a t o g r a p h i c or electrophoretic
separation).
References
1 D. H. Williamson, personal c o m m u n i c a t i o n , see G. Pfleiderer in H. U. Bergmeyer: M e t h o d e n der

enzymatischen Analyse, 1. E d n . , Verlag C h e m i e , Weinheim 1962, p . 382, footnote.
2 Y. Kojima & W. E. C. Wacker, J. L a b . Clin. M e d . 74, 521 [1969].
3 P. J. Buttery, Analyt. Biochem. 47, 294 [1972].
4 G. Pfleiderer, W. Gruber & Th. Wieland, Biochem. Z. 326, 446 [1955].
5 / . R. Stern, S. Ochoa & F. Lynen, J. biol. C h e m . 198, 313 [1952].
6 R. G. Wolfe & J. B. Neilands, J. biol. C h e m . 221, 61 [1956].
7 G. Pfleiderer & E. Hohnholz, Biochem. Z. 331, 245 [1959].
8 W. T. Jenkins, D. A. Yphantis & J. W. Sizer, J. biol. C h e m . 234, 51 [1951].
9 D. Cooney, R. Davis & G. van Atta, Analyt. Biochem. 40, 312 [1971].
10 S. R. Nahorski, Analyt. Biochem. 42, 136 [1971].
L-Lysine
Determination with Automated Analysers
G e o r g e E. Schaiberger
T h e p r o d u c t i o n of a m i n o acids in large a m o u n t s requires strict c o n t r o l of the process at all stages. This

results in a large n u m b e r of samples for analysis a n d consequently an a u t o m a t e d m e t h o d is of value.
T h e p h o t o m e t r i c m e t h o d described here is r a p i d , very a c c u r a t e , reliable a n d r e p r o d u c i b l e . W i t h slight
1,3
modifications ten o t h e r a m i n o acids can be analysed if the c o r r e s p o n d i n g decarboxylases are used (see
also p . 1663).
Principle
(1) H N(CH ) CHNH -COOH

2 2 4 2 dJ™^ H N(CH ) NH
2 2 5 2 + C0 2
The C 0 2 liberated is m e a s u r e d with p h e n o l p h t h a l e i n as indicator.
Equipment
Technicon-Autoanalyzer® with p h o t o m e t e r a n d recorder.
Reagents
6. Phenolphthalein
1. D i s o d i u m h y d r o g e n p h o s p h a t e ,
7. Methanol
Na HP0 H 0
2 4 2
8. W e t t i n g a g e n t
2. P o t a s s i u m d i h y d r o g e n p h o s p h a t e ,
e. g. D o w antifoam B.
KH P0 2 4
9. S o d i u m h y d r o x i d e , 1 N
3. L y s i n e d e c a r b o x y l a s e
10. P h o s p h o r i c a c i d , 0 . 4 N
acetone-dried p o w d e r , e. g. from Nutritional
Biochemicals C o . ; c o m m e r c i a l p r e p a r a t i o n , see 11. Merthiolate
p . 484. e.g. from Eli Lilly Co., Indianapolis, Indiana,

USA.
4. S o d i u m c a r b o n a t e , 0.1 M
5. Brij 3 5 * *
U s e o n l y fresh d i s t i l l e d w a t e r .
I. P h o s p h a t e buffer ( 0 . 3 M ; p H 4 . 3 5 ) :
B o i l 0 . 0 0 1 N H C 1 t o r e m o v e C 0 , a l l o w t o c o o l a n d d i s s o l v e 4 0 . 8 g. K H P 0
2 2 4 in t h i s
solution and m a k e up to 1 0 0 0 ml.
II. P h o s p h a t e buffer ( 0 . 3 M ; p H 8 . 8 5 ) :
B o i l 0 . 0 0 1 N H C 1 t o r e m o v e C 0 , a l l o w t o c o o l a n d d i s s o l v e 8 0 . 4 g.
2 Na HP0 -7H 0
2 4 2
in this s o l u t i o n a n d m a k e u p t o 1 0 0 0 m l . A l l o w t o c o o l a n d p r o t e c t f r o m C 0 . 2
* L-Lysine carboxy-lyase, E C 4.1.1.18.

** from Technicon C o r p o r a t i o n , T a r r y t o w n , N . Y., U S A .
III. Enzyme solution ( p H 6.5):

S u s p e n d 1.75 g. d e c a r b o x y l a s e ( a c e t o n e p o w d e r ) a n d 4 0 m g . m e r t h i o l a t e in p h o s p h a t e
buffer ( s o l u t i o n I) ( u s e h o m o g e n i z e r ) . F i l t e r s u s p e n s i o n t h r o u g h g l a s s w o o l , rinse w o o l
a n d h o m o g e n i z e r w i t h buffer a n d d i l u t e t o 2 9 5 m l . A d d 2 0 5 m l . p h o s p h a t e buffer ( s o l u t
i o n II) a n d 0.5 m l . D o w a n t i f o a m B w i t h v i g o r o u s s h a k i n g . P r o t e c t f r o m C 0 ; p r e p a r e
2
freshly e a c h d a y .
IV. C o l o u r r e a g e n t :
M i x t h o r o u g h l y in a b r o w n b o t t l e 1 0 0 0 m l . C 0 - f r e e distilled w a t e r , 1.8 m l . 0.1 M N a C 0
2 2 3
s o l u t i o n , 3.0 m l . 1 % p h e n o l p h t h a l e i n s o l u t i o n (in m e t h a n o l ) a n d 0.5 m l . Brij 3 5 ; p r e p a r e

freshly e a c h d a y . T h e s e n s i t i v i t y o f t h e c o l o u r r e a g e n t c a n b e a d j u s t e d b y v a r i a t i o n o f
the a m o u n t of N a C 0 2 3 added.
Procedure
F o r flow s y s t e m see F i g u r e l . N o t s h o w n is a s a m p l e r for 4 0 s a m p l e s . F o r t y d e t e r m i n a t i o n s

2
c a n b e carried o u t p e r h o u r w i t h a w a t e r w a s h i n g b e t w e e n e a c h d e t e r m i n a t i o n . T h e a u t o m a t i c
s y s t e m is c o n t r o l l e d b y a m u l t i - c h a n n e l p r o p o r t i o n i n g p u m p w h i c h d e l i v e r s k n o w n v o l u m e s
o f s a m p l e , d i l u e n t , e n z y m e s a n d r e a g e n t s t o o t h e r c o m p o n e n t s o f t h e s y s t e m . T h e flow rate
for e a c h r e a g e n t is d e t e r m i n e d b y t h e d i a m e t e r o f t h e t u b i n g .
A certain v o l u m e o f s a m p l e is s u c k e d u p , d i l u t e d w i t h w a t e r , s e p a r a t e d b y a C 0 - f r e e b u b b l e
2
] Upper "XÔSamples/hr.
Lower j plane ^
fDlane
(T) Size of tubing (inch)
^ 0.45 Sample
2j
Mixing spiral
0.56 P Q > Buffer
<£>,
0.65 Air I C 0 - f r e e )
2
0.35 Enzyme
<2> 0.25 Wetting agent
0.90
0.73 Colour reagent
WW (10)
Mixing spiral
Waste
©
Proportioning pump
Waste 3 Ht I T
I
Photometer Recorder
15-mm-Cuvette
550-nm-Filter
Fig. 1. Flow Scheme.
L-Lysine 1703
o f air a n d m i x e d w i t h a n e q u i v a l e n t v o l u m e o f buffered e n z y m e s o l u t i o n (III). T h e m i x t u r e

is i n c u b a t e d at 37 ° C t o f a v o u r t h e d e c a r b o x y l a t i o n . E n z y m e a n d s u b s t r a t e s o l u t i o n are
m i x e d b y r e p e a t e d p a s s a g e t h r o u g h t h e m i x i n g spiral. D u r i n g t h e p a s s a g e t h r o u g h t h e 37 ° C
heating bath the C 0 2 is t r a n s f e r r e d t o t h e air b u b b l e . In t h e l i q u i d / g a s s e p a r a t o r t h e l i q u i d
p h a s e is s e p a r a t e d a n d is d i s c a r d e d , t h e g a s p h a s e is p u m p e d further. T h i s g a s b u b b l e s e p a r a t e s
a stream o f colour reagent. The C 0 2 is a b s o r b e d b y t h e s o l u t i o n a n d t h e p H d e c r e a s e s , w h i c h
results in a d e c r e a s e in t h e c o l o u r o f t h e i n d i c a t o r . T h i s is m e a s u r e d at 5 5 0 n m i n a 15 m m .
f l o w - t h r o u g h c u v e t t e ; t h e i n t e n s i t y o f t h e c o l o u r is i n v e r s e l y p r o p o r t i o n a l t o t h e a m o u n t o f
l y s i n e in t h e s a m p l e .
Standard Curves
Prepare standard solutions o f L-lysine hydrochloride (125 mg. L-lysine hydrochloride are
e q u i v a l e n t t o 1 0 0 m g . L - l y s i n e ) . T h e s t a n d a r d c u r v e is r e p r o d u c i b l e ; f r o m d a y t o d a y t h e v a l u e s
v a r y w i t h i n ± 1 %.
Calculations
Plot the % transmission of the s t a n d a r d s on semi-logarithmic p a p e r against the L-lysine c o n c e n t r a t i o n s .

T h e values for transmission o b t a i n e d for the samples are read off from this s t a n d a r d curve to give m g .
lysine/100 ml.
T h e enzyme loses its activity if it is filtered completely clear. T h e suspended enzyme should be filtered
t h r o u g h glass wool t o r e m o v e large particles which block the n a r r o w passages of the glass/liquid separator.
C0 2 p r o d u c e d by bacterial c o n t a m i n a t i o n can displace the base line a n d increase the sensitivity of the
colour reagent. A d d i t i o n of m e r t h i o l a t e t o the enzyme solution a n d storage in ice prevents bacterial
growth. C o l o u r reagent a n d enzyme solution should be protected from C 0 . T h e containers should 2
therefore be filled to capacity.
Specificity
Lysine decarboxylase is specific for L-lysine.
References
1 G. E. Schaiberger & A. Ferrari, A u t o m a t i c Enzymatic Analysis for L-Lysine via D e c a r b o x y l a t i o n ,

A n n . N . Y. A c a d . Sci. 87, 8 0 0 - 8 9 3 [I960].
2 Technicon A u t o A n a l y z e r Bulletin L i : Lysine D e t e r m i n a t i o n . Technicon I n s t r u m e n t C o r p . , Tarrytown,
N e w York 10502.
3 L. M. White & A. M. Sauger, A n a l . C h e m . 39, 2 1 3 - 2 1 7 [1967].
L-Glutamate
UV-Assay with Gutamate Dehydrogenase and NAD
Erich Bernt a n d H a n s Ulrich Bergmeyer
Previous m e t h o d s for the determination of glutamic acid are based on reactions which are n o t specific a n d
are often very c o m p l i c a t e d . G l u t a m i c acid can be determined colorimetrically with ninhydrin after
1,2
c h r o m a t o g r a p h i c s e p a r a t i o n or manometrically by m e a n s of g l u t a m a t e d e c a r b o x y l a s e or L- o r D - g l u t a m a t e
3 4
o x i d a s e . However, the necessary enzymes are n o t easily available. T h e d e t e r m i n a t i o n of glutamate with

5
glutamate dehydrogenase, G 1 D H ( L - G l u t a m a t e : N A D ( P ) oxidoreductase, d e a m i n a t i n g , E C 1.4.1.3) is

easy to carry out, is precise a n d specific for the L-isomer. A D P activates a n d stabilizes the enzyme.
Application of Method: In biochemistry, clinical chemistry a n d food c h e m i s t r y 6 - 1 0

.
Principle
(1) L-Glutamate + N A D +
+ H 0 2 2-Oxoglutarate + N A D H + N H 4
+
T h e increase in N A D H , as m e a s u r e d by the extinction change at 340 (334, 365) n m , is p r o p o r t i o n a l to the

a m o u n t of L-glutamate.
The equilibrium of the reaction lies far to the l e f t . However, by t r a p p i n g the k e t o acid with hydrazine,
10
a n d by use of a large excess of N A D a n d an alkaline m e d i u m ( p H 9), L-glutamate can be quantitatively

oxidized to 2-oxoglutarate. U n d e r the conditions described below the reaction is stoichiometric u p t o
0.2 /rniole L-glutamate/cuvette. A D P activates a n d stabilizes the enzyme in the a s s a y . 11
Equipment
3 3 4 or 365 n m ; b e n c h c e n t r i f u g e .
Reagents
1. H y d r a z i n e h y d r a t e , c a . 2 4 % ( w / v ) A d d i t i o n a l for t h e a n a l y s i s o f s a m p l e s c o n
2. Glycine taining protein:
3. S u l p h u r i c a c i d , A . R . , 1 N
4. N i c o t i n a m i d e - a d e n i n e dinucleotide, N A D 6. P e r c h l o r i c a c i d , A . R . , s p . gr. 1.67; ca.
free acid; commercial p r e p a r a t i o n , see p . 545. 70%(w/w)
5. G l u t a m a t e d e h y d r o g e n a s e , G 1 D H 7. T r i p o t a s s i u m p h o s p h a t e , K P0 -3H 0
3 4 2
crystalline from ox liver; solution in 50% (v/v) 8. A d e n o s i n e - 5 ' - d i p h o s p h a t e , ADP

glycerol, free from a m m o n i u m s u l p h a t e ; ^30 disodium salt; commercial preparation, see
U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 525.
p. 461.
L-Glutamate 1705
Purity of Reagents
T h e enzyme m u s t be free from g l u t a m i n a s e a n d a m m o n i a ions. A D P - N a 2 should contain at least 8 0 %

A D P . All other reagents should be puriss. or A . R. grade.
P r e p a r e all s o l u t i o n s w i t h f r e s h , d o u b l y d i s t i l l e d w a t e r . Sterilize t h e c o n t a i n e r s t o p r e v e n t
bacterial c o n t a m i n a t i o n .
I. G l y c i n e - h y d r a z i n e buffer ( 0 . 5 M g l y c i n e ; 0 . 4 M h y d r a z i n e ; p H 9 ) :
D i s s o l v e 3.75 g. g l y c i n e a n d 5 . 5 0 g. c a . 2 4 % h y d r a z i n e h y d r a t e in d i s t i l l e d w a t e r , a d j u s t
to p H 9 with 1 N H S 0 2 4 a n d dilute t o 100 ml. with distilled water.
II. A d e n o s i n e - 5 ' - d i p h o s p h a t e ( 3 3 . 5 m M A D P ) :
D i s s o l v e 45 mg. A D P - N a 2 in 2.5 m l . d i s t i l l e d w a t e r .
III. N i c o t i n a m i d e - a d e n i n e dinucleotide (27 m M / ? - N A D ) :
D i s s o l v e 1 0 0 m g . N A D in 5 m l . d i s t i l l e d w a t e r .
I V . G l u t a m a t e d e h y d r o g e n a s e , G 1 D H ( c a . 10 m g . p r o t e i n / m l . ) :
If n e c e s s a r y , d i l u t e t h e s t o c k s o l u t i o n w i t h 5 0 % g l y c e r o l .
A d d i t i o n a l for the analysis o f samples c o n t a i n i n g p r o t e i n :

V . P e r c h l o r i c a c i d ( c a . 1.0 M ) :
D i l u t e 8.6 ml. 7 0 % perchloric acid w i t h distilled water t o 100 ml.
V I . Phosphate solution (1.93 M K P0 ):
3 4
D i s s o l v e 5 1 . 0 g. K P 0 - 3 H 0 i n d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
3 4 2
Store all solutions at 0 t o 4 °C. P r e p a r e the N A D a n d A D P solutions (II & III) freshly every two weeks.
Check the specific activity of the G 1 D H solution (IV) every m o n t h . T h e buffer a n d p h o s p h a t e solutions
are stable indefinitely.
Procedure
Dilute glutamic acid preparations, protein hydrolysates and other a m i n o acid mixtures so
that they c o n t a i n less t h a n 0.2 /zmole L - g l u t a m a t e / m l . To o b t a i n h o m o g e n e o u s solutions o f
m e a t e x t r a c t s , s o u p c u b e s , e t c . , h e a t in w a t e r u n t i l j u s t b o i l i n g , c o o l a n d filter. T h e fat r e m a i n s
o n t h e filter p a p e r .
R e m o v e a m m o n i u m ions (see "Sources o f Error") before the determination by lyophilizing
a s o l u t i o n o f t h e s a m p l e w h i c h h a s b e e n a d j u s t e d t o p H 9. If in s p i t e o f d i l u t i o n t h e s a m p l e is
strongly c o l o u r e d , isolate the g l u t a m a t e by a d s o r p t i o n o n an a n i o n e x c h a n g e resin (e.g. A m b e r -
lite I R A - 4 0 0 ) a n d e l u t i o n w i t h 1 N N a O H . T a k e 1.0 m l . o f t h e e l u a t e f o r t h e a s s a y .
C o l l e c t b l o o d w i t h o u t v e n e s t a s i s . A n t i c o a g u l a n t s d o n o t interfere. R e m o v e a n i m a l t i s s u e s a s
r a p i d l y a s p o s s i b l e a n d d e p r o t e i n i z e ; it is p r e f e r a b l e t o u s e " f r o z e n - s t o p " t o n g s t o c l a m p t i s s u e
(refer t o " C e l l a n d T i s s u e D i s i n t e g r a t i o n " , p . 4 0 0 ) .
Deproteinization:
W h o l e b l o o d : T h o r o u g h l y m i x 5.00 ml. b l o o d with 5.00 ml. perchloric acid (solution V) a n d

c e n t r i f u g e for 10 m i n . at 3 0 0 0 g. A d j u s t 5 m l . o f t h e s u p e r n a t a n t fluid t o p H 9 w i t h 1.0 m l .
p h o s p h a t e s o l u t i o n ( V I ) . A l l o w t o s t a n d 10 m i n . in a n ice b a t h a n d t h e n filter t h r o u g h a s m a l l ,
fluted filter p a p e r . A l l o w t o w a r m t o r o o m t e m p e r a t u r e a n d t a k e 1.00 m l . for t h e a s s a y .
Tissue: H o m o g e n i z e with 2 parts by weight o f perchloric acid (solution V) a n d centrifuge
for 10 m i n . at 3 0 0 0 g. A d j u s t 3 . 0 0 m l . s u p e r n a t a n t fluid t o p H 9 w i t h 1.00 m l . p h o s p h a t e
s o l u t i o n ( V I ) . A l l o w t o s t a n d 10 m i n . in a n ice b a t h a n d t h e n filter t h r o u g h a s m a l l , fluted
filter p a p e r . A l l o w t o w a r m t o r o o m t e m p e r a t u r e , d i l u t e 1 : 1 0 a n d t a k e 1.00 m l . for t h e a s s a y .
T h e g l u t a m a t e c o n t e n t o f b l o o d d e c r e a s e s b y 1 5 % in 2 4 hr. at 2 5 ° C , b u t d o e s n o t c h a n g e at 4 ° .
In a c i d o r n e u t r a l d e p r o t e i n i z e d e x t r a c t s o f b l o o d g l u t a m a t e is s t a b l e f o r at least 2 4 hr. at 25 ° C .
S i m i l a r results h o l d for t i s s u e s .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 o r H g 3 6 5 ) n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 3 . 3 5 m l . ; r o o m
t e m p e r a t u r e . F o r e a c h series o f m e a s u r e m e n t s p r e p a r e a r e a g e n t b l a n k c o n t a i n i n g w a t e r i n s t e a d
o f s a m p l e * . R e a d a g a i n s t air o r w a t e r .
Pipette successively into cuvettes: C o n c e n t r a t i o n in a s s a y m i x t u r e
G l y c i n e - h y d r a z i n e buffer (I) 2.00 ml. 300 m M glycine;

250 m M hydrazine
Sample 1.00 m l . u p t o 6 0 pM L - g l u t a m a t e
A D P solution (II) 0.10 ml. 1.0 m M A D P
N A D solution (III) 0.20 ml. 1.6 m M 0 - N A D
M i x , read extinction E . t
G 1 D H solution (IV) 0.05 ml. 155,ug./ml. ^ 4 . 5 U / m l .
Mix. A l l o w sample and blank to stand for ca. 45 m i n .

(for e a c h c u v e t t e there m u s t b e t h e s a m e i n t e r v a l
between adding the N A D solution a n d measuring E , 2
see " S o u r c e s o f E r r o r " ) . M e a s u r e e x t i n c t i o n E . 2
C a l c u l a t e t h e difference b e t w e e n E a n d E for s a m p l e
t 2
and blank. z l E . - z l E . = ^ E
s a m b l k is u s e d for
g l u t a m a t e
the calculations.
* N A D and hydrazine form a c o m p o u n d which a b s o r b s at 303 n m a n d which also has considerable

absorption at 365 n m . This b l a n k reaction is very rapid in t h e first few seconds (increase in extinction
of u p to 0.030 at 365 n m ) , then it creeps u p with a rate of A E/30 min. = 0.005 to 0.020. It is therefore
necessary to have the same reaction time after addition of N A D for the sample a n d blank cuvettes. This
also holds for all lengthy reactions which include N A D a n d hydrazine in the assay mixture, e.g. the
determination of L-lactate (p. 1464) a n d L-glycerol-3-phosphate (p. 1415).
L-Glutamate 1707
Calculations
U n d e r the described c o n d i t i o n s the reaction proceeds stoichiometrically. T h e calculation formula (2)

on p. 312 applies. T h e results are o b t a i n e d in jumole L-glutamate per ml. sample. This value m u s t be
multiplied by the a p p r o p r i a t e factor if the sample h a s been deproteinized, neutralized or diluted in any way.
In the case of whole b l o o d the specific gravity (ca. 1.06) a n d water c o n t e n t (ca. 80 %) m u s t be taken into
account. In this p r o c e d u r e for b l o o d where the dilution for deproteinization is 1 + 1 a n d for neutralization
is 3 + 1 a factor of 2.22 is o b t a i n e d . In the case of tissue where a 3 3 % h o m o g e n a t e ( / tissue) is used for 1
3
deproteinization, the dilution o n neutralization is 3 + 1 a n d t h e further dilution is 1 + 9, the factor is 40.

T h e following relationships hold for whole b l o o d o r tissue

Blood c = AE x 1.21 J E x 1.19 AE x 2.18 [//mole/ml.]
c = AE x 179 AE x 175 AE x 320 [pig./ml.]
Tissue c = AE x 21.97 AE x 21.5 AE x 39.41 [/imole/g.]
c = AE x 3232 AE x 3167 AE x 5799 [/ig./mg.]
W i t h a m e a n value of 2.90 m g . L - g l u t a m a t e / m l . s o u p seasoning the s t a n d a r d deviation was 0.06 m g .

a n d therefore the coefficient of variation is 2%.
N o r m a l Values
F o r the L-glutamate c o n t e n t of tissues of the cat, s e e . N o precise values are available for h u m a n b l o o d .
12
Interference in the assay system: A m m o n i u m ions interfere with the assay a n d therefore m u s t be removed
(see " P r o c e d u r e " ) . I n a c c u r a t e timing of the a d d i t i o n of the N A D solution t o the b l a n k a n d sample cuvettes
leads t o false values (Fig. 1).
0.150 h
Fig. 1. Progress curve of t h e extinction changes

in t h e d e t e r m i n a t i o n of g l u t a m a t e with G 1 D H .
Effect of inaccurate timing o n t h e A E values.
Curve I : Sample
Curve I I : Blank
A : M e a s u r e d t o o early in relation to the
60 min. b l a n k (A E t o o small)
B : M e a s u r e d at the right time
C : M e a s u r e d t o o late in relation to the
60 min. b l a n k cuvette (A E t o o large). M i n u t e s a f t e r a d d i t i o n of N A D - s o l u t i o n
Specificity
G l u t a m i n e , D - g l u t a m a t e , L-aspartate, pyrrolidonecarboxylic acid a n d other derivates of glutamic acid d o

not react.
References
1 B. A. Prescott & H. Waelsch, J. biol. C h e m . 164, 331 [1946].

2 P. P. Cohen, Biochem. J. 33, 551 [1939].
3 W. Troll ScR. K. Cannon, J. biol. C h e m . 200, 893 [1953].
4 A. Meister, H A. Sober & S. V. Tice, J. biol. C h e m . 189, 591 [1951].
5 A. E. Bender & H. A. Krebs, Biochem. J. 46, 210 [1950].
6 K. Mohler & W. Volley, Z . Lebensmittel U n t e r s . 140, 189 [1969].
7 M. Burba & M. Kastning, Z u c k e r 24, 386 [1971].
8 F. Drawert, H. Barton & W. Hagen, Brauwissenschaft 23, 345 [1970].
9 F. Drawert, H. Barton & W. Hagen, Brauwissenschaft 23, 432 [1970].
10 / . A. Olson & C. B. Anfinsen, J. biol. C h e m . 202, 841 [1953].
11 B. Eisenkraft, J. B. van Dorf & C. Veeger, Biochem. Biophys. A c t a 185, 9 [1969].
12 H. H. Tallan, W. H. Stein & S. Moore, J. biol. C h e m . 211, 927 [1954].
Determination with Glutamate Dehydrogenase,

Diaphorase, and Tetrazolium Salts
Hans-Otto Beutler and Gerhard Michal
T h e m e t h o d of determining g l u t a m a t e with d i a p h o r a s e * a n d tetrazolium salts overcomes the unfavourable

position of the equilibrium of the reaction because of the c o n t i n u o u s r e o x i d a t i o n of the N A D H formed.
Short reaction times are obtained. T h e sensitivity is increased, since the f o r m a z a n formed h a s a higher
extinction coefficient t h a n N A D H . T h e reagents a r e c h e a p . O n t h e o t h e r h a n d , the reagents a r e s o m e w h a t
light-sensitive a n d this m u s t be taken into a c c o u n t .
A similar m e t h o d , which involves I N T a n d phenazine m e t h o s u l p h a t e , h a s been described by Sowerby
and Ottaway .1
Application of Method: In biochemistry, clinical biochemistry, food chemistry, a n d pharmaceutical

chemistry.
Principle
(1) L-Glutamate+ N A D + H 0 ,+
2
G 1 P H
** » 2-Oxoglutarate+NADH+NH 4
+
(2) NADH+INT+H + d i a p h o r a s e
*> N A D +
+ Formazan
L - G l u t a m a t e is oxidatively d e a m i n a t e d to 2-oxoglutarate by n i c o t i n a m i d e - a d e n i n e dinucleotide ( N A D )

in the presence of g l u t a m a t e d e h y d r o g e n a s e ( G I D H ) . In the presence of d i a p h o r a s e , the resulting N A D H
reduces iodonitrotetrazolium chloride ( I N T ) t o a f o r m a z a n , w h o s e extinction is m e a s u r e d in the visible
region (492 n m ) . F o r details of the p r o c e d u r e , see p . 136.
2
* N A D H : lipoamide oxidoreductase, E . C . 1.6.4.3.

** L - G l u t a m a t e : N A D ( P ) oxidoreductase (deaminating), E . C . 1.4.1.3.
L-Glutamate 1709
Optimum Conditions for Measurements
A n equilibrium constant K H 2 G =4.5 x 10" 1 4

M (in tris buffer, 25 °C) has been reported for reaction (1);
2 3
the equilibrium thus lies well to the left. However, complete oxidation is achieved by the continuous removal
of the N A D H from the equilibrium by reaction (2), which is irreversible under the conditions used. The
colour intensity of the formazan formed has been found to depend slightly on the ammonia content of
the solution (a slight increase at low ammonia concentrations, followed by a decrease). Under the con
4
ditions of the assay with our I N T preparation the mean e 4 9 2 n m was 19.9 cm //*mole, and an error of < 3 %
2
resulted from up to a 32-fold molar excess of N H 3 over the quantity of glutamate used. Recalibration
with glutamic acid is advisable when a fresh I N T preparation is used (see below). The light-sensitivity
of I N T must be taken into account both in the storage of the solution and in the determination; direct
sunlight must be avoided at all times.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r c a p a b l e o f p r e c i s e m e a s u r e m e n t s at a b o u t
500 ( H g 492) n m ; laboratory centrifuge.
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 9. G l u t a m a t e d e h y d r o g e n a s e , G I D H
2. D i p o t a s s i u m h y d r o g e n phosphate, crystalline, from bovine liver, solution in 50%
K HP0 ,
2 4 A.R. glycerol, free from ammonium sulphate and
3. Potassium dihydrogen phosphate E D T A , approx. 100 U/mg. (25 °C, 2-oxo-
KH P0 , A.R. glutarate as substrate). Commercial prepara

2 4
tions, see p. 461.

4 . P o t a s s i u m h y d r o x i d e s o l u t i o n , A . R., 2 N
5. T r i t o n X - l 0 0 ® 10. L - ( - h ) - G l u t a m i c a c i d , A . R .
or "for biochemical use", C H N 0 content
6. N i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e , 5 9 4
>99%.
NAD
free acid, commercial preparations see p. 545.
A d d i t i o n a l reagents for the analysis of
7. I o d o n i t r o t e t r a z o l i u m c h l o r i d e , I N T
samples containing protein:
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-
tetrazolium chloride 11. Perchloric acid, A . R.
8. D i a p h o r a s e ( l i p o a m i d e dehydrogenase), sp. gr. = 1.67; approx. 70% (w/w)
DIA 12. T r i p o t a s s i u m p h o s p h a t e , K P0 -3H 0
3 4 2
isolated from pig heart; lyophilizate with stabil

izers, > 1 0 U / m g . (25 °C, Na-lipoate as sub
strate). Commercial preparations, see p. 448.
Purity of Reagents
G I D H must be free from glutaminase. Contamination by lactate dehydrogenase, malate dehydrogenase,

and alcohol dehydrogenase should not exceed 0.01 % of each.
M a k e u p all s o l u t i o n s w i t h freshly p r e p a r e d d o u b l y d i s t i l l e d w a t e r . Sterilize t h e flasks t o

prevent the growth o f micro-organisms.
I. T r i e t h a n o l a m i n e p h o s p h a t e b u f f e r ( 0 . 0 8 M t r i e t h a n o l a m i n e ; 0 . 0 2 M p o t a s s i u m p h o s
phate; p H 8.6):
a ) D i s s o l v e 1.86 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e i n w a t e r a n d a d j u s t t o p H 8 . 6 w i t h
a p p r o x . 4 . 4 m l . 2 N K O H ; a d d 0 . 6 3 m l . Triton X - 1 0 0 ® a n d m a k e u p t o 1 0 0 m l .
with water.
b) D i s s o l v e 0 . 8 6 g. d i p o t a s s i u m h y d r o g e n p h o s p h a t e a n d 7 m g . p o t a s s i u m d i h y d r o g e n
p h o s p h a t e in w a t e r a n d m a k e u p t o 1 0 0 m l .
Mix 20 ml. solution a) with 5 ml. solution b).
II. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 6 . 7 m M j f t - N A D ) :
D i s s o l v e 2 5 m g . N A D in 5 m l . w a t e r .
III. I o d o n i t r o t e t r a z o l i u m c h l o r i d e ( 1 . 1 9 m M ) :
D i s s o l v e 3 0 m g . I N T in 5 0 m l . w a t e r .
I V . D i a p h o r a s e (1 m g . p r o t e i n / m l . ) :
D i s s o l v e 3 m g . l y o p h i l i z a t e in 1 m l . w a t e r .
V. Glutamate dehydrogenase, G I D H (10 m g . protein/ml.):
U s e s t o c k s o l u t i o n in 5 0 % g l y c e r o l w i t h o u t d i l u t i o n .
VI. Standard glutamate solution (0.136 m M ) :
D i s s o l v e 5 0 . 0 m g . g l u t a m i c a c i d i n a p p r o x . 2 5 m l . w a t e r , a d j u s t t o p H 7.0 w i t h 2 N
K O H , a n d m a k e u p t o 50 m l . with water. D i l u t e 1 ml. o f this solution with 49 ml. water.
Additional solutions for the analysis o f samples containing protein:
V I I . P e r c h l o r i c a c i d ( a p p r o x . 1.0 M ) :
Dilute 8.6 ml. 7 0 % perchloric acid t o 100 ml. with water.
VIII. Phosphate (1.93 M K P 0 ) : 3 4
D i s s o l v e 5 1 . 0 g. K P 0 - 3 H 0 in w a t e r a n d m a k e u p t o 1 0 0 m l .
3 4 2
Store all solutions at 0 t o 4 °C. T h e I N T solution (III) is sensitive t o light a n d must always be kept in
dark bottles; it c a n be used for a b o u t 3 m o n t h s . T h e n i c o t i n a m i d e - a d e n i n e dinucleotide solution (II)
should be renewed after 2 weeks, a n d t h e d i a p h o r a s e solution (IV) after 3 weeks. T h e G I D H solution is
stable for 12 m o n t h s , a n d t h e other solutions keep indefinitely.
Procedure
Collection of sample: Dilute glutamic acid preparations, protein hydrolysates, a n d other

a m i n o a c i d m i x t u r e s s o t h a t t h e y c o n t a i n n o t m o r e t h a n 3 0 pg. o f L - g l u t a m a t e / m l . H e a t m e a t
extracts, s o u p c u b e s , e t c . , in w a t e r t o b o i l i n g in o r d e r t o p r e p a r e h o m o g e n e o u s s o l u t i o n s ,
t h e n c o o l a n d filter. F a t r e m a i n s o n t h e filter.
Take b l o o d without venestasis. T h e addition o f anticoagulants in the usual concentrations
d o e s n o t cause interference. Collect animal tissue samples a n d deproteinize as quickly as
L-Glutamate 1711
p o s s i b l e ; t h e s a m p l e s a r e b e s t t a k e n w i t h " q u i c k - f r e e z e " t o n g s (cf. " C e l l a n d T i s s u e D i s

integration", p. 400).
Deproteinization: W h o l e b l o o d : T h o r o u g h l y m i x 5.00 ml. w h o l e b l o o d with 5.00 ml. per

c h l o r i c a c i d ( V I I ) in a c e n t r i f u g e t u b e a n d c e n t r i f u g e f o r 10 m i n . a t 3 0 0 0 r p m . A d j u s t 5 m l .
s u p e r n a t a n t t o p H ~ 9 w i t h 1.0 m l . p h o s p h a t e s o l u t i o n ( V I I I ) , a l l o w t o s t a n d for 10 m i n .
in a n i c e b a t h , a n d filter t h r o u g h a s m a l l f o l d e d filter p a p e r . A f t e r a d j u s t m e n t o f t h e t e m p e r a t u r e
t o 2 5 ° C , u s e 0 . 5 0 m l . filtrate f o r t h e d e t e r m i n a t i o n .
H o m o g e n i s e t i s s u e w i t h t w i c e its w e i g h t o f p e r c h l o r i c a c i d ( V I I ) a n d c e n t r i f u g e for 10 m i n .
at 3 0 0 0 r p m . A d j u s t 3 . 0 0 m l . s u p e r n a t a n t t o p H — 9 w i t h 1.00 m l . p h o s p h a t e s o l u t i o n ( V I I I ) ,
a l l o w t o s t a n d f o r 10 m i n . i n a n i c e b a t h , a n d filter t h r o u g h a s m a l l f o l d e d filter p a p e r . A f t e r
adjustment o f the temperature t o 25 ° C , dilute 1 + 9 w i t h water a n d u s e 0.5 ml. for the determina
tion.
Stability of sample: T h e g l u t a m a t e c o n t e n t in b l o o d d e c r e a s e s b y 1 5 % a t 2 5 ° C a n d b y 0 %
at 4 ° C w i t h i n 2 4 h o u r s o f c o l l e c t i o n . G l u t a m a t e is s t a b l e f o r a t l e a s t 2 4 h o u r s at 2 5 ° C in t h e
a c i d extract after d e p r o t e i n i z a t i o n a n d in t h e n e u t r a l i z e d e x t r a c t . T h e s i t u a t i o n in t i s s u e s is
similar.
Assay System
W a v e l e n g t h : H g 4 9 2 n m ; light p a t h : 1 c m . ; final v o l u m e : 3.5 m l . ; r o o m t e m p e r a t u r e ; r e a d

a g a i n s t air.
M a k e u p a reagent blank with water instead o f s a m p l e for e a c h series o f determinations. D o
n o t leave I N T solution or assay mixture unprotected in daylight.
B e f o r e u s i n g a n e w I N T p r e p a r a t i o n , m e a s u r e its e x t i n c t i o n c o e f f i c i e n t a s f o l l o w s :
P r e p a r e a s s a y m i x t u r e w i t h 0.5 m l . s t a n d a r d g l u t a m a t e s o l u t i o n ( V I ) . T h i s c o n t a i n s 10 pg=
= 0 . 0 6 8 0 / / m o l e g l u t a m i c a c i d / 0 . 5 m l . M e a s u r e AE. £ =
^ Q Q ^ ^ [cm. //miole]. This value
2
is u s e d i n t h e c a l c u l a t i o n f o r m u l a g i v e n b e l o w , s s h o u l d b e b e t w e e n 1 9 . 0 a n d 2 0 . 0 c m / / / m o l e .
2
Buffer (I) 2.50 ml. 57 m M T R A , 14 m M p h o s p h a t e

N A D solution* (II) 0.20 ml. 0.38 m M N A D
I N T solution (III) 0.20 ml. 0.068 m M I N T
Diaphorase solution* (IV) 0.05 ml. 1 4 . 3 pg.jm\.>0.14 U/ml.
Sample 0.50 ml. u p t o 3 0 pM
Mix, read extinction E. l
G I D H solution 0.05 ml. 0 . 1 4 m g . / m l . > a p p r o x . 14 U / m l .
Mix. A l l o w sample a n d blank t o stand for approx.

15 m i n . R e a d e x t i n c t i o n E s e v e r a l t i m e s a t i n t e r v a l s
2
o f 3 m i n . D e t e r m i n e d i f f e r e n c e s E -E for s a m p l e a n d
2 1
blank.
A Esample ^ -
^Blank
=
A E G l u t a m a t e
* T h e commercially o b t a i n a b l e N A D / d i a p h o r a s e mixture (Boehringer M a n n h e i m ) m a y be used instead

of solutions (II) a n d (IV)
Calculations
F o r a given I N T p r e p a r a t i o n , the reaction proceeds stoichiometrically u n d e r the a b o v e conditions.

F o r m u l a (2) on p . 312 is therefore valid. T h e result is o b t a i n e d in //mole g l u t a m a t e p e r ml. of sample.
However, this value m u s t be multiplied by a factor if the sample h a s been deproteinized, neutralized, o r
otherwise diluted.
W h e n whole b l o o d is used, the w a t e r c o n t e n t m u s t also be taken into a c c o u n t . F r o m these d a t a a n d from
the dilution of 1 + 1 in the deproteinization a n d 5 + 1 in the neutralization, a factor of 2.21 is therefore
obtained for this p r o c e d u r e .
W h e n tissue is used, on the basis of an average water c o n t e n t of 8 0 % (v/v), a factor of 37.4 is o b t a i n e d
from the p r e p a r a t i o n of a 3 3 % h o m o g e n a t e (1/3 tissue), the neutralization of 3 + 1, a n d the dilution of
1+9.
This leads t o the following r e l a t i o n s h i p s :
In the sample solutions: c=A E x 7.00/e [//mole/ml.]

c=JExl030/£ [mg./ml. 1
In b l o o d : c=A E x 15.5/e [//mole/ml.]

c=A E x 2276/6 [mg./ml.]
In tissues: c=JEx262/£ [//mole/ml.]

c=JEx38520/e [mg./ml.]
e is the extinction coefficient of the f o r m a z a n as found by the a b o v e p r o c e d u r e .
With an average of 100 ug. L - g l u t a m a t e / m l . of sample solution, the s t a n d a r d deviation is 0.8 /zg L - g l u t a m a t e /
ml., a n d the coefficient of variation is 0 . 8 % .
N o r m a l Values
F o r the content in a n i m a l organs, s e e . Values of 14.0 + 1.1 mg./l. a n d 11.8 + 1.0 mg./l. have been f o u n d
5 6 , 7
for h u m a n serum (by c h r o m a t o g r a p h i c m e t h o d s ) .
Interference due to drugs or other therapeutic measures is u n k n o w n .
Sources of error in assay technique:

If the final value is not reached within 20 min., the following are possible r e a s o n s : a) excessively low enzyme
activity. M e a s u r e enzyme activity a n d use a larger q u a n t i t y of e n z y m e or, if necessary, fresh enzyme
preparations, b) Excessively high c o n t e n t of a m m o n i u m ions in t h e sample. To remove, m a k e the s a m p l e
alkaline and heat, then re-neutralize again. A m m o n i u m ions d o n o t interfere u p to an a p p r o x . 30-fold m o l a r
excess over the g l u t a m a t e content, c) Presence of high E D T A c o n c e n t r a t i o n s in the assay mixture. E D T A
inhibits the d i a p h o r a s e activity (above a b o u t 2 - 5 / / M ) . Use E D T A - f r e e reagents.
Reversal of the reaction or t o o low a final value is d u e t o precipitation of f o r m a z a n . C h e c k whether t h e
indicated quantity of Triton X-100 h a s been a d d e d .
Specificity of Method, see p . 1708.

L-Glutamate 1713
Other Determinations
L - G l u t a m i n e can be determined in the same assay mixture by a d d i t i o n of glutaminase or asparaginase

(which has a glutaminase side activity, a p p r o x . 2 % of the asparaginase activity).
References
1 J. M. Sowerby & J. H. Ottaway, Biochem. J. 79, 21 P [1961].

2 R. Kuhn & D. Jerchel, Ber. dt. chem. G e s . 74, 941 [1941].
3 C. Frieden in P. D. Boyer, H. A. Lardy & K. Myrback: T h e Enzymes, Vol. 7, A c a d e m i c Press, N e w
Y o r k 1963, p . 3.
4 H. O. Beutler & G. Michal, unpublished.
5 H. H. Tallau, W. H. Stein & S. Moore, J. biol. C h e m . 211, 927 [1954].
6 P. Hoecker x x x, Wiener Klin. Wschr. 83, 245 [1971].
7 W. L. Daniel & J. V. Higgins, A m . J. Diseases Child. 121, 401 [1971].
Determination with Glutamate Dehydrogenase and the 3-Acetylpyridine

Analogue of NAD (APAD)
Irene Witt*
Substitution of N A D by the 3-acetylpyridine analogue (3-acetylpyridine-adenine dinucleotide, A P A D )

in N A D - d e p e n d e n t enzyme reactions alters the position of their equilibria . T h e m o r e positive redox
1
potential of the A P A D / A P A D H system in c o m p a r i s o n to the N A D / N A D H system displaces the

+ +
equilibrium in favour of substrate oxidation. T h u s use of A P A D in enzymatic s p e c t r o p h o t o m e t r i c assays

avoids the necessity for t r a p p i n g the p r o d u c t of the oxidative reaction. T h e d e t e r m i n a t i o n of L-glutamate
with A P A D a n d g l u t a m a t e dehydrogenase, G I D H ( L - G l u t a m a t e : N A D oxidoreductase, E C 1.4.1.2)
described here is based o n t h a t of Holzer a n d Soling . 2
Application of Method: In biochemistry, clinical chemistry a n d foodstuff chemistry.
Principle
(1) L-Glutamate + A P A D +
+ H 0 ,
2
g l u t a m a t e
y 2-Oxoglutarate + A P A D H + NH^
dehydrogenase
The formation of A P A D H , as m e a s u r e d by the increase in extinction at H g 365 n m , is p r o p o r t i o n a l to

the a m o u n t of L-glutamate. T h e a b s o r p t i o n m a x i m u m of A P A D H is at 363 n m a n d the m o l a r extinction
3
coefficient, e is 9.1 x 1 0 c m . / m o l e at 363 n m . F o r the m e a s u r e m e n t at H g 365 n m a value of e = 9.1 x 1 0

6 2 3 6
c m . / m o l e is sufficiently a c u r a t e .
2 2
T h e equilibrium of the reaction is very d e p e n d e n t on the p H . A l t h o u g h substitution of A P A D for N A D

displaces the equilibrium in favour of substrate oxidation, it follows from the equilibrium c o n s t a n t t h a t
even with A P A D the equilibrium of the reaction lies on the left at p H 7 . 0 . To displace the equilibrium2
* This C h a p t e r was c o n t r i b u t e d in the first edition by H. Holzer, H. D. Soling a n d I. Witt.

completely in favour of the oxidized substrate, an alkaline p H , relatively high A P A D concentration

a n d absence of a m m o n i u m ions are required. As the affinity of G I D H for L-glutamate in the assay with
A P A D is low ( K is 4.5 m M ) , a high concentratio n of G I D H m u s t be used to obtain quantitative oxidation
M
of L-glutamate.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e for m e a s u r e m e n t s at 365 n m ; b e n c h
centrifuge.
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 4. G l u t a m a t e d e h y d r o g e n a s e , GIDH
K H P 0 , A . R.
2 4 (NH^-free)
2. D i s o d i u m h y d r o g e n p h o s p h a t e , from bovine liver, crystalline, solution in 5 0 %
N a H P 0 - 2 H 0 , A . R.
2 4 2 glycerol, ^ 90 U / m g . (25 °C). Commercial pre
3. 3 - A c e t y l p y r i d i n e - a d e n i n e d i n u c l e o t i d e , p a r a t i o n , see p 461.
APAD
commercial p r e p a r a t i o n , see p . 525.
P r e p a r e all s o l u t i o n s w i t h freshly p r e p a r e d , d o u b l y d i s t i l l e d w a t e r .
I. P h o s p h a t e buffer ( 6 6 m M , p H 8 . 2 ) :
D i s s o l v e 9 . 0 g. K H P 0 2 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r . D i s s o l v e 11.9 g. N a H P 0 - 2 H 0 in
2 4 2
1 0 0 0 ml. distilled water. M i x 4 parts by v o l u m e o f the K H P 0 2 4 solution with 96 parts

by v o l u m e o f the N a H P 0 2 4 solution and check p H with glass electrode.
II. 3 - A c e t y l p y r i d i n e - a d e n i n e d i n u c l e o t i d e (ca. 6.5 m M A P A D ) :
D i s s o l v e 5 m g . A P A D in 1.0 m l . d i s t i l l e d w a t e r .
III. G l u t a m a t e d e h y d r o g e n a s e , G I D H ( 1 0 m g . p r o t e i n / m l . ) :
U s e commercially available solution undiluted.
Store all solutions, stoppered, in a refrigerator at 0 - 4 °C. Solution I is stable for several weeks. P r e p a r e
solution II freshly each week. T h e enzyme solution is stable for a b o u t 6 m o n t h s .
Procedure
A s for t h e d e t e r m i n a t i o n o f L - l a c t a t e w i t h N A D , s e e p . 1 4 6 5 .
L-Glutamate 1715
Assay System
W a v e l e n g t h : H g 3 6 5 n m ; t o c o n s e r v e t h e A P A D , w h i c h is e x p e n s i v e , u s e c u v e t t e s w i t h s m a l l
v o l u m e s (V = 1.5 m l . ) ; l i g h t p a t h : 1 c m . ; final v o l u m e : 1.0 m l . ; r o o m t e m p e r a t u r e ; r e a d
a g a i n s t air.
Sample (deproteinized, neutralized) 0.10 ml. u p t o 0.1 m M

P h o s p h a t e buffer (I) 0.50 ml. 33 m M
A P A D solution (II) 0.05 ml. 0.33 m M
Distilled water 0.33 ml.
Cover the cuvettes with Parafilm and mix well.

F o l l o w t h e e x t i n c t i o n c h a n g e s u n t i l c o n s t a n t (ca. 3
min.) and then read extinction E.
1
G I D H solution (III) 0.02 ml. 0.20 mg./ml. = 18 U / m l .
M i x . R e a d t h e e x t i n c t i o n at 15, 2 0 a n d 2 5 m i n . If t h e
e x t i n c t i o n is c o n s t a n t r e a d e x t i n c t i o n E . E — E j =
2 2
= A E is u s e d for t h e c a l c u l a t i o n s .
D e t e r m i n e t h e e x t i n c t i o n c h a n g e d u e t o a d d i t i o n o f G I D H s o l u t i o n (III) b y m i x i n g in a further
0 . 0 2 m l . s o l u t i o n III at t h e e n d o f t h e r e a c t i o n . C o r r e c t E 2 for t h i s e x t i n c t i o n c h a n g e .
Calculations
T h e reaction proceeds stoichiometrically u n d e r the described conditions a n d therefore the calculation

formula (2) on p . 312 applies. T h e m o l a r extinction coefficient 2
e of A P A D H is 9.1 x 10 c m . / m o l e
6 2
at 365 n m .
It follows t h a t the c o n c e n t r a t i o n of L-glutamate in the sample is given b y :
AE
c = = l . i x AE Lumole/ml.l
9.1 x 0.1
This value m u s t be multiplied by a factor if the sample h a s been deproteinized, neutralized o r otherwise
diluted.
T h e accuracy of the m e t h o d was determined with an L-glutamate solution (L-glutamic acid, A grade, from
Calbiochem, Lucerne, Switzerland). A 10 m M solution gave a m e a n value of 10.1 ^ m o l e / m l . with a s t a n d a r d
deviation of 0.27 /^mole/ml. T h e coefficient of variation is 2 . 7 % .
N o r m a l Values
See Table, p . 2285.
References
1 N. O. Kaplan, M. M. Ciotti & F. E. Stolzenbach, J. biol. C h e m . 221, 833 [1956].

2 H. Holzer & H. D. Soling, Biochem. Z. 336, 201 [1962].
3 / . M. Siegel, G. A. Montgomery & R. M. Bock, A r c h . Biochem. Biophys. 82, 288 [1959].
L-Glutamine
Determination with Glutamine Synthetase
Dieter Mecke
M o s t of the present m e t h o d s for the d e t e r m i n a t i o n of glutamine are based o n the estimation of g l u t a m a t e

(see p . 1704) o r N H 3 (see p . 1802) after preliminary hydrolysis by boiling with acid or t r e a t m e n t with
g l u t a m i n a s e . These m e t h o d s are n o t applicable when the sample c o n t a i n s large a m o u n t s of g l u t a m a t e
1
and N H 4 or amide c o m p o u n d s which are readily hydrolysed. G l u t a m i n e can be determined by c h r o m a t o

graphic a n a l y s i s or the L-isomer, specifically, by microbiological m e t h o d s . These m e t h o d s are time-
2 3
c o n s u m i n g a n d require relatively expensive equipment. T h e following is a rapid a n d specific colorimetric

assay for the determination of L-glutamine based on the glutamyl transferase reaction catalysed by glutamine
synthetase ( L - G l u t a m a t e : a m m o n i a ligase ( A D P - f o r m i n g ) , E C 6.3.1.2). T h e enzyme from E. coli h a s high
transferase activity a n d is therefore particularly s u i t a b l e . 4
Application of Method: In biochemistry a n d clinical chemistry.
Principle
Glutamine + N H O H 2 s
8
^ a
n
s
e
e ) y-Glutamylhydroxamate + N H 3
The y-glutamylhydroxamate formed in the reaction gives a coloured complex with F e 3 +

salts, the c o n
centration of which can be determined at 500 n m or 546 n m . U n d e r the conditions of the assay the
equilibrium of the reaction lies virtually completely in favour of the h y d r o x a m a t e derivate.
T h e p H o p t i m u m of the enzyme is p H 7.7. T h e assay should be carried out in tris or imidazole buffer;
high concentrations of p h o s p h a t e (above 10 m M ) interfere. Sufficient enzyme should be a d d e d so t h a t
the reaction is complete within 15 min. because the h y d r o x a m i c acid formed is slowly hydrolysed. T h e
assay mixture should contain a b o u t 2 U of enzyme (ca. 0.1 mg.).
Equipment
Spectrophotometer or spectrum-line photometer suitable for accurate measurements at

500 n m or 546 n m ; bench centrifuge.
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris, 6. F e r r i c c h l o r i d e , F e C l 3 • 6 H 0 , A . R.
2
A . R. 7. P o t a s s i u m c a r b o n a t e , A . R .
2. S o d i u m h y d r o x i d e , 2 N , A . R . 8. H y d r o x y l a m i n e h y d r o c h l o r i d e ( h y d r o x y l
3. H y d r o c h l o r i c a c i d , c o n e , A . R . a m m o n i u m chloride), N H O H • HC1, 2
4. D i s o d i u m h y d r o g e n a r s e n a t e , A . R.
N a H A s 0 - 7 H 0 , A . R.
2 4 2
9. T r i c h l o r o a c e t i c a c i d , A . R .
5. M a n g a n e s e s u l p h a t e , M n S 0 4 • H 0,2
10. P e r c h l o r i c a c i d , A . R . , 7 0 % (w/w), sp.
A . R. gr. 1.67
L-Glutamine 1717
II. Adenosine diphosphate, A D P 12. G l u t a m i n e synthetase

disodium salt, A D P - N a ; commercial p r e p a r a t -
2 from E. coli; ^ 20 U / m g . (25 ° C ) ; for p r e p a -
ion, see p . 525. ration, see A p p e n d i x p . 1719.
P r e p a r e all s o l u t i o n s w i t h d o u b l y d i s t i l l e d w a t e r .
I. Tris buffer ( 0 . 8 M ; p H 7 . 7 ) :
D i s s o l v e 9 7 g. tris in 8 0 0 m l . w a t e r , a d j u s t w i t h c o n e . H C 1 t o p H 7.7 a n d d i l u t e w i t h
water to 1000 ml.
II. D i s o d i u m h y d r o g e n a r s e n a t e ( 0 . 2 M ) :
D i s s o l v e 6.2 g. N a H A s 0
2 4 • 7 H 0 in 100 ml. water.
2
III. M a n g a n e s e s u l p h a t e ( 0 . 2 M ) :
D i s s o l v e 3 . 4 g. M n S 0 4 • H 0 in 1 0 0 m l . w a t e r .
2
I V . A s s a y m i x t u r e f o r 10 d e t e r m i n a t i o n s :
M i x 1 3 9 m g . N H O H • H C 1 , 1 2 m g . A D P - N a , 5 m l . s o l u t i o n 1,1 m l . s o l u t i o n II, 0 . 0 5 m l .
2 2
s o l u t i o n III a n d 1 m l . 2 N N a O H .
V . G l u t a m i n e s y n t h e t a s e (1 m g . p r o t e i n / m l . ; 2 0 U / m l . ) :
D i l u t e s t o c k s o l u t i o n w i t h 0.1 M tris buffer, p H 7.7.
VI. Ferric chloride reagent:
D i s s o l v e 5 g. F e C l 3 • 6 H 0 , a n d 10 g. t r i c h l o r o a c e t i c a c i d in w a t e r , a d d 2 5 m l . c o n e .
2
HC1 and dilute to 300 ml. with water.
Store all solutions, well stoppered, at 0 to 4 °C.

Solutions I to III a n d solution VI are stable indefinitely. Solution IV should be p r e p a r e d freshly each week.
Solution V is stable for at least 3 m o n t h s .
Procedure
G l u t a m i n e is e a s i l y h y d r o l y s e d in a q u e o u s s o l u t i o n , t h e r e f o r e s t o r e t h e s a m p l e s at 0 t o 4 ° C .
Immediately deproteinize samples containing protein with perchloric acid and neutralize
w i t h K C 0 . C a r e m u s t b e t a k e n t h a t t h e s a m p l e s are d i l u t e d a s little a s p o s s i b l e . T h e g l u t a m i n e
2 3
c o n t e n t o f s a m p l e s d e c r e a s e s b y a b o u t 5 % after 2 4 hr. a t 4 ° C .
Assay System
W a v e l e n g t h : 5 0 0 o r H g 5 4 6 n m ; light p a t h : 1 c m . ; i n c u b a t i o n v o l u m e : 2.0 m l . ; final v o l u m e :

4 . 0 m l . ; i n c u b a t i o n a t 37 ° C ( w a t e r b a t h ) ; m e a s u r e m e n t s at r o o m t e m p e r a t u r e . R e a d a g a i n s t
a blank containing water instead of sample.
Pipette into a centrifuge tube: C o n c e n t r a t i o n in a s s a y
Assay mixture (IV) 0.70 ml. 2 0 0 m M t r i s ; 100 m M NH OH;2
10 m M N a H A s 0 ; 1.5 m M
2 4
A D P ; 0.5 m M MnS0 4
Sample + water 1.20 m l . 0.1 t o 2.5 m M g l u t a m i n e

Glutamine synthetase (V) 0.10 ml. 50 /ig./ml. = l U / m l .
M i x , a l l o w t o i n c u b a t e f o r 3 0 m i n . at 37 ° C .
Ferric chloride reagent (VI) 2.00 ml.
M i x , c e n t r i f u g e , p o u r t h e s u p e r n a t a n t fluid i n t o a
cuvette and read extinction against blank.
It is r e c o m m e n d e d t o c h e c k p o s s i b l e i n t e r f e r e n c e in t h e a s s a y b y t h e a d d i t i o n o f a k n o w n
amount of glutamine to a sample.
Calculations
U n d e r the conditions of the assay the reaction is stoichiometric. 1 /imole of glutamine gives an extinction
compared to the blank of 0.129 at 500 n m or 0.109 at 546 nm. Hence for measurements at 500 n m the
glutamine concentration of the 4 ml. assay mixture is:
c = AE x 7.75 [/imole/ml.]
c = AE x 1133 [Mg./ml.]
Over the range of 0.5 to 5.0 /rniole glutamine/assay the mean s t a n d a r d deviation for several series of
measurements was 5 % . This gives a coefficient of variation of 3.4%.
N o r m a l Values
H u m a n serum contains 0.4 to 1 jimole g l u t a m i n e / m l . . T h e glutamine content of cerebral cortex is a b o u t

1
5 to 10 /miole/g. fresh w t . . 5
Effects of drugs and other therapeutic measures: N o effects k n o w n . A d d i t i o n of citrate, oxalate or fluoride
(all 10 m M ) to the sample does not affect the result.
Interference in the assay technique: L o w activity of the glutamine synthetase p r e p a r a t i o n results in t o o low
glutamine values. The samples should be read within at least 30 min. after addition of the F e C l reagent, 3
otherwise too low values will be obtained.

L-Glutamine 1719
Strongly coloured c o m p o u n d s , a n d substances which form complexes with iron, interfere in the deter
m i n a t i o n . Labile acyl c o m p o u n d s such as acyl p h o s p h a t e s , anhydrides a n d esters can simulate high
values.
Spe c ific ity
The enzymatic reaction is specific for L-glutamine. D - G l u t a m i n e does not interfere in the assay. T h e pre
sence of asparagine, g l u t a m a t e , a s p a r t a t e a n d N H 4 also have n o effect.
Appendix
Glutamine Synthetase from E. coli 6
The enzyme can be obtained in high yield from cells of E. coli which have been grown in an a m m o n i a -
free culture m e d i u m . A m e d i u m of the following c o m p o s i t i o n is suitable: 136 g. K H P 0 ; 35 g. K O H ;
2 4
20 g. N a g l u t a m a t e ; 20 g. glycerol; 2 g. M g S 0 4 and 5 mg. F e S 0 4 in 10 1. water.

The purification p r o c e d u r e includes the following steps: disintegration of the cells a n d extraction with
water, precipitation of nucleic acids with streptomycin sulphate (100 m g . streptomycin sulphate/g. wet
wt. cells), precipitation of inactive protein at p H 5.0 a n d finally precipitation of the enzyme at p H 4.4.
T h e active precipitate is dissolved in 0.1 M tris buffer ( p H 7.7) a n d after a d d i t i o n of 1 m M M g S 0 is heated 4
for 10 min. at 60 °C.

If necessary, the enzyme can be obtained in a m o r e concentrated state by precipitation with acetone to
4 0 % (v/v) a n d dissolving the precipitate in a small a m o u n t of tris buffer.
References
1 H. Kluge, H. Hermann & V. Wieczorek, Z. klin. C h e m . u. klin. Biochem. 5, 86 [1967].

2 J. V. Benson, jr., M. J. Gordon & J. A. Patterson, A n a l . Biochem. 18, 228 [1967].
3 J. A. Roper & H. Mcllwain, Biochem. J. 42, 485 [1948].
4 C. Gancedo & H. Holzer, E u r o p e a n J. Biochem. 4, 190 [1968].
5 S. Berl&J. G. McMurtry, Arch. Biochem. Biophys. 118, 645 [1967].
6 D. Mecke, K. Wulff, K. Liess & H. Holzer, Biochem. Biophys. Res. C o m m u n . 24, 452 [1966].
Determination with Glutaminase and Glutamate Dehydrogenase

Patricia L u n d
L-Glutamine is formed in m a n y tissues from L-glutamate, a m m o n i a a n d A T P by the enzyme glutamine

synthetase ( L - G l u t a m a t e : a m m o n i a ligase, A D P - f o r m i n g , E C 6.3.1.2). It is also a p r o d u c t of protein
degradation. T h e c o n c e n t r a t i o n of glutamine in m a m m a l i a n blood is higher t h a n that of any other a m i n o
acid. It is usually determined by m e a s u r e m e n t of g l u t a m a t e or a m m o n i a before a n d after acid hydrolysis o r 1
enzymatic h y d r o l y s i s ' . Acid hydrolysis is not specific. M a n y other tissue constituents yield a m m o n i a
2 3
on acid hydrolysis, others, for example glutathione, give g l u t a m a t e . Therefore after acid hydrolysis values
which are t o o high are obtained regardless of whether g l u t a m a t e or a m m o n i a is determined.
Enzymatic hydrolysis with purified glutaminase ( L - G l u t a m i n e a m i d o h y d r o l a s e , EC 3.5.1.2) from E. coli
followed by s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of g l u t a m a t e (see p . 1704) has the a d v a n t a g e of simplicity
a n d specificity.
Application of Method: In biochemistry a n d in clinical chemistry.

Principle
(1) L-Glutamine + H 0 2
g l u t a m i n a s e
> L-Glutamate + NH 3
I 1
(2) L-Glutamate + H 0 + N A D 2
+
^ ^ e l e » 2-Oxoglutarate + N A D H + N H 4
+
Reaction (1) is allowed to reach completion, before a p o r t i o n of the reaction mixture is taken for the
glutamate assay.
G l u t a m i n a s e from E. coli has its p H o p t i m u m at p H 5.0; therefore the hydrolysis is carried o u t in acetate
buffer at this p H .
Equipment
3 3 4 o r 3 6 5 n m ; f r o z e n - s t o p e q u i p m e n t ( s e e p. 4 0 0 ) ; c o n s t a n t t e m p e r a t u r e w a t e r b a t h at
3 7 - 4 0 °C.
Reagents
1. S o d i u m a c e t a t e , C H C O O N a - 3 H 0 , A . R .
3 2 5. P e r c h l o r i c a c i d , A . R . , 6 0 % ( w / w ) , s p . g r .
2. A c e t i c a c i d , A . R., c a . 9 9 % ( w / w ) 1.54
3. L - G l u t a m i n e 6. P o t a s s i u m h y d r o x i d e , A . R . , 3 0 % ( w / v )
4. Glutaminase
from E. coli, lyophilized p o w d e r , ca. 4.0 U / m g .
(37 °C). C o m m e r c i a l p r e p a r a t i o n , see p . 465.
Purity of Reagents
T h e glutaminase should be free from g l u t a m a t e decarboxylase. This should be checked with each new
batch of enzyme by testing the recovery of g l u t a m a t e after a one h o u r incubation at 37 °C u n d e r the
conditions given for hydrolysis of glutamine. C o n t a m i n a t e d p r e p a r a t i o n s of glutaminase can be used if
hydroxylamine, a powerful inhibitor of g l u t a m a t e decarboxylase, is included (final concentration 2 m M )
in the incubation during glutamine hydrolysis.
I. A c e t a t e buffer ( 0 . 5 M ; p H 5 . 0 ) :
a) D i s s o l v e 6.8 g. C H C O O N a - 3 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
3 2
b ) D i l u t e 2 . 9 m l . a c e t i c a c i d t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r . M i x 6 7 . 8 m l . a) w i t h 3 2 . 2 m l . b ) .
II. L - G l u t a m i n e s t a n d a r d s o l u t i o n ( 1 0 m M ) :
D i s s o l v e 1 4 . 6 m g . L - g l u t a m i n e in d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
III. G l u t a m i n a s e ( 0 . 5 m g . p r o t e i n / m l . ) :
D i s s o l v e 0.5 m g . g l u t a m i n a s e p o w d e r in 1 m l . d i l u t e a c e t a t e buffer p H 5 ; this a m o u n t is
sufficient f o r 18 a s s a y s .
I V . P e r c h l o r i c a c i d ( 1 0 % w / w ; c a . 1.52 M ) :
D i l u t e 10 m l . 6 0 % p e r c h l o r i c a c i d t o 6 0 m l . w i t h d i s t i l l e d w a t e r .
L-Glutamine 1721
Store L-glutamine solution (II) at - 1 5 °C a n d acetate buffer (I) in a refrigerator (ca. 4 °C). T h e glutaminase
solution (III) can also be stored at - 1 5 °C.
Procedure
Collect b l o o d without venestasis into a heparinized syringe. Collect tissues o f laboratory ani
m a l s with quick-freeze c l a m p s (see "Cell a n d Tissue D i s i n t e g r a t i o n " p. 400).
Deproteinization:
Pipette a v o l u m e of 1 0 % perchloric acid (IV) approximately equal to the b l o o d sample into

a centrifuge tube and w e i g h tube. A d d b l o o d and determine weight of b l o o d . M i x t h o r o u g h l y
a n d c e n t r i f u g e for 10 m i n . at 3 0 0 0 g. N e u t r a l i z e a p o r t i o n o f t h e s u p e r n a t a n t fluid w i t h 3 0 %
K O H ( u n i v e r s a l i n d i c a t o r ) . A l l o w t o s t a n d for 10 m i n . in a n ice b a t h a n d t h e n c e n t r i f u g e
for 10 m i n . at 3 0 0 0 g. M e a s u r e t h e i n c r e a s e in v o l u m e o n n e u t r a l i z a t i o n . Treat s e r u m in t h e
same way. T h e m e t h o d for preparation o f tissue extracts has been described by Williamson
et a l . .
4
G l u t a m i n e is s t a b l e in n e u t r a l s o l u t i o n at —15 ° C . T o a v o i d t h e p o s s i b i l i t y o f h y d r o l y s i s o f
glutamine exposure o f s a m p l e t o acid or alkaline c o n d i t i o n s s h o u l d be reduced to a m i n i m u m .
I n c u b a t i o n v o l u m e : 1.0 m l . ; i n c u b a t i o n t e m p e r a t u r e : 3 7 - 4 0 ° C .
Pipette into small stoppered C o n c e n t r a t i o n in h y d r o

Blank Standard Test
tubes : lysis mixture
A c e t a t e buffer* (I) 0.50 ml. 0.50 ml. 0.50 ml. 250 m M

Glutaminase solution (III) 0.10 ml. 0.10 ml. 0.10 ml. 0.05 mg. protein/ml.
L-Glutamine standard
solution (II) — 0.05 ml. — 0.5 m M
Sample — — 0.10 to
0.40 ml. 0.2-0.4 mM
Distilled water 0.40 ml. 0.35 ml. t o 1.0 m l .
M i x , i n c u b a t e for 1 hr. a n d t h e n c o o l t u b e s in a n ice b a t h .

T a k e 0.5 m l . o f t h e B l a n k a n d Test, a n d 0 .2 m l . o f S t a n d a r d
( c o r r e s p o n d i n g t o 0.1 / z m o l e ) for t h e g l u t a m a t e a s s a y (see
p . 1 7 0 4 ) . It is n o t n e c e s s a r y t o d e p r o t e i n i z e t h e s a m p l e s b e f o r e
the glutamate assay.
* 0.1 ml. of the acetate buffer should be replaced by 0.1 ml. 20 m M h y d r o x y l a m i n e solution if the gluta
minase p r e p a r a t i o n is c o n t a m i n a t e d with g l u t a m a t e decarboxylase. See 'Purity of R e a g e n t s ' .
Enzymatic Determination of Glutamate
S e e C h a p t e r " G l u t a m a t e " , p. 1 7 0 4 . A n a l y s e a s e c o n d test 2 w h i c h h a s n o t b e e n h y d r o l y s e d

t o o b t a i n t h e initial g l u t a m a t e c o n t e n t o f t h e s a m p l e . It is r e c o m m e n d e d t o carry o u t t h e g l u t a
m a t e a s s a y in t h e p r e s e n c e o f 0.3 m M A D P ; t h e latter a c t i v a t e s G I D H 5
and decreases the
reaction time.
Calculations
See C h a p t e r on " G l u t a m a t e " , p . 1704. T h e value obtained for the total g l u t a m a t e plus glutamine after
hydrolysis must be multiplied by 2 because only 5 0 % of the sample is t a k e n for the glutamate assay.
Subtract the value for test 2 from test 1; the difference is the glutamine content of the sample.
N o r m a l V a l u e s , A c c u r a c y and P r e c i s i o n
T h e following values were obtained in fed rats (mean + s t a n d a r d deviation, n u m b e r of m e a s u r e m e n t s

in brackets): arterial b l o o d : 0.57 ± 0.03 /imole/ml. (6); liver: 4.70 ± 0.93 /miole/g. (14); b r a i n : 4.51 ±
± 0 . 7 1 /miole/g. (6). T h e coefficient of variation is ca. 3.9%.
References
1 H. B. Vickery, G. R. Pucher, H. E. Clark, A. C Chibnall & R. G. Westall, Biochem. J. 29, 2710 [1935].
2 H. A. Krebs, Biochem. J. 43, 51 [1948].
3 L. Goldstein, Amer. J. Physiol. 210, 661 [1966].
4 D. H. Williamson, P. Lund & H. A. Krebs, Biochem. J. 103, 514 [1967].
5 G. M. Tomkins, K. L. Yielding, N. Talal & J. F. Curran, Cold Spring H a r b o u r Symp. q u a n t . Biol. 28,
461 [1963].
L- Hydroxyproline
C a r m e n L. R o s a n o
Hydroxyproline is an a m i n o acid which is found virtually exclusively in collagen; occasionally small

a m o u n t s of free hydroxyproline occur in blood a n d urine. T h e major p r o p o r t i o n of hydroxyproline is
peptide or polypeptide b o u n d .
F o r the enzymatic determination of hydroxyproline an enzyme mixture is used which specifically converts
the cyclic imino acid hydroxyproline to the nitrogen-containing c o m p o u n d s , a m m o n i a (ca. 95%) a n d
glutamic acid. The d e g r a d a t i o n of hydroxyproline by enzymes from Pseudomonas fluorescens may be
similar to t h a t with enzymes from Ps. striata .
1
Application of Method: In clinical biochemistry, biochemistry a n d in foodstuff chemistry.
Principle
(1) L-Hydroxyproline , HYDROXYPROLINE

~ ~ ^ D-Allohydroxyproline
2
epimerase*
i
(2) D-Allohydroxyproline ^ / / ^ ^ ^ e ^ ' zP-PyiToHne-4-hydroxy-2-carboxylate
^ (unknown)
(3) Pyrroline-4-hydroxy-2-carboxylate—> —> —>
(L-Glutamate _ = i l ^ i £ = = ± 2-Oxoglutarate + NH ) 3
dehydrogenase
T h e incubation is carried out at r o o m t e m p e r a t u r e u n d e r microaerophilic conditions. After the enzymatic

reaction a lower intensity of colour with the ninhydrin reaction modified according to Piez et a l . is obtained 2
than with an untreated sample. T h e difference in extinction at 350 n m is p r o p o r t i o n a l to the a m o u n t of

hydroxyproline present.
T h e m e t h o d with acid ninhydrin described by Piez et al. has been modified; instead of a 2 hr. incubation
2
at r o o m t e m p e r a t u r e 30 min. at 37 °C is used.
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for p r e c i s e m e a s u r e m e n t s at 3 5 0 n m ( e . g . B e c k m a n D U ) ; l a b o r
a t o r y a u t o c l a v e ; 37 ° C w a t e r b a t h .
Reagents
1. H y d r o c h l o r i c a c i d , A . R., ca. 3 7 % w / w , 2. P o t a s s i u m d i h y d r o g e n p h o s p h a t e ,
s p . g r . 1.185 KH P0 ,2 4 A.R.
* E C 5.1.1.8.
** N o E C n u m b e r .
3. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , 7. A c e t i c a c i d , A . R .
K HP0 ,
2 4 A.R. 8. NoriteA
4. Hydroxyproline 9. E n z y m e e x t r a c t f r o m Ps.fluorescens
5. P h e n a z i n e m e t h o s u l p h a t e , P M S F o r p r e p a r a t i o n , see p. 1726.
6. 1 , 2 , 3 - T r i k e t o h y d r i n d e n e , n i n h y d r i n
P r e p a r a t i o n of S o l u t i o n s
I. P o t a s s i u m p h o s p h a t e buffer ( 0 . 2 5 M ; p H 7 . 8 ) :
D i s s o l v e 0.341 g. K H P 0 2 4 i n d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l . a n d d i s s o l v e 4 . 3 6 g.
K HP02 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l . M i x 9 2 m l . K H P 0 2 4 solution with
8 ml. K H P 0 2 4 solution.
II. N i n h y d r i n r e a g e n t ( 0 . 1 5 % w / v ; 8.4 m M ) :
D i s s o l v e 1 5 0 m g . n i n h y d r i n in 100 m l . a c e t i c a c i d .
III. P h e n a z i n e m e t h o s u l p h a t e ( 3 . 2 7 m M ) :
D i s s o l v e 10 m g . P M S in 10 m l . d i s t i l l e d w a t e r .
IV. E n z y m e extract (ca. 5 m g . p r o t e i n / m l . ) :
A d d e n z y m e extract undiluted to the assay.
V. Hydroxyproline standard (1.0 m M ) :
D i s s o l v e 13.1 m g . h y d r o x y p r o l i n e in 1 0 0 m l . distilled w a t e r .
Store solution I a n d V, stoppered, in a refrigerator at 0 - 4 ° C ; they are stable indefinitely. Store solutions
III and IV at —10 ° C ; solution III is stable for ca. 14 days, and solution IV for at least 3 m o n t h s . Solution II
must be prepared freshly each day.
Procedure
S a m p l e s o f differing p u r i t y c a n b e a n a l y s e d . If h y d r o x y p r o l i n e is t o b e d e t e r m i n e d in u r i n e ,
c o l l e c t the 2 4 hr. u r i n e u n d e r a l a y e r o f t o l u e n e t o a v o i d b a c t e r i a l d e c o m p o s i t i o n .
Hydrolysis:
F o r t h e d e t e r m i n a t i o n o f p e p t i d e - b o u n d h y d r o x y p r o l i n e (in u r i n e 9 0 % o f t h e t o t a l a m o u n t )
h y d r o l y s i s is n e c e s s a r y . T h e m e t h o d for u r i n e is g i v e n b e l o w . A d d 5 m l . 12 N H C 1 (ca. 3 7 %
w / w ) t o 5 m l . u r i n e , k e e p for 3.5 hr. u n d e r 1 a t m . in a n a u t o c l a v e . A d d 0.5 g. N o r i t e A t o 5 m l .
o f t h e h y d r o l y s e d m i x t u r e t o r e m o v e c h a r r e d p r o d u c t s . S h a k e t h e m i x t u r e a n d filter t h r o u g h
W h a t m a n filter p a p e r N o . 4 0 . E v a p o r a t e 3 m l . o f t h e filtrate t o d r y n e s s at 4 0 ° C , t a k e u p t h e
r e s i d u e in 3 m l . w a t e r a n d b r i n g t o d r y n e s s a g a i n t o r e m o v e t h e e x c e s s H C 1 . D i s s o l v e t h e d r y
r e s i d u e in 3 . 0 m l . p h o s p h a t e buffer (I). U n d e r n o r m a l c o n d i t i o n s t a k e 0 . 1 - 0 . 2 m l . f o r t h e a s s a y .
L-Hydroxyproline 1725
Assay System
Wavelength: 350 n m ; light p a t h : 1 c m . ; incubation v o l u m e : 0.56 m l . ; r o o m temperature;

assay v o l u m e : 3.56 ml.
P r e p a r e s t a n d a r d s w i t h h y d r o x y p r o l i n e s t a n d a r d s o l u t i o n ( V ) i n s t e a d o f s a m p l e . F o r e a c h test
and standard prepare a blank (containing water instead of enzyme), and also t w o reagent
blanks (no sample, with and without enzyme).
P i p e t t e i n t o 12 m l . c e n t r i f u g e t u b e s C o n c e n t r a t i o n in assay mixture
Sample up to 0.2 ml.

P h o s p h a t e buffer (I) t o 0.5 ml. 223 m M
P M S solution (III) 0.01 m l . 58 pM
E n z y m e extract (IV) or water 0.05 ml. 4 4 7 fig. protein/ml.
I n c u b a t e t h e s o l u t i o n w i t h v i g o r o u s s h a k i n g for 9 0 m i n .
at r o o m t e m p e r a t u r e .
Ninhydrin solution (II) 3.0 ml. 7.0 m M
Mix vigorously, centrifuge the tubes containing the

e n z y m e for 10 m i n . at 3 0 0 0 r p m a n d p o u r the super
n a t a n t fluid i n t o c l e a n t u b e s . I n c u b a t e all s o l u t i o n s f o r
3 0 m i n . at 37 ° C . R e a d t h e e x t i n c t i o n s a g a i n s t t h e c o r
responding reagent blanks.
Calculate the differences E b l a n k - E t e s t or E b l a n k - E s t a n d a r d . T h e s e v a l u e s are u s e d for t h e

calculations.
Calculations
Plot the extinction differences of the s t a n d a r d s (ordinate) against the a m o u n t s of hydroxyproline (abscissa).
T h e s t a n d a r d curve is linear for 1 - 1 0 ug. hydroxyproline/assay. O b t a i n the a m o u n t s of h y d r o x y p r o l i n e
corresponding to the corrected values from the s t a n d a r d curve.
W i t h a m e a n value of 19 mg. total hydroxyproline in 24 hr. urine a s t a n d a r d deviation of 0.8 mg. was
found. T h e coefficient of variation is 4 . 2 % .
N o r m a l Values
T h e total a m o u n t of free a n d b o u n d hydroxyproline in 24 hr. urine varies between 10 a n d 28 mg.
Intake of foodstuffs containing gelatine should be avoided before the collection of urine.
The natural imino acids proline, DL-pipecolic acid a n d 5-hydroxypipecolic acid d o not affect the reaction of
the enzyme extract from Ps. fluoresceins with hydroxyproline.
Appendix
Enzyme extract from Ps.fluorescens
Inoculate 1.8 1. of minimal m e d i u m with 200 ml. of an overnight culture of Ps fluoresceins on minimal
m e d i u m ( 0 . 1 % hydroxyproline, 0 . 1 % M g S 0 - 7 H 0 , 0.2% K H P 0
4 2 2 4 ; p H 7.8). I n c u b a t e at 30 °C until the
cell count reaches approximately 3 x 1 0 / m l . Centrifuge at 6000 r p m for 20 min., wash the cell paste
8
three times with 1% KC1 a n d then suspend in 10 ml. tris buffer (10 m M , p H 7.8; 6 m M m e r c a p t o e t h a n o l ) .
Disintegrate cells in a F r e n c h press at 680 a t m . a n d remove cell debris by centrifugation at 7000 g for
30 min. Clarify s u p e r n a t a n t fluid by centrifugation for 30 min. at 25 000 g. Adjust the enzyme extract with
tris buffer (10 m M , p H 7.8; 30% glycerol) to 5 mg. protein/ml. Store at - 1 0 °C.
References
1 E. Adams, J. biol. C h e m . 234, 2073 [1959].

2 K. A. Piez, R. Irrevere & H. L. Wolff, J. biol. C h e m . 223, 687 [1956].
DL-Serine and DL-Threonine
Serine a n d t h r e o n i n e are usually determined by chemical m e t h o d s involving c h r o m a t o g r a p h i c separation

followed by t r e a t m e n t with ninhydrin. T h e enzymatic p r o c e d u r e described here is a rapid a n d specific
m e t h o d for o b t a i n i n g the sum of serine plus t h r e o n i n e . 1
Principle
(la) Serine p e r i o d a t e
> Glyoxylate + Formaldehyde
(lb) Threonine p e r i o d a t e
> Glyoxylate + Acetaldehyde
(2) Glyoxylate + N A D H + H +
Glycollate + N A D +
Serine a n d t h r e o n i n e are oxidized by p e r i o d a t e at neutral p H t o give glyoxylate a n d the c o r r e s p o n d i n g

aldehyde. After removal of excess p e r i o d a t e with glycerol, the glyoxylate is reduced t o glycollate with
N A D H a n d lactate d e h y d r o g e n a s e , L D H (L-Lactate: N A D oxidoreductase, E C 1.1.1.27). T h e decrease
in extinction at 340 (334, 365) n m d u e to the oxidation of N A D H is p r o p o r t i o n a l to the a m o u n t of serine
(threonine) present.
A l t h o u g h glyoxylate is oxidized by p e r i o d a t e to formic acid a n d C 0 , u n d e r the conditions described

2
below this reaction is relatively slow a n d recovery of serine a n d threonine is consistently 8 8 - 9 0 % , providing
t h a t the time intervals are strictly a d h e r e d t o .
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r f o r a c c u r a t e m e a s u r e m e n t s at 3 4 0 , 3 3 4 o r
365 n m ; c h r o m a t o g r a p h y c o l u m n s (1 c m . x 10 c m . ) .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 9. D L - S e r i n e , A . R.
K H P 0 , A . R.
2 4 10. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o
2. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , tide, N A D H
K H P 0 , A . R.
2 4 d i s o d i u m salt, N A D H - N a 2 ; commercial p r e p
3. P e r c h l o r i c acid, A. R., sp. gr. 1.54; a r a t i o n , see p . 545.
ca. 6 0 % (w/w). 11. Lactate d e h y d r o g e n a s e , L D H
4. A m m o n i a s o l u t i o n , A . R . , s p . gr. 0 . 8 8 . from rabbit m u s c l e ; crystalline suspension in
5. P o t a s s i u m h y d r o x i d e , K O H , A . R . 3.2 M a m m o n i u m sulphate s o l u t i o n ; ^360
6. A m b e r l i t e I R - 1 2 0 ( H ) , A . R.
+
U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , see
7. S o d i u m m e t a p e r i o d a t e , N a I 0 , A . R.
4 p. 481.
8. G l y c e r o l , A . R .
Purity of Reagents
T h e L D H should n o t contain appreciable a m o u n t s of A D H .
P r e p a r e all s o l u t i o n s w i t h d o u b l e d i s t i l l e d o r d e i o n i z e d w a t e r .
I. P h o s p h a t e buffer (0.1 M ; p H 6 . 8 ) :
a) D i s s o l v e 1 3 . 6 g. K H P 0 2 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r .
b ) D i s s o l v e 1 7 . 4 g. K H P 0
2 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r .
M i x s o l u t i o n s a) a n d b ) in t h e r a t i o o f 39 : 61 p a r t s b y v o l u m e . C h e c k t h e p H w i t h a
glass electrode.
II. P e r c h l o r i c a c i d (ca. 3 0 % w / v ) :
Dilute 40 ml. 6 0 % H C 1 0 4 t o 120 ml. with distilled water.
III. P o t a s s i u m h y d r o x i d e (ca. 2 0 % w / v ) :
D i s s o l v e 2 0 g. K O H in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
IV. A q u e o u s a m m o n i a (ca. 2 N ) :
D i l u t e 2 0 m l . a m m o n i a s o l u t i o n ( s p . gr. 0 . 8 8 ) t o 1 2 0 m l . w i t h d i s t i l l e d w a t e r .
V . G l y c e r o l (1 M ) :
D i s s o l v e 0 . 9 2 g. g l y c e r o l in d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
VI. S o d i u m m e t a p e r i o d a t e (ca. 0.2 M ) :
D i s s o l v e 0 . 4 2 6 g. N a I 0 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
VII. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 6 m M j ? - N A D H ) :
VIII. DL-Serine standard solution (1.0 m M ) :
D i s s o l v e 10.5 m g . D L - s e r i n e in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
I X . L a c t a t e d e h y d r o g e n a s e , L D H (5 m g . p r o t e i n / m l . ) :
U s e t h e s t o c k s u s p e n s i o n in 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
P r e p a r e the s o d i u m m e t a p e r i o d a t e solution (VI) freshly each day. Store the N A D H solution at - 1 5 °C.
T h e L D H suspension is stable for m o n t h s at 0 - 4 °C.
Procedure
Collection, Treatment and Stability of the Sample
Collection:
T h i s m e t h o d h a s o n l y b e e n t e s t e d o n p u r e s o l u t i o n s a n d o n e x t r a c t s o f liver t i s s u e p r e p a r e d
as d e s c r i b e d b e l o w . O b t a i n t i s s u e s f r o m e x p e r i m e n t a l a n i m a l s w i t h freeze c l a m p s (refer t o
p. 4 0 0 ) .
Deproteinization :
P u l v e r i z e f r o z e n liver in a m o r t a r t o a fine p o w d e r , w i t h f r e q u e n t a d d i t i o n s o f l i q u i d N .2
Transfer t h e p o w d e r ( 1 - 2 g.) t o a w e i g h e d p l a s t i c c e n t r i f u g e t u b e c o n t a i n i n g 2 m l . o f f r o z e n
DL-Serine and DL-Threonine 1729
3 0 % perchloric acid solution (II), care being taken n o t t o allow t h a w i n g t o occur. A d d 5 ml.
i c e - c o l d d i s t i l l e d w a t e r a n d i m m e d i a t e l y h o m o g e n i z e t h e m i x t u r e in t h e c e n t r i f u g e t u b e w i t h
a g l a s s p e s t l e d r i v e n b y a l o w - s p e e d m o t o r . R e m o v e p r o t e i n b y c e n t r i f u g a t i o n i n t h e c o l d at
3 0 0 0 g f o r 10 m i n . M e a s u r e v o l u m e o f t h e s u p e r n a t a n t fluid a n d a d j u s t t o p H 5 - 6 w i t h K O H
s o l u t i o n (III). A l l o w t o stand for 30 m i n . in a n ice b a t h a n d then centrifuge off t h e precipitate
o f K C 1 0 . M e a s u r e t h e v o l u m e o f s u p e r n a t a n t fluid a n d u s e f o r i s o l a t i o n o f t h e a m i n o a c i d
4
fraction.
Isolation of amino acid fraction:
T h e e n z y m a t i c m e t h o d f o r t h e d e t e r m i n a t i o n o f serine p l u s t h r e o n i n e r e q u i r e s t h e i r p r e l i m i n
ary s e p a r a t i o n f r o m i n t e r f e r i n g s u b s t a n c e s . A b s o r b 5 m l . o f t h e n e u t r a l , d e p r o t e i n i z e d liver
e x t r a c t o n a c o l u m n (1 c m . x 5 c m . ) o f A m b e r l i t e I R - 1 2 0 ( H ) . W a s h t h e c o l u m n w e l l w i t h
+
distilled water a n d elute the a m i n o acids with 2 0 m l . a q u e o u s a m m o n i a solution (IV). Take

t h e e l u a t e t o d r y n e s s in vacuo at 5 0 ° C , d i s s o l v e r e s i d u e i n d i s t i l l e d w a t e r a n d u s e d i r e c t l y f o r
the d e t e r m i n a t i o n o f serine plus threonine.
S e r i n e a n d t h r e o n i n e a r e s t a b l e p r o v i d i n g b a c t e r i a l c o n t a m i n a t i o n is a v o i d e d .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 3 . 1 9 m l . ; m e a s u r e
a g a i n s t a c u v e t t e c o n t a i n i n g d i s t i l l e d w a t e r . A s e r i n e s t a n d a r d (0.1 m l . s o l u t i o n V I I I ; 0.1 / x m o l e )
m u s t b e carried t h r o u g h t h e p r o c e d u r e w i t h e a c h series o f m e a s u r e m e n t s t o c h e c k t h e r e c o v e r y
of glyoxylate.
Pipette into cuvettes: Concentration in assay mixture
A m i n o acid sample 2.0 m l . 1 0 - 1 0 0 pM s e r i n e p l u s t h r e o n i n e

P h o s p h a t e buffer s o l u t i o n (I) 1.0 m l . 33 m M
Sodium metaperiodate solution (VI) 0.020 ml. c a . 1.3 m M
Exactly 5 m i n . later m i x i n
Glycerol solution (V) 0.050 ml. c a . 16 m M
Exactly 10 m i n . later m i x i n
N A D H solution (VII) 0.1 m l . ca. 0.2 m M
R e a d t h e initial e x t i n c t i o n E x a n d m i x in
L D H suspension (IX) 0.020 ml. c a . 3 0 pg. p r o t e i n / m l . c a . 2 . 2 U / m l .
R e a d extinction at 20, 30 a n d 4 0 m i n . ; extrapolate

to determine extinction E . E 2 t —E 2 = AE is u s e d
used for the calculations.
T h e e x t i n c t i o n c h a n g e s o c c u r r i n g o n a d d i t i o n o f L D H s u s p e n s i o n ( I X ) are m e a s u r e d b y a d d i t i o n
of 0.02 ml. L D H to a cuvette containing water instead o f sample.
Calculations
U n d e r the conditions of the assay the reduction of glyoxylate is quantitative with the stoichiometric
formation of an equivalent a m o u n t of N A D . T h e calculation formula ( 2 ) on p . 312 applies. A correction
must be applied for the recovery of the serine s t a n d a r d in the assay. T h e results are o b t a i n e d in pmole
serine plus t h r e o n i n e / m l . sample. This value m u s t b e multiplied by the a p p r o p r i a t e dilution factor F d u e
t o the preliminary t r e a t m e n t of the sample.
The percentage recovery of serine ( 0 . 5 - 1 . 5 //mole) a d d e d to frozen liver p o w d e r was 96 + 11 (S. D . ) % .

The coefficient of variation is 5 % .
N o r m a l Values
Values for serine plus t h r e o n i n e for livers from fed rats are 1.27 + 0.34 ^ m o l e / g . fresh wt. of liver; starved
(48 hr.) rats, 0.93 ± 0.27; alloxan-diabetic rats, 0.49 ± 0 . 1 3 . 1
Interference in the assay: Failure t o r e m o v e excess m e t a p e r i o d a t e with glycerol can lead t o destruction
of N A D H . This is shown by a slow decrease in extinction before addition of L D H .
Preliminary isolation of the a m i n o acid fraction confers specificity t o the m e t h o d , because n o other a m i n o
acids apart from serine a n d t h r e o n i n e yield glyoxylate on periodate oxidation. T h e m e t h o d does n o t differ
entiate between the stereoisomers of the two a m i n o acids.
Other Methods of Measurement
Theoretically it should be possible t o differentiate t h r e o n i n e from serine by subsequent d e t e r m i n a t i o n of

the acetaldehyde formed (equation l b ) from the former with alcohol d e h y d r o g e n a s e (see p . 1506).
Serine (threonine) d e h y d r a t a s e (L-serine hydrolase (deaminating) E C 4.2.1.13) catalyses the dehydrative
d e a m i n a t i o n of serine a n d t h r e o n i n e t o give the c o r r e s p o n d i n g 2-oxo acid, p y r u v a t e a n d 2 - o x o b u t y r a t e .
2
T h e keto acids can be d e t e r m i n e d by reduction with L D H a n d N A D H .
References

2 A. S. M. Selim & D. M. Greenberg, J. biol. C h e m . 234, 1474 [1959].
3-Hydroxykynurenine
Helmut Schievelbein and Karin Loschenkohl
3-Hydroxykynurenine ( 3 - O H - K ) is an intermediate in the catabolism of t r y p t o p h a n a n d is excreted in

h u m a n urine. T h e present m e t h o d s for its d e t e r m i n a t i o n give variable recoveries; they depend on ex
traction of urine with organic solvents, separation by c o l u m n or thin-layer c h r o m a t o g r a p h y , elution a n d
colorimetric o r fluorimetric assay.
In the enzymatic m e t h o d described here 3-hydroxykynurenine ( 3 - O H - K ) is converted to 3-hydroxy-
anthranilic acid a n d alanine with kynureninase (L-Kynurenine hydrolase, E C 3.7.1.3). 3-Hydroxyanthranilic
acid (3-OH-A) is converted to 2-amino-3-carboxymuconate semialdehyde with 3-hydroxyanthranilic acid
oxidase ( 3 - H y d r o x y a n t h r a n i l a t e : oxygen 3,4-oxidoreductase, decyclizing, E C 1.13.11.6). T h e principle of
the two m e t h o d s has been used by Wiss et a l . ' for m e a s u r e m e n t s of the activity of 3-OH-A oxidase a n d
1 2
kynureninase.
Application of Method: In clinical chemistry a n d biochemistry.
Principle
^ C O O H
CI NH 2
+ H 0
2 kynuren.nase^ ^ T
NH 2
+ CH3-CH-COOH
NH 2
OH OH
(3 - H y d r o x y k y n u r e n i n e ) (3-Hydroxy (Alanine)
anthranilic acid)
.COOH COOH
+ 2 0 2 + 2 H 02 + 2 H 0 2 2
NH 2
H O COOH
OH
(3-Hydroxyanthranilic acid) (2 - A m i n o - 3 - c a r b o x y -
muconate semialdehyde)
The p r o d u c t can be determined by its a b s o r p t i o n m a x i m u m at 360 n m . T h e present m e t h o d is based on

the determination of 3 - O H - A ; the d e t e r m i n a t i o n of 3 - O H - K was first described by us in 1968 .
3 4
T h e conditions described for the m e t h o d are o p t i m u m . See also " 3 - H y d r o x y a n t h r a n i l i c acid", p . 1736.
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for m e a s u r e m e n t s at 3 6 0 n m . P h o t o m e t e r s w i t h filters at 365 n m

are a l s o s u i t a b l e p r o v i d e d t h a t it is p o s s i b l e t o e x p a n d t h e s c a l e t o o b t a i n e x a c t r e a d i n g s
b e c a u s e the i n c r e a s e s in e x t i n c t i o n m a y b e s m a l l . A n i n s t r u m e n t w i t h a c o n s t a n t t e m p e r a t u r e
c u v e t t e h o l d e r is p r e f e r a b l e . A t h e r m o s t a t i c a l l y - c o n t r o l l e d w a t e r b a t h e q u i p p e d for s h a k i n g
a n d a p H m e t e r are a l s o r e q u i r e d .
Reagents
1. D i s o d i u m h y d r o g e n p h o s p h a t e , Weber & Wiss . See appendix p . 1735. O u r lyo

1
Na HP0 -2H 0
2 4 2
philized p r e p a r a t i o n s h a d a specific activity with
2. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , kynurenine as substrate of60.74 //U/mg. (25 °C).
According to the activity of the p r e p a r a t i o n 5-15
KH P0 2 4
mg. enzyme are required for each assay and this

3. 3 - H y d r o x y k y n u r e n i n e , 3 - O H - K
is a d d e d as solid to the assay mixture.
e. g. from C a l b i o c h e m , Los Angeles, U S A
11. 3-Hydroxyanthranilic acid oxidase,
4. Pyridoxal-5 '-phosphate, P L P
3-OH-A oxidase
5. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris
purified from pig liver as described by Weber &
6. F e r r o u s a m m o n i u m s u l p h a t e ,
Wiss .1
Isolation, see p . 1739. O u r p r e p a r a t i o n s
F e S 0 ( N H ) S 0 - 6 H 0 , A . R.
4 4 2 4 2
had specific activities of 1 9 . 6 - 6 5 . 3 m U / m g . (25
7. C y s t e i n e h y d r o c h l o r i d e °C). This activity is sufficient for the conversion
8. H y d r o c h l o r i c a c i d , 0.1 N of 3 - 1 0 /ig. 3 - O H - A / m g . protein/min.
9. S o d i u m h y d r o x i d e , 0.1 N a n d 1 N 12. /?-Glucuronidase/arylsulphatase,
10. Kynureninase GRD/ARS
purified from pig liver (including the fractiona- from snails, stabilized enzyme solution; ^ 5 U
tion with a m m o n i u m sulphate) as described by G R D / m l . (25 °C), ^ 2 . 5 U A R S / m l . (25 °C).
C o m m e r c i a l p r e p a r a t i o n , see p . 460.
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y d i s t i l l e d w a t e r .
I. P h o s p h a t e buffer (33 m M : p H 7 . 4 ) :
D i s s o l v e 4 . 8 0 5 g. N a H P 0 - 2 H 0 + 0 . 8 1 7 g. K H P 0
2 4 2 2 4 in d i s t i l l e d w a t e r a n d m a k e u p
to 1 0 0 0 ml.
II. 3 - O H - K S t a n d a r d s o l u t i o n
a) S t o c k s o l u t i o n ( 0 . 1 3 4 m M ) : d i s s o l v e 1.5 m g . 3 - O H - K in 5 0 m l . 0.1 N H C 1
b) Working standard (13.4 pM):
N e u t r a l i z e 5 m l . s t o c k s o l u t i o n w i t h 5 m l . 0.1 N N a O H a n d d i l u t e w i t h buffer
( s o l u t i o n I) t o 5 0 m l .
III. P y r i d o x a l - 5 ' - p h o s p h a t e ( 0 . 2 3 m M ) :
D i s s o l v e 6 m g . P L P in 100 m l . buffer ( s o l u t i o n I).
I V . Tris buffer ( 8 . 3 m M ; p H 7 . 4 ) :
D i s s o l v e 9 9 8 m g . tris in 2 5 0 m l . distilled w a t e r , a d d 6 9 m l . 0.1 N H C 1 a n d d i l u t e w i t h
distilled w a t e r t o 1 0 0 0 m l .
V. Ferrous a m m o n i u m sulphate (5.36 m M ) :

D i s s o l v e 2 1 0 m g . F e S 0 ( N H ) S 0 - 6 H 0 in 1 0 0 m l . tris buffer ( s o l u t i o n I V ) .
4 4 2 4 2
VI. Cysteine (13.66 m M ) :

D i s s o l v e 2 4 0 m g . c y s t e i n e H C 1 in 100 m l . tris buffer ( s o l u t i o n I V ) .
VII. Cysteine-iron solution (6.83 m M cysteine; 2.68 m M F e 2 +
):
M i x 1 ml. of solutions V and IV.
VIII. 3-Hydroxyanthranilic acid oxidase, 3 - O H - A oxidase:
D i s s o l v e 5 - 2 0 m g . o f t h e l y o p h i l i z e d p r e p a r a t i o n in 1 m l . buffer ( s o l u t i o n I).
3-Hydroxykynurenine 1733
S t o r e all s o l u t i o n s , s t o p p e r e d , in a refrigerator at 0 t o 4 ° C . T h e 3 - O H - K w o r k i n g s t a n d a r d
( l i b ) m u s t b e p r e p a r e d freshly e a c h d a y ; t h e s t o c k s o l u t i o n ( I I a ) is s t a b l e for s e v e r a l w e e k s
in a refrigerator. T h e P L P s o l u t i o n is s t a b l e for a b o u t a w e e k in a refrigerator. T h e c y s t e i n e -
i r o n s o l u t i o n ( V I I ) m u s t b e p r e p a r e d freshly e a c h d a y . T h e o x i d a s e s o l u t i o n ( V I I I ) m u s t b e
p r e p a r e d freshly e a c h d a y a n d k e p t in a n ice b a t h .
Procedure
3 - O H - K is u n s t a b l e at n e u t r a l a n d a l k a l i n e p H , t h e r e f o r e u r i n e s a m p l e s s h o u l d b e a n a l y s e d
as s o o n as p o s s i b l e after c o l l e c t i o n . O t h e r w i s e , a d j u s t 2 4 hr. u r i n e s a m p l e s w i t h H C 1 t o p H 4
o r b e l o w . If u r i n e s a m p l e s m u s t b e s t o r e d for a n y p e r i o d , freeze t h e m .
Hydrolysis :
F o r t h e d e t e r m i n a t i o n o f t o t a l 3 - O H - K (free a n d esterified) p r o c e e d a s f o l l o w s : i n c u b a t e
10 ml. urine w i t h 0 . 0 4 m l . / ? - g l u c u r o n i d a s e / a r y l s u l p h a t a s e s o l u t i o n at r o o m t e m p e r a t u r e f o r
2 hr. T h e n n e u t r a l i z e 10 m l . o f h y d r o l y s e d a n d 10 m l . o f n o n - h y d r o l y s e d u r i n e t o p H 7.4 w i t h
1 N N a O H a n d filter.
Assay System
Incubation with kynureninase:
2 5 m l . E r l e n m e y e r flasks w i t h g r o u n d - g l a s s s t o p p e r s ; 38 ° C ( c o n s t a n t t e m p e r a t u r e bath
e q u i p p e d for s h a k i n g ) .
Pipette into Experimental Standard Endogenous* C o n c e n t r a t i o n in

flasks: (A) (A+S) 3 - O H - A (E) assay mixture
P h o s p h a t e buffer (I) 2.00 ml. — 2.00 ml. 16.1 m M

S a m p l e (urine) 2.00 ml. 2.00 ml. 2.00 ml. u p t o c a . 1 pg.
3-OH-K/ml.
3 - O H - K (standard
solution l i b ) — 2.00 ml. — 6 . 5 4 pM
PLP solution (III) 0.10 ml. 0.10 ml. 0.10 ml. 5.51 pM
Kynureninase 15 m g . 15 m g . — 3.66 m g . / ml. =
2 2 0 juU/ml.
I n c u b a t e for 3 hr., c o o l t h e flasks in a w a t e r b a t h t o 2 0 ° C a n d

filter. D e t e r m i n e t h e 3 - O H - A c o n t e n t in e a c h o f t h e s e m i x t u r e s .
* The " e n d o g e n o u s " 3 - O H - A is that already present in the sample, a n d because of its lability this m u s t
be treated in the same way a n d the value obtained must be subtracted from the other analytical results
in the calculations.
Determination with 3-OH-A Oxidase
W a v e l e n g t h : 3 6 0 ( H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 2 2 m l . ; 25 ° C . R e a d a g a i n s t
a b l a n k c o n s i s t i n g o f 1.0 m l . u r i n e + 1 m l . buffer (I).
Filtered incubation solution 2.00 ml.

Fe-cysteine solution (VII) 0.02 ml. 6 1 . 5 fiM cysteine
2 4 /nM F e 2 +
3-OH-A oxidase (VIII) 0.20 ml. 9-30 mU/ml.
R e a d the extinction immediately and record the in

c r e a s e A E until n o further rise o c c u r s . M i x t h e c u v e t t e
contents thoroughly during the measurements.
Calculations
AE -AE A E
x 3 [^g-/ml.;
AE A+S —A E A
AE -AE
A E w 3 [^mole/ml.]
AE sA +
—
AE A 224.1
To calculate the 24 hr. excretion multiply by the total a m o u n t of urine in ml. If the concentration of the
standard solution is altered the factors 3 in the above formula m u s t be changed accordingly.
T h e addition of 1 - 4 fig. 3 - O H - K / m l . to urine resulted in direct p r o p o r t i o n a l i t y between the concentration

and the extinction readings. C o m p l e t e conversion of the substrate is therefore assured.
Replicate determinations (10) on a urine sample gave a concentration of 1.32 ± 0.019 ug. 3 - O H - K / m l .
T h e coefficient of variation is 4.46. Recovery experiments with addition of 1 fig. a n d 3 fig. 3-OH-K gave
recoveries of 1.0 ± 0 . 0 2 2 ug. a n d 3.0 + 0.016 fig. respectively. T h e coefficients of variation are 6.61 and 1.62,
respectively.
N o r m a l Values
T h e following values have been found in healthy subjects: total 3-OH-K 0.554 mg./24 hr., free 3-OH-K
0.360 mg./24 hr. (mean values, n = 13). It should be n o t e d that these values are n o t representative, because
only a relatively small p o p u l a t i o n has been studied. T h e values in the old literature are significantly
higher; this is p r o b a b l y d u e to the lack of specificity of the earlier m e t h o d s . 5
Effects of drugs and other therapeutic agents: D r u g s , e.g. sulphonamides, which m a y result in an alkaline
urine, d o not interfere in the m e t h o d , b u t d o interfere in the quantitative recovery of the excreted 3-OH-K,
because it is very unstable a b o v e p H 7. Possible interference by drugs or metabolites excreted in the
urine is corrected for by inclusion of a s t a n d a r d plus sample in the assay. Acid hydrolysis is not r e c o m m e n -
3-Hydroxykynurenine 1735
ded, because the high electrolyte c o n c e n t r a t i o n m a y inhibit the enzyme activity so m u c h that the assay
is impossible.
Kynureninase only reacts with 3 - O H - K a n d k y n u r e n i n e ; the succeeding enzyme, however, only reacts
with the 3-OH-A formed so that absolute specificity is assured.
Appendix
Kynureninase from Pig Liver 1
Extract 250 g. acetone-dried p o w d e r of pig liver, with stirring, for 15 min. in the cold with 3.75 litres
66 m M p h o s p h a t e buffer, p H 7.4. Centrifuge off the insoluble residue. W a r m small p o r t i o n s of the extract
in a boiling water b a t h to 6 5 - 6 6 °C with vigorous stirring, then keep for 3 min. in a t h e r m o s t a t at 68 °C.
C o o l rapidly a n d centrifuge (3-5-fold purification of kynureninase in the s u p e r n a t a n t fluid with 9 2 %
yield). A d d 25.34 g. a m m o n i u m sulphate/100 ml. s u p e r n a t a n t fluid ( 4 4 % s a t u r a t i o n ) and stir for 20 min.
in the cold. Centrifuge off the precipitate a n d increase the a m m o n i u m sulphate c o n c e n t r a t i o n in the super
n a t a n t fluid t o 5 2 % (4.73 g./ml.). After 60 min. (slow stirring) centrifuge off the protein precipitate, dissolve
in 6 0 - 8 0 ml. 10 m M p h o s p h a t e buffer, p H 6.0 a n d dialyse against this buffer for 1 8 - 2 0 hr. in the cold
with several changes of buffer.
References
1 F. Wiss & O. Wiss in Hoppe-Seyler-Thierfelder (Eds.), Physiologisch- u n d pathologisch-chemische

Analyse. 10. Edition. Vol. V I / B , p . 806. Springer Verlag Berlin-Heidelberg-New Y o r k 1966.
2 O. Wiss, Z. N a t u r f o r s c h g . lib, 54 [1956].
3 H. Schievelbein & Ellen Buchfink, Clin. chim. A c t a ( A m s t e r d a m ) 18, 291 [1967].
4 H. Schievelbein & Karin Loschenkohl, Z. klin. Chemie a n d klin. Biochemie 6\ 138 [1968].
5 CA. Benassi, F.M. Veronese & A. de Antoni, Clin. chim. A c t a ( A m s t e r d a m ) 17, 383 [1967].
3-Hydroxyanthranilic Acid
Helmut Schievelbein and Karin Loschenkohl
3-Hydroxyanthranilic acid ( 3 - O H - A ) is a n intermediate in the catabolism of t r y p t o p h a n a n d is excreted

in urine. It has been suggested t h a t this substance, which is carcinogenic in a n i m a l experiments, m a y
be involved in the pathogenesis of bladder cancer in h u m a n s . Until n o w determination of 3-OH-A has
1
required its isolation by extraction of the urine a n d separation by c h r o m a t o g r a p h i c m e t h o d s followed

by colorimetric o r fluorimetric assay with variable recoveries.
T h e enzymatic m e t h o d described here is a slight modification of t h e m e t h o d originally described by u s . 2
Principle
C
^COOH 3 _ O H _ A ^ . C O O H
( 1 )
L I + 2 O z + 2 H 0
2 __ i A _ > ^ ff_ + 2 H O
2 z
— C-NH 2
H b
X
COOH
N
O H|^ + 2 0 2 + 2 H 0
2 • J, !L
( 3 - H y d r o x y a n t h r a n i l i c acid) (2-Amino-3-carboxy-
muconate semialdehyde)
3-OH-A is oxidized by 3-hydroxyanthranilic acid oxidase, 3 - O H - A O ( 3 - H y d r o x y a n t h r a n i l a t e : oxygen 3,4-

oxidoreductase, decyclizing, E C 1.13.11.6) to 2-amino-3-carboxymuconate semialdehyde. T h e p r o d u c t can
be determined by its a b s o r p t i o n m a x i m u m at 360 n m . T h e principle of the m e t h o d has already been used
by Wiss to determine the activity of 3 - O H - A O .
3
The conditions described for the m e t h o d are o p t i m u m . T h e use of very highly purified enzyme p r e p a r a t i o n s
is of no advantage, because the purified enzyme is inactivated within minutes in solution at p H 8.0. A n t h r a -
nilic acid, which is also present in urine, is a competitive inhibitor, but is n o t a s u b s t r a t e ; for this and other 4
reasons a s t a n d a r d must be included in the assay. The p H o p t i m u m for 3 - O H - A O is between 6.5 and 8.0.
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e f o r m e a s u r e m e n t s at 3 6 0 n m . P h o t o m e t e r s w i t h filters at 365 n m
are a l s o s u i t a b l e p r o v i d e d t h a t it is p o s s i b l e t o e x p a n d t h e s c a l e t o o b t a i n e x a c t r e a d i n g s ,
b e c a u s e the i n c r e a s e s in e x t i n c t i o n m a y b e s m a l l . A n i n s t r u m e n t w i t h a c o n s t a n t t e m p e r a t u r e
c u v e t t e h o l d e r is p r e f e r a b l e . A p H m e t e r is a l s o r e q u i r e d .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 4. 3 - H y d r o x y a n t h r a n i l i c a c i d , 3 - O H - A
2. Cysteine hydrochloride 5. S o d i u m h y d r o x i d e , 0.1 N
3. F e r r o u s a m m o n i u m s u l p h a t e , A . R., 6. H y d r o c h l o r i c a c i d , 0.1 N
FeS0 (NH ) S0 -6
4 4 2 4 H 0
2
3-Hydroxyanthranilic Acid 1737
7. / ? - G l u c u r o n i d a s e / a r y l s u l p h a t a s e 8. 3 - H y d r o x y a n t h r a n i l i c a c i d o x i d a s e ,
GRD/ARS 3-OH-AO
from snails, stabilized enzyme solution; ^ 5 U p r e p a r e d from pig liver according to Wiss et a l . . 5
G R D / m l . (25 °C), ^ 2 . 5 U A R S / m l . (25 °C). O u r p r e p a r a t i o n s h a d specific activities of 19.6

C o m m e r c i a l p r e p a r a t i o n , see p . 460. to 53 m U / m g . (25 °C). This activity is sufficient
for the conversion of 3 - 1 0 /ig. 3-OH-A/mg.
protein/min. a n d is therefore suitable for assays
in urine. F o r isolation of the enzyme, see the
A p p e n d i x , p . 1739.
I. Tris buffer ( 8 . 3 m M ; p H 7 . 4 ) :
D i s s o l v e 9 9 8 m g . tris in 2 5 0 m l . d i s t i l l e d w a t e r , a d d 6 9 m l . 0.1 N H C 1 a n d d i l u t e w i t h
distilled water to 1 0 0 0 ml.
II. C y s t e i n e ( 1 3 . 7 m M ) :
D i s s o l v e 2 4 0 m g . c y s t e i n e h y d r o c h l o r i d e in 100 m l . tris buffer ( s o l u t i o n I).
III. F e r r o u s a m m o n i u m s u l p h a t e ( 5 . 4 m M ) :
D i s s o l v e 2 1 0 m g . f e r r o u s a m m o n i u m s u l p h a t e in 100 m l . tris buffer ( s o l u t i o n I).
I V . C y s t e i n e / f e r r o u s a m m o n i u m s u l p h a t e ( 6 . 8 m M c y s t e i n e , 2.7 m M F e 2 +
):
M i x e q u a l v o l u m e s o f s o l u t i o n s II a n d III.
V. 3 - O H - A Standard solution (0.39 m M ) :
D i s s o l v e 1.2 m g . 3 - O H - A in 2 0 m l . 0.1 N H C 1 . T h e H C 1 is n e u t r a l i z e d b y N a O H in t h e
cuvette.
VI. 3 - O H - A oxidase ( 5 - 2 0 mg. protein/ml.):
A c c o r d i n g to the activity o f the preparation dissolve 5 to 20 mg. of the lyophilized e n z y m e
in 1.0 m l . tris buffer ( s o l u t i o n I).
Store all solutions in a refrigerator at 0 to 4 °C. Solutions IV a n d V I m u s t be p r e p a r e d freshly each d a y

a n d kept in a n ice b a t h .
Procedure
Collection and hydrolysis
If p o s s i b l e u s e a p o r t i o n o f a 2 4 - h r . u r i n e s a m p l e . D u r i n g t h e c o l l e c t i o n t h e u r i n e s h o u l d b e
adjusted to b e l o w p H 4 with c o n e . HC1. Immediately before the assay neutralize the urine
t o p H 7.4 w i t h 2 N N a O H a n d filter.
F o r t h e d e t e r m i n a t i o n o f free a n d esterified 3 - O H - A ( t o t a l 3 - O H - A ) a d d 0 . 0 2 m l . /?-glucuroni-
d a s e / a r y l s u l p h a t a s e s o l u t i o n t o 5 m l . u r i n e a n d i n c u b a t e for 2 h o u r s at r o o m t e m p e r a t u r e .
L o n g e r p e r i o d s o f i n c u b a t i o n result in l o w v a l u e s for t h e free c o m p o u n d .
Stability of sample
3 - O H - A is a v e r y l a b i l e c o m p o u n d a n d at p H 8.0 it u n d e r g o e s r a p i d , s p o n t a n e o u s o x i d a t i o n .
T h e r e f o r e a c i d i f i c a t i o n is a b s o l u t e l y n e c e s s a r y , e s p e c i a l l y a s u r o l o g i c a l p a t i e n t s o f t e n h a v e
urinary tract i n f e c t i o n s a n d e x c r e t e s t r o n g l y a l k a l i n e u r i n e d u e t o b a c t e r i a l p r o d u c t i o n o f
a m m o n i a . A c c o r d i n g t o Pipkin
6
et al? o r a l a d m i n i s t r a t i o n o f a s c o r b i c a c i d l e a d s t o stabili
z a t i o n o f 3 - O H - A in u r i n e .
Assay System
W a v e l e n g t h : 3 6 0 ( H g 3 6 5 ) n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 2 . 3 2 m l . ; t e m p e r a t u r e : 25 ° C .
A s t a n d a r d a n d a b l a n k is r e q u i r e d for e a c h d e t e r m i n a t i o n . R e a d a g a i n s t a b l a n k .
R e p e a t t h e d e t e r m i n a t i o n o f t o t a l 3 - O H - A w i t h h y d r o l y s e d urine.
Pipette successively Experimental Standard C o n c e n t r a t i o n in

Blank
into cuvettes: (A) ( A + S) assay mixture
Tris buffer (I) 1.00 m l . 1.00 m l . 1.00 m l . 3.58 m M

S a m p l e (urine) 1.00 m l . 1.00 m l . 1.00 m l . u p t o c a . 1 pg.
3-OH-A/ml.
Standard solution(V) — 0.05 ml. — 8.45 pM
0.1 N N a O H — 0.05 ml. —
Cysteine/Fe solution 0.02 ml. 0.02 ml. — 5 8 . 8 pM cysteine
(IV) 23.1 pM Fe 2 +
Oxidase solution (VI) 0.20 ml. 0.20 ml. — ca. 2 - 4 m U / m l .

Distilled water 0.10 ml. — 0.32 ml.
M i x a n d r e a d e x t i n c t i o n s o f A a n d A + S a g a i n s t t h e b l a n k until
c o n s t a n t ( c a . 15 m i n . ) .
I m m e d i a t e l y after a d d i t i o n o f t h e e n z y m e s o l u t i o n r e a d i n g s are m a d e a g a i n s t the b l a n k .

T h e e x t i n c t i o n is r e c o r d e d as l o n g as it c o n t i n u e s t o i n c r e a s e ( 1 0 - 2 0 m i n . ) . R e a d i n g s c a n
b e m a d e e v e r y m i n u t e or e v e r y t w o m i n u t e s a n d all the c u v e t t e s m u s t b e w e l l m i x e d d u r i n g
this period.
Calculations
T h e extinction of the experimental cuvette is c o m p a r e d to that of the s t a n d a r d :
C =
AT: AV X 3
bg/ ]
m l
AE -AE
A+S A
c = x —-— [umole/ml.]
J E A + S - ^ E A 153.4
If the concentration of the s t a n d a r d solution is altered the factor 3 in the a b o v e formula m u s t be changed
accordingly. To calculate the 24 hr. excretion multiply by the total a m o u n t of urine in ml. If m o r e t h a n a
few d r o p s of N a O H are required t o neutralize the urine, this m u s t be allowed for in the calculations.
3-Hydroxyanthranilic Acid 1739
Replicate determinations (10) on a urine containing 0.45 ug. 3 - O H - A / m l . gave a s t a n d a r d deviation of

0.025. The coefficient of variation is 5.68. Recovery experiments a d d i n g 3 fig. 3 - O H - A / m l . urine gave
100 ± 2.6%.
N o r m a l Values
T h e urinary excretion of n o r m a l subjects of total 3 - O H - A is 0.456 mg./24 hr. a n d of free 3 - O H - A is 0.235

mg./24 hr. (mean values, n = 27).
These values are not representative because the m e t h o d has only been used on a small p o p u l a t i o n sample.
T h e values in the old literature are significantly higher; this is p r o b a b l y d u e to the lack of specificity of
the m e t h o d s used.
Effects of drugs and other therapeutic agents: D r u g s , e. g. s u l p h o n a m i d e s , which m a y result in an alkaline

urine, can cause the alkaline hydrolysis of 3-OH-A. It is also possible t h a t d r u g s o r metabolites excreted
in the urine m a y act as inhibitors. This is corrected for by the inclusion of a s t a n d a r d plus sample in t h e
assay.
Interference in the assay technique: Acid hydrolysis is not r e c o m m e n d e d because the high electrolyte con
centration often inhibits the enzyme activity so strongly that the assay is impossible.
T h e specificity is a b s o l u t e ; n o other e n d o g e n o u s substrate for the enzyme is k n o w n .
Appendix
3-Hydroxyanthranilic Acid O x i d a s e from Pig Liver 3
Extract acetone-dried p o w d e r of pig liver with 10 volumes 0.1 M tris buffer, p H 7.4 for 15 min. Precipitate
inactive protein by addition of solid a m m o n i u m sulphate ( 3 0 % s a t u r a t i o n ) to the s u p e r n a t a n t fluid a n d
remove by centrifugation. Precipitate the active protein with a m m o n i u m sulphate ( 4 5 % saturation).
Dissolve the precipitate o b t a i n e d by centrifugation in the m i n i m u m a m o u n t of 33 m M tris buffer, p H 7.4
and dialyse for several h o u r s against this buffer. O b t a i n a dry enzyme p o w d e r by lyophilization; the
activity is maintained for several weeks.
References
1 H. Schievelbein & Th. Zickgraf'm: N i k o t i n , P h a r m a k o l o g i e u n d Toxikologie des T a b a k r a u c h e s , Thieme-

Verlag Stuttgart, p. 242 [1968].
2 H. Schievelbein & Ellen Buchfink, Clin. chim. Acta ( A m s t e r d a m ) 18, 291 [1967].
3 O. Wiss, Z. Naturforschg. lib, 54 [1956].
4 F. Weber & O. Wiss i n : Hoppe-Seyler-Thierfelder: Physiologisch- u n d pathologisch-chemische Analyse.
10th. edn., Vol. V I / B , p . 806. Springer Verlag B e r l i n - H e i d e l b e r g - N e w Y o r k 1966.
5 O. Wiss,H. Simmer &H. Peters, Hoppe-Seyler's Z. physiol. C h e m i e 304, 221 [1956].
6 G. Pipkin & J. U. Schlegel, Proc. Soc. exp. Biol. M e d . 120, 592 [1965].
7 G. Pipkin, R. Nishimura, L. Bunowsky & I. U. Schlegel, Proc. Soc. exp. Biol. M e d . 126, 702 [1967].
Spermidine
Uriel Bachrach
Spermidine is widely distributed in m a m m a l i a n tissues, bacteria a n d viruses. T h e m e t h o d described here

for the enzymatic determination of spermidine depends on its oxidation by a specific bacterial enzyme,
spermidine oxidase* . 1
Application of Method: In microbiology a n d in biochemistry.
Principle
(1) NH (CH ) NH(CH )4NH

2 2 3 2 2 + % 0 2 NH (CH ) NH
2 2 3 2 +
N
Spermidine Propane-1,3-diamine ^-Pyrroline
T P
* to
N ^NH OH"
(2) rn
, L V
i; i
A -Pyrroline o-Aminobenzaldehyde
T h e pyrroline formed in the oxidation gives a yellow c o l o u r with o - a m i n o b e n z a l d e h y d e . T h e extinction 2
of this dye is the measure of the spermidine present.
T h e o p t i m u m p H for the oxidation of spermidine is p H 6.5. Between 0.05 a n d 0.5 /miole spermidine the
assay is p r o p o r t i o n a l to the a m o u n t of sample a d d e d .
Equipment
Spectrophotometer or spectrum-line p h o t o m e t e r s u i t a b l e for a c c u r a t e measurements at

435 n m ; bench centrifuge; water bath (37 °C); p H meter.
Reagents
1. S o d i u m d i h y d r o g e n p h o s p h a t e , 5. D i e t h y l e t h e r
N a H P 0 - 2 H 0 , A.R.
2 4 2 6. F r e e z e - d r i e d cells f r o m Serratia
2. D i s o d i u m h y d r o g e n p h o s p h a t e , marcescens
N a H P 0 - 7 H 0 , A.R.
2 4 2 p r e p a r a t i o n , see A p p e n d i x , p . 1742.
3. S o d i u m c h l o r i d e , A . R. 7. o-Aminobenzaldehyde
4. Trichloroacetic acid
* N o System n u m b e r has yet been assigned by the E n z y m e C o m m i s s i o n of the I U B .

Spermidine 1741
D i s s o l v e 0 . 3 g. N a H P 0 - 2 H 0 a n d 1.3 g. N a H P 0 - 2 H 0 in 5 0 m l . d i s t i l l e d w a t e r ,
2 4 2 2 4 2
check p H with a glass electrode and dilute to 100 ml. with distilled water.
II. S o d i u m c h l o r i d e ( 0 . 1 5 M ) :
D i s s o l v e 0 . 8 5 g. N a C l in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
III. o - A m i n o b e n z a l d e h y d e ( 0 . 1 % ) :
D i s s o l v e 10 m g . o - a m i n o b e n z a l d e h y d e in 10 m l . d i s t i l l e d w a t e r .
I V . Cell s u s p e n s i o n o f Serratia marcescens (10 mg./ml.):
S u s p e n d 2 0 m g . f r e e z e - d r i e d cells o f Serratia marcescens in 2 m l . p h o s p h a t e buffer ( s o l u
t i o n I).
V. Trichloroacetic acid (0.4 N ) :
D i s s o l v e 6 . 5 2 g. t r i c h l o r o a c e t i c a c i d in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
Store solutions I, II a n d IV in glass-stoppered flasks at 4 °C. Freeze-dried cells of Serratia marcescens keep
their enzyme activity in the frozen state ( — 20 °C) for 12 m o n t h s ; they should be suspended just before use
in p h o s p h a t e buffer (solution I). Solution III is stable for 2 weeks at —20 °C.
Procedure
If t h e s a m p l e c o n t a i n s free s p e r m i d i n e in s o l u t i o n , n o s p e c i a l p r e t r e a t m e n t is r e q u i r e d , o t h e r
wise extract p o l y a m i n e s by the m e t h o d described o n p. 1745.
T r i c h l o r o a c e t i c a c i d e x t r a c t s c a n b e s t o r e d indefinitely at —20 ° C w i t h o u t l o s s o f s p e r m i d i n e .
Assay System
W a v e l e n g t h : 4 4 5 n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 1.0 m l . ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t
b l a n k c o n t a i n i n g d i s t i l l e d w a t e r i n s t e a d o f s a m p l e ; i n c u b a t i o n t e m p e r a t u r e 37 ° C .
Sample + N a C l solution (II) 0.4 ml. u p t o 0.5 ^ m o l e s p e r m i d i n e

Serratia cell s u s p e n s i o n (IV) 0.5 m l .
o-Aminobenzaldehyde (III) 0.1 m l .
I n c u b a t e for 6 0 m i n . at 37 ° C , t h e n transfer t h e c o n
tents o f the tubes to cuvettes a n d measure the ex
tinctions.
Calculations
T h e extinction coefficient of pyrroline-o-benzaldehyde at 435 n m is £ = 1.85 x 10 cm. /yumole. U n d e r 6 2
the above conditions the reaction proceeds stoichiometrically a n d therefore the calculation formula (2)
o n p . 312 applies. T h e results are obtained in ,umole spermidine/ml. sample. This value must be multi
plied by a factor if the sample has been deproteinized, neutralized or diluted in a n y way. U n d e r the con
ditions described here the following relationship h o l d s :
AE
c = [wmole/ml.l
1.85 x v L M 1 1
v = sample volume in assay [ml.]
In rat liver a m e a n value of 1.2 + 0.1 ^ m o l e spermidine/g. fresh wt. was found. T h e coefficient of variation
is 0.67%.
N o r m a l Values, s e e p . 1 7 4 7 .
Serratia marcescens should be grown under the conditions described below. Changes in the composition
of the culture m e d i u m can result in loss of the specificity of the enzyme. G r o w t h at s u b o p t i m u m tempera
tures can cause decreased oxidase activity.
The m e t h o d is specific for spermidine. Spermine and diamines, like, putrescine (butane-l,4-diamine),
cadaverine (pentane-l,5-diamine) are not oxidized. 3,3'-Diaminodipropylamine is oxidized by the enzyme,
but does not give z^-pyrroline so n o reaction occurs with o-aminobenzaldehyde.
Appendix
Isolation of Spermine O x i d a s e from Serratia marcescens2
Reagents
Difco yeast extract*

Dipotassium hydrogen p h o s p h a t e , K H P 0 - 3 H 0 2 4 2
Potassium dihydrogen p h o s p h a t e , K H P 0 2 4
Glucose
M a g n e s i u m sulphate, M g S 0 • 7 H 0 4 2
Spermidine-3 HC1
Method
Strain: Serratia marcescens J (or A T T C * * 8195). Culture m e d i u m : dissolve 0.5 g. yeast extract, 2.0 g.
K H P 0 - 3 H 0 , 1.0 g. K H P 0 , 1.0 g. glucose, 0.2 g. M g S 0 - 7 H 0 and 0.1 g. spermidine in distilled
2 4 2 2 4 4 2
water and m a k e up to 1000 ml.; adjust to p H 7.0 with N a O H and sterilize in an autoclave.
* Difco Laboratories Inc. Detroit, Michigan, U S A

** American Type Culture Collection, 2112 M Street, N o r t h , Washington, 7 D . C . , U S A
Spermidine 1743
Inoculate the culture m e d i u m with a p o r t i o n of the subculture (1 : 5 v/v) a n d incubate for 20 hr. at 30 °C
with vigorous shaking. Collect the cells by centrifugation a n d lyophilize; store at - 2 0 °C in vacuo.
References
1 U. Bachrach, J. biol. C h e m . 237, 3443 [1962].

2 U. Bachrach & /. S. Oser, J. biol. C h e m . 238, 2098 [1963].
Spermine and Spermidine
Uriel Bachrach
Spermine and spermidine occur in m a m m a l i a n tissues, bacteria a n d viruses. T h e m e t h o d described here

for the determination of polyamines d e p e n d s on their stoichiometric oxidation by the specific a m i n e
oxidase ( A m i n e : oxygen oxidoreductase, deaminating, flavin-containing, E C 1.4.3.4).
Application of Method: In microbiology and in biochemistry.
Principle
(1) NH (CH2)3NH(CH2)4NH(CH2)3NH + 2 0
2 2 2 + 2 H 0 2
Spermine
a
—^C(CH )2NH(CH2)4NH(CH )2C
2 + 2 NH 2
PX 3 + 2 H 0 2 2
H H
(2) NH (CH2)3NH(CH )4NH2 + 0

2 2 2 + H 0 2 —* ^(CH ) NH(CH2)4NH
2 2 2 + NH 3 + 2 H 0 2 2
H
Spermidine
CH 3 / CH 3 CH 3
(3) 2
N
C=N-NH + RC
P [o] r < V ^ -> V^
I IQJL C - N - N - C R - N - N - C ^ J.CJJ
N
The oxidation of spermine or of spermidine leads to the formation of i m i n o a l d e h y d e s . These are deter 1
mined according to equation (3) by reaction with N-methyl-2-benzothiazolone h y d r a z o n e to give coloured

derivatives . T h e increase of colour at 660 n m is measured.
2
T h e o p t i m u m p H for the oxidation is p H 7.4. T h e colour formed in the d e t e r m i n a t i o n is linearly p r o

portional to the polyamine concentration between 0.01 and 0.1 pinole.
Equipment
S p e c t r o p h o t o m e t e r or s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e for p r e c i s e m e a s u r e m e n t s at 6 6 0 n m ;
bench centrifuge; water bath (37 °C); p H meter.
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 3. N - M e t h y l - 2 - b e n z o t h i a z o l o n e hydrazone
2. S o d i u m c h l o r i d e , A . R. hydrochloride ( N B T H )
Spermine a n d Spermidine 1745
4. F e r r i c c h l o r i d e , a n h y d r o u s , A . R. 8 Amine oxidase
5. T r i c h l o r o a c e t i c a c i d from bovine s e r u m or plasma, purified accord
6. D i e t h y l e t h e r ing t o . F o r isolation, see p . 1747.
3
7. H y d r o c h l o r i c a c i d , A . R . , 1 N
Purity of Reagents
T h e enzyme should be purified 60-fold a n d the specific activity should be ca. 80 U / m g . (30 °C). If necessary,
sheep serum or p l a s m a can be used as a source.
Only use distilled water.

I. Tris buffer ( 0 . 2 M ; p H 7 . 4 ) :
D i s s o l v e 2 . 4 2 g. tris i n 5 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7 . 4 ( p H - m e t e r ) w i t h c a . 1 7 . 5 m l .
1 N HC1 and dilute to 1 0 0 0 ml. with distilled water.
II. S o d i u m c h l o r i d e ( 0 . 1 5 M ) :
D i s s o l v e 0 . 8 5 g. N a C l in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
III. N - M e t h y l - 2 - b e n z o t h i a z o l o n e h y d r a z o n e h y d r o c h l o r i d e , N B T H ( 0 . 4 % ) :
D i s s o l v e 0 . 2 g. N B T H in d i s t i l l e d w a t e r a n d m a k e u p t o 5 0 m l .
I V . Ferric c h l o r i d e ( 0 . 2 % ) :
D i s s o l v e 0 . 2 g. F e C l 3 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
V. Trichloroacetic acid (0.4 N ) :
D i s s o l v e 6 . 5 2 g. t r i c h l o r o a c e t i c a c i d in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
VI. A m i n e oxidase (ca. 200 U / m l . ) :
U s e the e n z y m e s o l u t i o n prepared according to p. 1747.
Store solutions III a n d IV in b r o w n , stoppered, bottles for not longer t h a n 10 days. T h e a m i n e oxidase
is stable for ca. 12 m o n t h s at - 2 0 °C.
Procedure
If t h e s a m p l e c o n t a i n s free p o l y a m i n e s in s o l u t i o n n o p r e t r e a t m e n t is n e c e s s a r y ; o t h e r w i s e
e x t r a c t c e l l u l a r m a t e r i a l w i t h t r i c h l o r o a c e t i c a c i d as f o l l o w s : To e a c h 1 g. c e l l u l a r m a t e r i a l
a d d 3 m l . t r i c h l o r o a c e t i c a c i d ( s o l u t i o n V ) a n d h e a t for 10 m i n . in a s t e a m b a t h . S e p a r a t e off
i n s o l u b l e m a t e r i a l b y c e n t r i f u g a t i o n (15 m i n . at 3 0 0 0 g ) , e x t r a c t s u p e r n a t a n t fluid w i t h 10 m l .
ether, r e p e a t e x t r a c t i o n t w i c e m o r e w i t h e t h e r a n d r e m o v e r e s i d u a l e t h e r b y b l o w i n g air t h r o u g h
the solution.
T r i c h l o r o a c e t i c a c i d e x t r a c t s c a n b e s t o r e d indefinitely at — 2 0 ° C w i t h o u t l o s s .
Assay System
W a v e l e n g t h : 6 6 0 n m ; light p a t h : 1 c m . ; i n c u b a t i o n v o l u m e : 0 . 2 m l . ; i n c u b a t i o n t e m p e r a t u r e :
37 ° C ; final v o l u m e : 3.2 m l . ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t b l a n k c o n t a i n i n g N a C l s o l u t i o n
(II) i n s t e a d o f s a m p l e .
F o r e a c h series o f m e a s u r e m e n t s a s s a y a c o n t r o l v a l u e c o n t a i n i n g N a C l s o l u t i o n (II) i n s t e a d
of enzyme.
P i p e t t e i n t o test t u b e s : C o n c e n t r a t i o n in assay mixture
Sample + N a C l solution (II) 0.08 ml. u p t o 0.1 / / m o l e s p e r m i n e

up to 0.2 //mole spermidine
Tris buffer (I) 0.10 ml. 0.1 M
Enzyme solution (VI) 0.02 ml. ca. 2 0 U / m l .
M i x a n d i n c u b a t e for 4 hr. at 37 ° C .
N B T H solution (III) 0.5 ml.
M i x a n d i n c u b a t e for 3 0 m i n .
F e C l solution
3 (IV) 2.5 ml.
A l l o w t o s t a n d for 2 5 m i n . at r o o m t e m p e r a t u r e a n d
measure extinctions. The difference AE between
e x t i n c t i o n s o f t h e test a n d c o n t r o l are u s e d for t h e
calculations
Calculations
T h e extinction coefficient £ of the oxidation p r o d u c t at 660 n m is 12.5 x 1 0 cm?/mole for spermine 6
and 6.25 x 10 c m . / m o l e for spermidine. U n d e r the above conditions the reaction proceeds stoichio-
6 2
metrically and therefore the calculation formula (2) on p . 312 applies. T h e result is obtained as /imole
spermine or spermidine/ml. sample. This value m u s t be multiplied by a factor if the s a m p l e h a s been
deproteinized, neutralized or diluted in any way. T h e following relationship holds for the m e t h o d des
cribed h e r e .
AExV , . f
c = [umole/ml.l
£ xv
T h e a b o v e calculation is correct when the sample contains either spermine o r spermidine. If b o t h poly-
amines are present the determination is not correct because of the different £ values. In this case spermidine
must be determined separately according to p . 1740. T h e spermine c o n c e n t r a t i o n of the sample is then
calculated as follows:
=^ "" 2.T ""° T" ["

E r m e ) X m o l e / m l ]
Cs „
P m i „ e 1
A Es ermidine
P
= = e X
Spermidine =
6.25 X Cs p e r m i d i n e
S p e r m i d i n e = A * ° l spermidine/ml. sample determined according to p . 1740.

m e
Spermine a n d Spermidine 1747
In yeast extract a mean value of 1.68 ± 0.025 //mole spermine/g. has been found. T h e coefficient of
variation w a s 1.21 %.
Normal Values
H. Tabor a n d C. W. Tabor* found the following values:
Spermine Spermidine
Organ Species
/zmole/g. fresh wt.
Prostate Man 2.40

Rat 5.70 7.70
Pancreas Man 0.55
Mouse 1.00 2.80
Rat 1.00 8.60
G u i n e a pig 1.40 2.10
Liver Man 0.28
Mouse 1.10 1.40
Rat 1.20 1.60
Kidney Man 0.15
Mouse 0.70 0.50
M a m m a r y carcinoma Mouse 0.70 1.20
Sources of Error
Purified enzyme should be used, a l t h o u g h ox or sheep p l a s m a or serum can be used. I n c o m p l e t e extraction

of the trichloroacetic acid with ether can considerably decrease the sensitivity of the assay system.
A m i n e oxidase from serum is specific for polyamines. Diamines, such as putrescine (butane-1,4-diamine),
cadaverine ( p e n t a n e - l , 5 - d i a m i n e ) a n d histidine are n o t oxidized a n d therefore d o n o t interfere even when
present in excess. Synthetic polyamines such as a m i n o p r o p y l - e t h a n e - l , 2 - d i a m i n e a n d a m i n o p r o p y l -
h e p t a n e - l , 7 - d i a m i n e are n o t oxidized u n d e r these conditions.
Appendix
Isolation of Amine Oxidase 3
Reagents
Potassium dihydrogen phosphate, K H P 0 2 4 S o d i u m citrate-5.5 H 02
Dipotassium hydrogen phosphate, K H P 0 2 4 A m m o n i u m sulphate

Benzylamine S o d i u m acetate
Sulphuric acid, 2 N M a n g a n o u s chloride, M n C l - 4 H 0
2 2
Ethyl alcohol, absolute Ox b l o o d

Citric acid
Solutions
I. P h o s p h a t e buffer (0.2 M ; p H 7.4):

Dissolve 2.91 g. K H P 0 2 4 and 0.466 g. K H P 0 2 4 in distilled water and m a k e u p t o 100 ml.
II. Benzylamine (0.1 M ) :
A d d 1.07 g. distilled benzylamine to 5 ml. 2 N H S 0 2 4 a n d dilute to 100 ml. with distilled water.
III. Citrate (0.12 M ) :
Dissolve 8 g. citric acid in distilled water, add 26.7 g. sodium citrate and dilute to 1000 ml. with distil
led water.
IV. A m m o n i u m sulphate, s a t u r a t e d :
Dissolve 800 g. ( N H ) S 0 in distilled water and m a k e u p to 100 ml. H e a t to boiling and allow to
4 2 4
cool to r o o m t e m p e r a t u r e .
V. S o d i u m acetate (10 m M ) :
Dissolve 0.82 g. sodium acetate in distilled water and m a k e u p to 1000 ml.
VI. M a n g a n o u s chloride (0.1 M ) :
Dissolve 1.98 g. M n C l - 4 H 0 in distilled water a n d m a k e u p to 100 ml.
2 2
VII. E t h a n o l (25%):
Mix 75 ml. distilled water with 25 ml. alcohol.
Procedure
A d d 100 ml. citrate solution (III) to 600 ml. ox blood and obtain the plasma by centrifugation. To 100 ml.
plasma add 54 ml. ( N H ) S 0 4 2 4 solution (IV) and cool to 4 °C. R e m o v e the precipitate by filtration. To
the filtrate add 96 ml. ( N H ) S 0 solution (IV) and dissolve the precipitate in 20 ml. distilled water.
4 2 4
F r a c t i o n a t e this solution by addition of the following volumes of a m m o n i u m sulphate solution (IV) a n d

collect the precipitate each time by filtration: I. 4.1 ml., II. 2.1 ml., III. 1.9 ml., IV. 2.5 ml., V. 2.5 ml.
Dissolve each precipitate in water a n d determine the activity. C o m b i n e fractions III a n d IV, which are
usually the most active, and dialyse against 1000 ml. s o d i u m acetate solution (V).
H e a t 5 ml. portions of the dialysed solution in a water b a t h at 65 °C for 10 min., rapidly cool to 0 °C a n d
to each portion add 0.5 ml. M n C l solution (VI). F r a c t i o n a t e with e t h a n o l at —10° as follows: I. 2.5 ml.,
2
II. 1.25 ml., III. 1.25 ml., + 0 . 2 5 ml. absolute ethanol, IV. 1.25 ml. absolute e t h a n o l , V. 1.0 ml. absolute
ethanol. Centrifuge off the precipitate each time and dissolve in cold water.
D e t e r m i n a t i o n of activity: Pipette into a q u a r t z cuvette 1.0 ml. p h o s p h a t e buffer (solution I), 0.1 ml.
benzylamine (solution II) a n d 0.05 ml. enzyme solution and dilute to 3 ml. R e a d the extinction every 5 min.
at 250 n m against a blank without benzylamine. A s p e c t r o p h o t o m e t r i c unit is the enzyme activity which
in the initial phase decreases the extinction at 250 n m by 0.001 /min. A s p e c t r o p h o t o m e t r i c unit corresponds
to 0.077 International U n i t s (U).
References
1 C. W. Tabor, H. Tabor & U. Bachrach, J. biol. C h e m . 239, 2194 [1964].

2 U. Bachrach & B. Reches, Analyt. Biochem. 17, 38 [1966].
3 C. W. Tabor, H. Tabor & S. M. Rosenthal in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology.
Academic Press, New Y o r k 1955, Vol. / / , p . 390.
4 H. Tabor & C W. Tabor, P h a r m a c o l . Rev. 16, 245 [1964].
Carbamoylphosphate
Mary Ellen Jones
T h e occurence of c a r b a m o y l p h o s p h a t e ( C A P ) in higher plants a n d animals has excited little interest as yet.

There are two c a r b a m o y l p h o s p h a t e synthetases responsible for the synthesis in vertebrates. C a r b a m o y l
p h o s p h a t e synthetase I is located in high activity in the liver of adult urea-forming v e r t e b r a t e s 1 - 3
; it
provides C A P for the synthesis of urea. This enzyme is not rate-limiting in the biosynthesis of urea, a n d it
is possible, t h o u g h n o t certain, t h a t free C A P occurs in liver cells. C a r b a m o y l p h o s p h a t e synthetase I I 4 , 5
is
found in the soluble fraction of the tissues of m a n y (probably all) vertebrates, a n d a p p e a r s to be responsible
for the synthesis of C A P for the biosynthesis of pyrimidines. T h e latter enzyme is p r o b a b l y rate-limiting in
the biosynthesis of p y r i m i d i n e s ; the enzyme activity in tissues is l o w ' . It is therefore unlikely t h a t free
6,7 5 7
c a r b a m o y l p h o s p h a t e occurs in extra-hepatic tissues of n o r m a l animals. C A P is not found in the b l o o d of

normal rabbits . 8
C A P can be determined specifically a n d quantitatively by the enzymatic synthesis of citrulline or c a r b a m o y l -

aspartate if highly purified ornithine t r a n s c a r b a m o y l a s e 9
or a s p a r t a t e t r a n s c a r b a m o y l a s e 10
is used.
T h e use of radioactively labelled L-ornithine or L-aspartate considerably increases the sensitivity of the
m e t h o d ' ' . T h e p r o c e d u r e described here for serum m a k e s use of a s p a r t a t e t r a n s c a r b a m o y l a s e (Car
8 1 1 1 2
b a m o y l p h o s p h a t e : L - a s p a r t a t e carbamoyltransferase, E C 2.1.3.2.) with radioactive L-aspartate as the

c a r b a m o y l acceptor.
Principle
(1) C a r b a m o y l p h o s p h a t e + [ C ] - L - A s p a r t a t e - ^ ^ ^ ^
14
[ C]-Carbamoyl- L-aspartate + P
14
s
T h e radioactivity i n c o r p o r a t e d into c a r b a m o y l - L-aspartate ( C A A ) is p r o p o r t i o n a l to the q u a n t i t y of C A P .

[ C ] - C A A is separated from [ C ] - a s p a r t a t e by c o l u m n c h r o m a t o g r a p h y ; " c o l d " C A A is a d d e d . The
14 14
quantity of C A P is calculated from the specific radioactivity of C A A in the eluates, the specific radioacti
vity of the substrate [ C ] - L - a s p a r t a t e , a n d the q u a n t i t y of " c o l d " C A A a d d e d as the carrier.
14
The experimental conditions must be so chosen that the labile substrate, c a r b a m o y l p h o s p h a t e , is protected
a n d the requirements of the enzyme are met. T h e enzyme catalyses the reaction between p H 7 a n d p H 9.
Since C A P decomposition increases with increasing p H above 8.5 1 3
, the m e a s u r e m e n t s should not be
carried out at p H values higher t h a n 8.5. Samples m u s t be cooled (ice), a n d C A P m u s t be converted as
rapidly as possible into C A A . T h e equilibrium position is strongly in favour of C A A . F o r a s p a r t a t e trans
c a r b a m o y l a s e from E. coli, the Michaelis c o n s t a n t with respect to C A P is 0.2 m M . In view of the low C A P
1 4
concentrations in nearly all biological material, relatively large quantities of p u r e enzyme must be used
to bring the reaction to completion in a reasonable time in the presence of the anions of the sample, e. g.
chloride or p h o s p h a t e , which have an inhibitory a c t i o n . 15
Equipment
P h o t o m e t e r for m e a s u r e m e n t s at o r n e a r 5 6 0 n m ; l a b o r a t o r y c e n t r i f u g e , chromatography
c o l u m n s (1 c m . d i a m e t e r , w i t h s i n t e r e d g l a s s p l a t e at t h e b o t t o m ) ; G e i g e r c o u n t e r o r s c i n t i l l a t i o n
counter; constant-temperature water bath.
Reagents
For the incubation mixture
1. [ C ] - L - A s p a r t i c a c i d
1 4
5. P e r c h l o r i c a c i d , A . R . , s p . g r . 1 . 6 7 ; 7 0 %
labelled on C-3 o r C-4, specific radioactivity (w/w)
^ 1 mCi/mmole. 6. P o t a s s i u m d i h y d r o g e n p h o s p h a t e ,
2. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris KH P0 2 4
3. A s p a r t a t e t r a n s c a r b a m o y l a s e 7. S o d i u m h y d r o x i d e s o l u t i o n , 2 N
from Escherichia coli ' ,
10 16
^ 9 0 U / m g . (28 °C 8. H y d r o c h l o r i c a c i d , 1 N
activity m e a s u r e d a c c o r d i n g t o Gerhard and 9. Ethylenediaminetetra-acetate, E D T A
Holoubek ); 10
^ 1.6 m g . of p r o t e i n / m l . d i s o d i u m salt, E D T A - N a H • 2 H 0
2 2 2
4. Carbamoylphosphate, C A P 10. 2-Mercaptoethanol

dilithium salt; for commercial preparation, see
p. 528; keep in desiccator
For the ion exchange chromatography ' 8 12
1 1 . D o w e x - 1 x 10, 1 0 0 - 2 0 0 m e s h 13. F o r m i c a c i d , A . R . , s p . gr. 1 . 2 ; 8 8 % ( w / v )

14. P o t a s s i u m h y d r o x i d e s o l u t i o n , 1 N
12. D o w e x - 5 0 x 10, 1 0 0 - 2 0 0 m e s h
15. p-Dimethylaminobenzaldehyde,
For the colour test for CAA ' 8 11
16. S u l p h u r i c a c i d , H S 0 , A . R.
2 4
19. Brij 3 5
17. D i p h e n y l a m i n e - p - s u l p h o n i c a c i d e. g. from A t l a s Chemical C o .
s o d i u m salt, e. g. from E a s t m a n Organic 20. P o t a s s i u m persulphate, K S 0 , A . R. 2 2 8
Chemicals 21. Carbamoyl-DL-aspartic acid,

• 18. D i a c e t y l m o n o x i m e ( 2 , 3 - b u t a n e d i o n e purest commercial g r a d e , e. g. from N u t r i t i o n a l
2-oxime) Biochemicals
purest available, m . p . 7 4 - 7 5 ° C ; e.g. from
E a s t m a n Organic Chemicals
Purity of Reagents
The enzyme must be free from p h o s p h a t a s e activity t o w a r d s C A P a n d from any enzyme t h a t causes t r a n s
formation of a s p a r t a t e .
U s e o n l y freshly distilled water.
I. [ C ] - A s p a r t a t e ( 0 . 2 M ; > 1 m C i / m o l e ) :
1 4
Dissolve 26.6 mg. o f solid [ 14

C ] - a s p a r t i c a c i d h a v i n g a n a c t i v i t y o f 1 m C i / m m o l e in 0 . 2 m l .
o f N K O H + 0.81 m l . d i s t i l l e d w a t e r ; t h e p H s h o u l d b e b e t w e e n 5 a n d 8 ( i n d i c a t o r p a p e r ) .
Dilute commercial [ 14
C]-aspartate solutions appropriately, and adjust t o p H values
between 5 and 8 with N K O H * .
* T h e exact specific radioactivity m u s t be k n o w n , since this solution is used for the d e t e r m i n a t i o n of C A P

in the sample. To determine a 10 m M C A P solution, a [ C ] - a s p a r t a t e solution having a specific r a d i o
14
activity of at least 1 m C i / m m o l e is necessary. If lower C A P c o n c e n t r a t i o n s a r e t o b e d e t e r m i n e d , t h e

specific radioactivity m u s t be correspondingly increased while the a s p a r t a t e c o n c e n t r a t i o n r e m a i n s the
same.
Carbamoylphosphate 1751
If t h e r a d i o a c t i v i t y is h i g h e r t h a n 1 m C i / m m o l e , d i l u t e w i t h 0 . 2 M [ C ] - a s p a r t a t e s o l u
12
tion (prepared as described a b o v e ) . K e e p the solution at —20 °C.

II. Tris b u f f e r (1 M ; p H 7 . 8 ) :
D i s s o l v e 12.1 g. tris i n 6 6 . 7 m l . N H C 1 , a n d m a k e u p t o 100 m l . w i t h d i s t i l l e d w a t e r ; s t o r e at
+ 4 °C.
III. Aspartate t r a n s c a r b a m o y l a s e ( 1 . 6 - 2 m g . p r o t e i n / m l . ) :
Store the e n z y m e obtained according t o 1 0
in 4 0 m M p o t a s s i u m p h o s p h a t e s o l u t i o n
( p H 7 . 0 ; 2 m M 2 - m e r c a p t o e t h a n o l , 0 . 2 m M E D T A ) at - 20 °C.
I V . C a r b a m o y l p h o s p h a t e (0.1 M ) :
D i s s o l v e 157 m g . c a r b a m o y l p h o s p h a t e d i l i t h i u m salt in 10 m l . o f i c e - c o l d d i s t i l l e d w a t e r
( t h e s e w e i g h t s are for a p r e p a r a t i o n w i t h 8 0 % c a r b a m o y l p h o s p h a t e , 9 % Li, a n d 6 %
H 0 , a n d s h o u l d b e s u i t a b l y a d j u s t e d f o r p r e p a r a t i o n s w i t h different a n a l y t i c a l d a t a ) . If
2
the preparation c o n t a i n s a large quantity o f L i P 0 , this can be r e m o v e d by centrifugation

3 4
o r r a p i d v a c u u m filtration, b e c a u s e it is i n s o l u b l e .
S i n c e t h i s s o l u t i o n is a l s o u s e d a s t h e s t a n d a r d s o l u t i o n , t h e c o n t e n t m u s t b e d e t e r m i n e d
b y p h o s p h a t e a n a l y s i s , b y c h e m i c a l c o n v e r s i o n i n t o u r e a , o r b y t h e c o l o u r test a s
1 3 1 3
described o n p. 1755*.
C A P s o l u t i o n s a r e s t a b l e a t — 2 0 ° C . H o w e v e r , t h e y d e c o m p o s e o n t h a w i n g ; t h e half-life
is 4 0 m i n . at 3 7 ° C , a p p r o x . 160 m i n . at r o o m t e m p e r a t u r e , a n d 10 hr. at 0 ° C . T h a w s t o c k
s o l u t i o n s i m m e d i a t e l y b e f o r e u s e , a n d d o n o t k e e p l o n g e r t h a n 1 hr. at 0 ° C .
D i l u t e standard s o l u t i o n s : dilute the stock solution as required with ice-cold distilled
w a t e r , u s e i m m e d i a t e l y , a n d d i s c a r d a n y t h a t is n o t r e q u i r e d .
V. Perchloric acid ( I N ) :
D i l u t e 11.7 m l . 7 0 % H C 1 0 4 to 100 ml. with distilled water.
VI. D o w e x - 1 (charged with formate):
W a s h D o w e x - 1 t w o t o t h r e e t i m e s , in p o r t i o n s , w i t h 10 v o l u m e s o f 1 N K O H , a n d r e m o v e
t h e a l k a l i b y w a s h i n g w i t h 1 0 v o l u m e s o f w a t e r u n t i l p H < 9. Treat t h e r e s i n t h r e e t i m e s
for several hours or overnight with 1 N H C O O H , and w a s h with water until the p H o f the
w a s h w a t e r is p H > 4. In t h e w a s h e s w i t h w a t e r , t h e resin s h o u l d n o t settle for l o n g e r t h a n
2 0 o r 3 0 m i n . s o t h a t t h e v e r y fine p a r t i c l e s are d e c a n t e d off w i t h t h e w a s h w a t e r . S t o r e t h e
r e s i n u n d e r d i s t i l l e d w a t e r at r o o m t e m p e r a t u r e .
VII. D o w e x - 5 0 ( H +
form):
W a s h D o w e x - 5 0 as described a b o v e , but with water; impurities are eliminated in this w a y ,
a n d t h e final p r o d u c t is D o w e x - 5 0 i n t h e H +
f o r m . S t o r e t h e resin u n d e r d i s t i l l e d w a t e r at
r o o m temperature.
V I I I . F o r m a t e buffer (0.1 M ; p H = 3.2):
D i l u t e 4 . 3 6 m l . f o r m i c a c i d a n d 2 0 m l . N N a O H t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r ; s t o r e at
4°C.
IX. p-Dimethylaminobenzaldehyde : 1 8
D i s s o l v e 2 g. p - d i m e t h y l a m i n o b e n z a l d e h y d e in 5 0 m l . 1 N H C 1 . M a k e u p fresh s o l u t i o n
daily.
* N o t m o r e t h a n 1 //mole of C A P should be used in this case, since the C A A formed can be determined in
the c o l o u r test only in the range between 0.05 a n d 1 /miole. [ C ] - A s p a r t a t e m a y be used as the substrate.
12
F o r smaller quantities of C A P , t h e d e t e r m i n a t i o n must be carried out by the radioactive m e t h o d des

cribed below.
X . S u l p h u r i c a c i d ( a p p r o x . 12 M ) :
A l l o w 330 ml. cone. H S 0 2 4 t o run slowly into 170 ml. ice-cold distilled water in an ice bath
w i t h stirring ( c a u t i o n ) ; s t o r e at r o o m t e m p e r a t u r e .
X I . D i p h e n y l a m i n e - p - s u l p h o n a t e (4.1 m M ) :
D i s s o l v e 1 1 4 m g . s o d i u m salt in 1 0 0 m l . 0 . 1 N H C 1 . S t o r e at 4 ° C i n b r o w n b o t t l e s o r b o t t l e s
w r a p p e d in a l u m i n i u m foil.
XII. Diacetyl monoxime (2.25% w/v):
D i s s o l v e 2 . 2 5 g. d i a c e t y l m o x i m e in 1 0 0 m l . d i s t i l l e d w a t e r ; m a k e u p freshly e v e r y w e e k ,
a n d s t o r e at 4 ° C in b r o w n b o t t l e s o r b o t t l e s w r a p p e d in a l u m i n i u m foil.
X I I I . Brij 3 5 :
A d d 5 g. Brij t o 2 5 m l . d i s t i l l e d w a t e r , a n d a l l o w t h e m i x t u r e t o s t a n d o v e r n i g h t at 4 ° C
w i t h o u t s h a k i n g ; s t o r e at 4 ° C . M a k e u p freshly e v e r y t w o m o n t h s .
XIV. Potassium persulphate (approx. 9 m M ) :
Dissolve 250 mg. K S 0 2 2 8 in d i s t i l l e d w a t e r t o 1 0 0 m l . ; s t o r e at 4 ° C , a n d p r e p a r e fresh
solution every t w o m o n t h s .
X V . C A A colour reagent:
Introduce 3 v o l u m e s sulphuric acid (X), 1 v o l u m e diphenylamine-p-sulphonate solution
( X I ) , 1 v o l u m e d i a c e t y l m o n o x i m e s o l u t i o n ( X I I ) , a n d 0 . 0 1 6 v o l u m e Brij s o l u t i o n ( X I I I )
i n t o a c o n t a i n e r in this o r d e r a n d m i x .
P r e p a r e fresh s o l u t i o n d a i l y . F r e s h r e a g e n t ( X I ) o c c a s i o n a l l y h a s a p a l e g r e e n c o l o u r a n d
g i v e s a p u r p l e - r e d c o l o u r in s u l p h u r i c a c i d ; this u s u a l l y d i s a p p e a r s o n a d d i t i o n o f s o l u t
i o n s ( X I I ) a n d ( X I I I ) if t h e m i x t u r e is h e a t e d for 5 m i n . at 6 0 ° C . If t h e c o l o u r d o e s n o t
d i s a p p e a r , m a k e u p fresh s o l u t i o n s ( X I ) a n d ( X I I ) .
X V I . C a r b a m o y l a s p a r t a t e , C A A ( s t o c k s o l u t i o n 10 m M ) :
S l o w l y a d d 10 m l . 0.1 N K O H t o 8 8 . 0 m g . N - c a r b a m o y l - L - a s p a r t i c a c i d in a 5 0 m l .
g r a d u a t e d flask u n t i l s o l u t i o n o c c u r s . M a k e u p t o 5 0 m l . w i t h d i s t i l l e d w a t e r ; k e e p f r o z e n .
D i l u t e 1 : 5 0 w i t h distilled w a t e r i m m e d i a t e l y b e f o r e u s e a s t h e s t a n d a r d s o l u t i o n for t h e
C A A c o l o u r test ( s o l u t i o n X V I a ) .
Procedure
Collection and treatment of sample
P l a s m a : I n t r o d u c e 0 . 1 3 3 m l . o f t h e f o l l o w i n g s o l u t i o n i n t o c e n t r i f u g e t u b e s i n a n i c e b a t h for
8
e a c h m l . o f b l o o d : 15 m g . d e x t r a n 4 0 , 1 5 m g . g l u c o s e , 4 . 2 5 m g . N a C l , a n d 15 m g . E D T A - N a H 2 2
• 2 H 0 per m l . , p H 7.4. I n t r o d u c e freshly c o l l e c t e d b l o o d i n t o t h e t u b e s , m i x w i t h t h e a q u e o u s

2
s o l u t i o n b y s h a k i n g , c e n t r i f u g e for 2 0 m i n . at 0 ° C t o r e m o v e c e l l s , d e c a n t t h e p l a s m a , a n d a n a l y s e
i m m e d i a t e l y . If p l a s m a m u s t b e s t o r e d , e v e n for o n l y a f e w m i n u t e s , it m u s t b e p l a c e d in ice.
T i s s u e : W e h a v e s o far i n v e s t i g a t e d o n l y r a b b i t p l a s m a . H o w e v e r , it s h o u l d b e p o s s i b l e t o u s e a
d e p r o t e i n i z e d t i s s u e h o m o g e n a t e if it is c a r e f u l l y p r e p a r e d . H o m o g e n a t e s t r e a t e d w i t h p e r c h l o r i c
acid and carefully neutralized with K H C 0 3 s h o u l d b e s u i t a b l e . N o t e t h e f o l l o w i n g p o i n t s in t h e
p r e p a r a t i o n . A f t e r c o l l e c t i o n , r a p i d l y c o o l t h e t i s s u e in i c e - c o l d 2 0 m M tris buffer, p H 7.8. S i n c e
t h e C A P c o n c e n t r a t i o n in t i s s u e s is l o w , t h e t i s s u e s s h o u l d b e h o m o g e n i z e d at a h i g h c o n c e n t r a
t i o n , e . g . 1 g. o f tissue per 3 m l . o f i c e - c o l d 2 0 m M tris buffer, p H 7.8. D e p r o t e i n i z e t h e h o m o -
genates immediately, since tissue extracts contain C A P - h y d r o l y s i n g p h o s p h a t a s e s . To a v o i d 1 9
further d i l u t i o n , u s e 0.1 v o l u m e o f i c e - c o l d 5 N H C 1 0 ; c o o l w e l l . I m m e d i a t e l y after c e n t r i f u g a

4
t i o n , carefully n e u t r a l i z e t h e e x t r a c t w i t h 2.5 M K C 0 2 3 solution (pH 6 - 8 ) to avoid chemical

hydrolysis of C A P 1 3
. Again cool well; remove K 0 0 4 in t h e c o l d . T h e t o t a l t i m e f r o m t h e c o l l e c
tion o f the tissue until the i n c u b a t i o n with aspartate t r a n s c a r b a m o y l a s e s h o u l d n o t exceed 3 0 -
4 0 m i n . U n l i k e p l a s m a , o t h e r t i s s u e s c o n t a i n c o n s i d e r a b l e q u a n t i t i e s o f free a s p a r t i c a c i d , w h i c h
r e d u c e s t h e specific r a d i o a c t i v i t y o f [ C ] - a s p a r t a t e ; it is t h e r e f o r e p r o b a b l y n e c e s s a r y t o d e t e r
1 4
m i n e t h e a s p a r t a t e c o n t e n t s e p a r a t e l y in t h e p r o t e i n - f r e e e x t r a c t s .
C A P - e n r i c h e d p l a s m a a n d t i s s u e e x t r a c t s : S i n c e t h e r e c o v e r y o f a d d e d C A P (final c o n c e n t r a t i o n
4 x 10 ~ o r 4 x 1 0 ~ M ) w a s o n l y 7 5 %, t h e r e c o v e r y s h o u l d a l w a y s b e c h e c k e d . I n t h e a n a l y s i s
5 6
o f p l a s m a , C A P s o l u t i o n d i l u t e d w i t h 0 . 8 5 % i c e - c o l d N a C l s o l u t i o n is a d d e d t o t h e b l o o d b e f o r e
c e n t r i f u g a t i o n . T h e C A P c o n c e n t r a t i o n e x p e c t e d i n t h e p l a s m a o r t i s s u e e x t r a c t , e. g. 5 0 pM or
l e s s , s h o u l d b e a d d e d a s a s t a n d a r d s o l u t i o n in a v o l u m e c o r r e s p o n d i n g t o 1 o r 2 % o f t h e s a m p l e
v o l u m e . In the case o f tissue extracts, standard C A P solution should be a d d e d before deprotein
ization.
Stability of sample
Samples should not be stored, but c a r b a m o y l p h o s p h a t e should be converted enzymatically into

[ C ] - C A A a s q u i c k l y a s p o s s i b l e . A f t e r this e n z y m a t i c r e a c t i o n , d e p r o t e i n i z a t i o n , a n d n e u t r a l
1 4
ization, the samples can be stored.

Assay System
Enzymatic reaction
I n c u b a t i o n t e m p e r a t u r e : 37 ° C ; i n c u b a t i o n v o l u m e : 1 m l . , final v o l u m e : 4 m l . T h r e e m i x t u r e s
are n e c e s s a r y ; t u b e 1: s a m p l e ; t u b e 2 : s a m p l e + a d d e d C A P ; t u b e 3 : b l a n k w i t h s a m p l e .
Pipette into centrifuge tubes 1 2 3 C o n c e n t r a t i o n in

assay mixture

Sample + C A P * 0.85 ml.
[ C]-Aspartate solution
14
(I) 0.05 ml. 0.05 ml. 10 m M
Tris buffer (II) 0.05 ml. 0.05 ml. 0.05 ml. 50 m M
ATCase solution (III) 0.05 ml. 0.05 ml. 0.05 ml. approx. 8 U/ml.
M i x , i n c u b a t e f o r 15 m i n . a n d t h e n c o o l in ice.
C C A solution (XVI) 1.00 m l . 1.00 m l . 1.00 m l . 2.5 m M

[ C]-Aspartate solution
14
(I) 0.05 ml. 2.5 m M
H C 1 0 solution
4 (V) 2.00 ml. 2.00 ml. 2.00 ml. 0.5 N
M i x , c e n t r i f u g e , d e c a n t s u p e r n a t a n t fluids i n t o c o n i c a l c e n t r i f u g e t u b e s
w i t h 0.1 m l . g r a d u a t i o n s , w a s h e a c h s e d i m e n t w i t h 2 m l . o f 0 . 4 N
HCIO4, c e n t r i f u g e , c o m b i n e s u p e r n a t a n t s w i t h first s u p e r n a t a n t s . N e u
tralize w i t h 2 N K O H ( p H 6 - 8 ) , c o o l t o 0 ° C , c e n t r i f u g e ( o r d e c a n t ) , u s e
supernatant**.
* According t o p . 1753.
** This neutral solution can be kept indefinitely at - 2 0 °C. In the unneutralized solution, C A A would
cyclize to form h y d a n t o i n .
Separation of[ CJ-CAA

14
[ C ] - C A A m u s t be separated from [ C ] - a s p a r t a t e a n d from other p r o d u c t s formed from [ C ] - a s p a r t a t e

14 14 14
d u r i n g the enzymatic reaction (e. g. pyruvate, oxaloacetate). M o r e t h a n 9 8 % of [ C ] - a s p a r t a t e is adsorbed

14
o n Dowex-50 ( H f o r m ) ; [ C ] - C A A passes t h r o u g h . T h e remaining 2 % of [ C ] - a s p a r t a t e is removed on

+ 14 14
passage t h r o u g h Dowex-1 (aspartate h a s a charge close t o zero at p H 3.2; C A A is negatively charged).

T h e fractions a r o u n d the C A A peak are determined in the colour test a n d by radioactivity measurements.
T h e colour test determines the " c o l d " carrier C A A (solution X V I ) a d d e d at the end of the reaction,
while the radioactivity m e a s u r e m e n t determines the quantity of [ C ] - C A P that has reacted enzyma-
12
tically with [ C ] - a s p a r t a t e to form [ C 1 - C A A . T h e specific radioactivity of C A A should theoretically be

14 14
constant in t h e fractions. However, since a dicarboxylic acid is formed from a s p a r t a t e in serum a n d is eluted
immediately after C A A , the specific radioactivity of [ C ] - C A A is constant only in the first half of the p e a k .
14 8
To ensure t h a t only p u r e fractions with constant radioactivities are used for the calculation, radioactivity
m e a s u r e m e n t s m u s t be carried o u t in all fractions.
Introduce a layer o f water over the D o w e x - 5 0 c o l u m n ( H +
form),* i n s i d e d i a m e t e r 1 c m . ,
packing depth 9 - 1 0 c m . , a n d also over a D o w e x - 1 c o l u m n . A d d the neutralized extract o f the
incubation mixtures, including 5 ml. o f w a s h water, o n t o the D o w e x - 5 0 c o l u m n . Rinse the
c o l u m n w i t h 2 0 m l . d i s t i l l e d w a t e r ; [ C ] - C A A p a s s e s t h r o u g h . C o l l e c t all t h e w a t e r r u n n i n g
1 4
f r o m t h e c o l u m n in a 5 0 m l . E r l e n m a y e r flask. If t h e p H o f t h e e l u a t e is b e l o w 7.0, n e u t r a l i z e
w i t h K O H . I n t r o d u c e all t h e e l u a t e o n t o t h e D o w e x - 1 c o l u m n , a n d rinse t h e E r l e n m e y e r flask
a n d t h e s i d e s o f t h e c o l u m n w i t h w a t e r . E l u t e w i t h 3 5 0 m l . 0.1 N f o r m a t e s o l u t i o n ( V I I I ) .
C o l l e c t t h e e l u a t e s in 10 m l . f r a c t i o n s ( 3 5 f r a c t i o n s ) , a n d s h a k e w e l l . A p p l y o n e d r o p f r o m
e a c h f r a c t i o n t o filter p a p e r , a l l o w t o d r y , a n d s p r a y w i t h p - d i m e t h y l a m i n o b e n z a l d e h y d e s o
l u t i o n ( I X ) . C a r r y o u t t h e c o l o u r test f o r C A A a n d t h e r a d i o a c t i v i t y m e a s u r e m e n t o n t h e frac
tion giving the strongest colour (usually fraction 22), as well as the previous 8 fractions and the
4 subsequent fractions.
Radioactive measurements
F o r m e a s u r e m e n t s with the G e i g e r counter, apply 0.5 ml. o f e a c h fraction t o etched glass
p l a t e s ( d u p l i c a t e d e t e r m i n a t i o n s ) . W h e n a s c i n t i l l a t i o n c o u n t e r is u s e d , i n t r o d u c e 0.5 m l . o f
e a c h f r a c t i o n i n t o 1.5 m l . d i s t i l l e d w a t e r , a n d a d d 18 m l . d i o x a n e - n a p h t h a l e n e s c i n t i l l a t i o n l i q u i d .
Determine the n u m b e r o f counts per min. from [ C]-aspartate as follows. M i x an appropriate
14
a m o u n t o f the [ C ] - a s p a r t a t e standard w i t h s o l u t i o n VIII a n d c o u n t in the s a m e w a y as [ C ] -

14 1 4
C A A . T h e specific a c t i v i t i e s o f [ C ] - a s p a r t a t e a n d [ C ] - C A A are e x p r e s s e d a s d i s i n t e g r a t i o n s
14 1 4
per m i n . per //mole.
CAA Colour Test

Wavelength: approx. 560 n m ; light p a t h : 1 c m . ; test v o l u m e 6.5 m l . ; 2 2 ° C ; m e a s u r e against
w a t e r . C a r r y o u t i n c u b a t i o n s a n d c o o l i n g i n t h e d a r k , s i n c e t h e c o l o u r is s e n s i t i v e t o light.
P r e p a r e a b l a n k w i t h w a t e r i n s t e a d o f s a m p l e for e a c h series o f m e a s u r e m e n t s .
S t a n d a r d s : U s e 0.1 t o 0 . 5 m l . o f C A A s t a n d a r d s o l u t i o n ( X V I a ) - f w a t e r t o 0 . 5 m l . i n s t e a d o f
s a m p l e ( c o r r e s p o n d i n g t o 0 . 0 2 t o 0.01 / / m o l e C A A ) .
P i p e t t e i n t o test t u b e s :
Sample 0.5 ml.

Colour reagent (XV) 5.0 m l .
M i x , c o v e r test t u b e s w i t h g l a s s s p h e r e s , a n d h e a t f o r
3 0 m i n . in 6 0 ° C w a t e r b a t h ; c o o l in i c e a n d k e e p i n 2 2 ° C
water bath.
Persulphate solution (XIV) 1.0 m l .
M i x ; after e x a c t l y 4 0 m i n . , t a k e t h e t e s t t u b e s f r o m t h e
the b a t h in the order o f addition o f persulphate a n d
measure the extinctions; subtract extinction o f blank;
J E is o b t a i n e d .
Calculations
Colour Test for CAA

T h e C A A concentration in the sample is determined by c o m p a r i s o n with s t a n d a r d s , a n d is given by
c = ^ W e x 2 xc S t a n d a r d [/miole/ml.]
^Standard
= concentration of the s t a n d a r d [^mole/ml.]

Cstandard
According to , A E = 0.700 at 560 n m for 0.1 umole of C A A in the assay mixture.

1 1
Specific Radioactivity
Divide the readings in c p m for the 0.5 ml. fractions by the n u m b e r of /rniole C A A / 0 . 5 m l . ; the result is the
specific radioactivity of [ C ] - C A A [cpm//miole].
14
T h e C A P c o n t e n t in the sample (plasma), simplified in accordance with p . 313, e q u a t i o n (6), is:
/rniole C A P - ^ m o l e
[ 1 2
C]- ) ( P - radioactivity of [ C ] - C A A )
C A A x s e c 14
Spec, radioactivity of [ C ] - a s p a r t a t e
14
10 /*mole of C A A [ C ] are a d d e d to the incubation mixture. After allowance for the sample volume
12
(0.85 ml.) a n d dilution of the sample with anticoagulants by a factor of 1.133, we o b t a i n
Spec, radioactivity of [ C ] - C A A14
/rniole c a r b a m o y l p h o s p h a t e / m l . p l a s m a = 13.3 x o _
Spec, , . ^ of [ C ] - a s p a r t a t e
radioactivity
A r 1 4 14
This very long m e t h o d is n o t very a c c u r a t e ; it does not even detect 100% of the C A P c o n t e n t in the plasma.
N o C A P is found in n o r m a l rabbit p l a s m a . O n a d d i t i o n of 0.04 o r 0.004 /rniole of
8 1 2
C - C A P to 1 ml.
of plasma, only 6 2 - 7 5 % was subsequently found (0.04 /rniole of C A P gave four fractions with 0.026,
0.020,0.025, a n d 0.028 /rniole, average 0.025). Better precision is p r o b a b l y possible if [ C ] - a s p a r t a t e having 14
a higher specific activity is u s e d . 8
T h e three tubes indicated in the pipetting scheme a b o v e give the following values: Tube 1 gives the C A P con
tent in the tissue, less the loss d u r i n g the p r e t r e a t m e n t of the tissue before a d d i t i o n of a s p a r t a t e a n d aspar
tate t r a n s c a r b a m o y l a s e to convert the unstable C A P into stable C A A . Tube 2 gives the fraction of the a d d e d
[ C ] - C A P t h a t is recovered together with the C A P c o n t e n t from tube 1. It allows the correction of the
12
C A P content found in tube 1. Tube 3 with [ C ] - a s p a r t a t e (added after the incubation) serves to check t h a t
14
the [ C ] - L - a s p a r t a t e c o n t a i n s n o radioactive impurity t h a t is eluted from Dowex-1 like C A A , a n d is

14
necessary because a few c o u n t s per min. are found in practice in all the fractions of the C A A peak. This is a
non-specific b l a n k , but a true " b a c k g r o u n d v a l u e " . T h e specific radioactivity of the C A A peak should be
subtracted from the values of the C A A p e a k s from tubes 1 a n d 2.
Normal Values
As was m e n t i o n e d earlier, the m e t h o d has been carried out only in n o r m a l r a b b i t plasma, in which n o C A P
was detected.
Effects due to therapeutic measures: None known.
Sources of error in the assay technique: 1. Chemical or enzymatic hydrolysis of C A P during the treat
ment of the s a m p l e ; this is taken into a c c o u n t by t u b e 2. 2. T h e formation of foreign substances from [ C ) - 14
a s p a r t a t e t h a t are eluted with C A A o n c h r o m a t o g r a p h y ; the purity of the C A A fractions in the peak is

checked by the symmetry of radioactivities a n d colours formed. 3. In tissues other t h a n plasma, the
a s p a r t a t e c o n c e n t r a t i o n can be so high t h a t the specific activity of [ C l - a s p a r t a t e is significantly reduced
14
by [ C ] - a s p a r t a t e in the extract. This is n o p r o b l e m in n o r m a l plasma, which contains less t h a n 0.1 //mole of

12
L-aspartate/ml. In clinical material in which the a s p a r t a t e concentration in the p l a s m a is increased, or in the

extracts of m o s t tissues with a s p a r t a t e c o n c e n t r a t i o n s of a r o u n d 100 /zmole/g. fresh weight, however, the
aspartate content of the tissue m a y be higher t h a n the 10 pinole of a d d e d [ C ] - L - a s p a r t a t e . This excess of

14
a s p a r t a t e does n o t inhibit the a s p a r t a t e t r a n s c a r b a m o y l a s e from E. coli, b u t it reduces t h e specific activity

of [ C ] - a s p a r t a t e . This value m u s t therefore be accurately k n o w n in o r d e r to be able to calculate the C A P
14
content of the tissue, a n d since a decrease in the specific radioactivity of [ C ] - a s p a r t a t e decreases the sensi
14
tivity of the m e t h o d .
Dicarboxylic acids such as maleate or succinate inhibit a s p a r t a t e t r a n s c a r b a m o y l a s e from E. coli, a n d these
acids should n o t therefore be a d d e d to the tissues or tissue e x t r a c t s . C T P is a s t r o n g inhibitor of the enzyme
17
1 7
. Fluoride, arsenate, a n d p h o s p h a t e inhibit the e n z y m e ; these ions should be avoided in the assay mixture
15
A c e t y l p h o s p h a t e can serve as the substrate instead of C A P , so t h a t this process is n o t really a test for C A P
in cells t h a t synthesize a c e t y l p h o s p h a t e . H o w e v e r , since a c e t y l p h o s p h a t e is n o t k n o w n t o occur in m a m
19
malian tissues, this is n o t a p r o b l e m ; o n the other h a n d , it limits the applicability of the m e t h o d to bacteria.
The specificity of the enzyme for L-aspartate is m a r k e d , t h o u g h erythro-/?-OH-aspartate can also serve as
the s u b s t r a t e ; however, this is rarely e n c o u n t e r e d in practice.
20
References
1 S. Grisolia & P. P. Cohen, J. biol. C h e m . 204, 753 [1953].

2 P. P. Cohen & G. W. Brown, jr. in M. Florkin & H. S. Mason, C o m p a r a t i v e Biochemistry, Vol. II,
p. 161, Academic Press, N e w York, 1960
3 R. T. Schimke, J. biol. C h e m . 237, 459 [1962].
4 S. E. Hager & M. E. Jones, J. biol. C h e m . 242, 5667 [1967].
5 M. Tatibana & K. Ito, Biochem. Biophys. Res. C o m m u n s , 26, 221 [19671
6 S. E. Hager & M. E. Jones, J. biol. C h e m . 240, 4 5 5 6 [1965].
7 S. E. Hager & M. E. Jones, J. biol. C h e m . 242, 5 6 7 4 [1967].
8 A. Herzfeld, S. E. Hager & M. E. Jones, A r c h . Biochem. Biophys. 707, 544 [1964].
9 M. Nakamura & M. E. Jones in H. Tabor & C W. Tabor, M e t h o d s of E n z y m o l o g y , Vol. 17 A, p . 286.
Academic Press, N e w York.
10 J. C Gerhart & H Holoubeck, J. biol. C h e m . 242, 2 8 8 6 [1967].
11 H. Reichard&P. Reichard, J. L a b . Clin. M e d . 52, 709 [1958].
12 L. H Smith, jr. & F. A. Baker, J. Clin. Invest. 38, 798 [1959].
13 CM. Allen & M. E. Jones, Biochemistry 3, 1238 [1964].
14 M. R. Bethell & M. E. Jones, P r o c . N a t l . A c a d . Sci., U . S. 60, 1442 [1968].
15 K. Kleppe, Biochim. Biophys. A c t a 122, 450 [1966].
16 M. Shepherdson & A. B. Pardee, J. biol. C h e m . 235, 3 3 3 3 [I960].
17 / . C. Gerhart & A. B. Pardee, J. biol. C h e m . 237, 891 [1962].
18 D.M. P. Philips, Biochim. Biophys. A c t a 13, 560 [1954].
19 S. Grisolia & L. Raijman, Advances in Chemistry 44, 128 [1964].
20 M. L. Kornguth & H. J. Sallach, A r c h . Biochem. Biophys. 21, 39 [I960].
Carnitine and Acylcarnitines
D a v i d J. P e a r s o n , P h i l i p K. T u b b s a n d J a m e s F . A . C h a s e t
Carnitine (3-hydroxy-4-trimethylaminobutyric acid) a n d its o-acyl esters a r e found in m a n y a n i m a l

tissues, particularly muscle, a n d in p l a n t s a n d y e a s t s . Only the l a e v o r o t a r y isomer occurs in N a t u r e .
1 2
T h e reversible acylation of (-)-carnitine proceeds in principle according to e q u a t i o n (1). T h e enzyme car

nitine acetyltransferase ( A c e t y l - C o A : carnitine O-acetyltransferase, E C 2.3.1.7) is specific for the transfer
of m o n o c a r b o x y l i c acid residues (2 to 10 C a t o m s 3 , 4
) , while carnitine palmitoyltransferase ( P a l m i t o y l - C o A :
L-carnitine (^-palmitoyltransferase, E C 2.3.1.21) transfer longer-chain acyl g r o u p s . Carnitine acetyl- 5
transferase p r e p a r a t i o n s active e n o u g h t o allow the enzymatic d e t e r m i n a t i o n of carnitine a n d its derivatives

have been available for s o m e years.
Application of Method: In biochemistry, p l a n t physiology, a n d clinical biochemistry for d e t e r m i n a t i o n of

carnitine a n d its derivatives in a n y biological material.
Unesterified Carnitine
In a system t h a t contains acetyl coenzyme A in excess a n d carnitine acetyltransferase, carnitine is acetylated;
a stoichiometric a m o u n t of coenzyme A is formed. If coenzyme A is allowed to react further in a coupled
irreversible action, carnitine can be d e t e r m i n e d quantitatively. Two m e t h o d s have proved suitable.
T h e extinction coefficient 2
of s o r b y l - C o A at 300 n m is 23.5 + 0.5 c m . / ^ m o l e . According t o eqn. (2) on
2
T h e thiokinase m e t h o d (Acid: C o A ligase ( A M P - f o r m i n g ) , E C 6.2.1.3) is very sensitive, a n d is particularly

suitable for tissue extracts, t h o u g h it can also be used for the d e t e r m i n a t i o n of carnitine in solutions.
However, purified thiokinase is required, a n d the assay system is relatively complicated.
T h e D T N B m e t h o d (5,5'-dithiobis-(2-nitrobenzoate)) is less sensitive b u t simple a n d fast. It is the m e t h o d
of choice for the d e t e r m i n a t i o n of carnitine in solution. Its use for the m e a s u r e m e n t of carnitine content in
tissue is limited by the fact t h a t tissue extracts contain substances t h a t reduce D T N B . These can m a k e the
extinction t o o high before the enzyme reaction. Marquis a n d Fritz 6
have r e c o m m e n d e d t h a t the deproteini
zed extract at p H 8.5 be heated for 5 m i n u t e s at 90 °C to minimize this effect; this t r e a t m e n t oxidizes inter
fering thiols. However, liver extracts c a n n o t be analysed by this m e t h o d even after such a preliminary
treatment, because their thiol c o n t e n t is t o o high. M o r e o v e r , significant hydrolysis of acetylcarnitine
occurs o n heating even in mild alkali.
(—)-Carnitine by the Thiokinase Method

Principle
(1) Ac-CoA + Carnitine , c a r n i t i n e

- Acetylcarnitine + C o A S H
acetyltransferase .
I
(2) C o A S H + A T P + Sorbate t h i o k i n a s e
> Sorbyl-CoA + A M P + PPj
T h e increase in s o r b y l - C o A , as m e a s u r e d by the increase in extinction at 300 n m , is p r o p o r t i o n a l to the

7
a m o u n t of carnitine present.
Deceased.
Carnitine a n d Acylcarnitines 1759
Reaction (2) is irreversible with excess A T P a n d sorbate. p H Values a b o v e 8.5 should be avoided, because
carnitine acetyltransferase is otherwise very rapidly i n a c t i v a t e d . T h e e n z y m e is also strongly inhibited by
3
metal i o n s ; the metal c o n t e n t of tissue extracts is often sufficient. This inhibition is prevented by the
addition of 1.25 m M E D T A .
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at 3 0 0 n m , p r e f e r a b l y w i t h c o u p l e d
recorder. Cuvette holder with exact constant-temperature c o n t r o l .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 10. Perchloric acid, A . R . , 7 0 % ( w / w ) , sp.gr.

2. M a g n e s i u m chloride, A . R., MgCl 2 1.67
6H 0 2 11. Acetyl coenzyme A, A c - C o A
3. S o r b i c a c i d , C H C H = C H • C H =
3 F o r commercial p r e p a r a t i o n s * as
= CHCOOH A c - C o A - L i H - 3 H 0 , see p . 524. O r p r e p a r e d
3 2
recrystallized twice from w a t e r from C o A by t r e a t m e n t with acetic a n h y d r i d e

4. H y d r o c h l o r i c acid, 1 N according t o . 8
5. P o t a s s i u m h y d r o x i d e , 1 N 12. Carnitine acetyltransferase, A C T

6. P o t a s s i u m h y d r o g e n c a r b o n a t e , K H C 0 3 crystallized from pigeon pectoral muscle, sus
7. P o t a s s i u m d i h y d r o g e n phosphate, pension in a m m o n i u m sulphate. A p p r o x . 120
KH P0 2 4 U / m g . (25 ° C ) . F o r c o m m e r c i a l p r e p a r a t i o n ,
9
8. A d e n o s i n e t r i p h o s p h a t e , A T P see p . 438.
d i s o d i u m salt, A T P - N a H - 3 H 0 ; for
2 2 2 com 13. Thiokinase, T K
mercial p r e p a r a t i o n , see p . 527. from bovine liver m i t o c h o n d r i a ; ^ 0.6 U / m g .
9. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA, (25 °C). P r e p a r e d a c c o r d i n g t o 1 0
u p to puri
as d i s o d i u m salt, E D T A - N a H - 2 H 0 . 2 2 2 fication step C ; further t r e a t m e n t with calcium
p h o s p h a t e gel a n d c h r o m a t o g r a p h y on D E A E -
cellulose to r e m o v e interfering e n z y m e s . 2
Purity of Reagents
Carnitine acetyltransferase should be free from acetyl-CoA hydrolase. T h i o k i n a s e m u s t n o t contain

carnitine acetyltransferase a n d c o n t a m i n a t i o n with acetyl-CoA hydrolase should be less t h a n 0.05 %.
Preparation of Solution
U s e water distilled from glass.

I. Tris buffer ( 0 . 4 M ; p H 8 . 2 ; 2 0 m M e a c h o f s o r b a t e , M g C l , A T P ) : 2
a) D i s s o l v e 4.8 g. tris a n d 1.0 g. M g C l 2 • 6 H 0 in a p p r o x . 3 0 m l . d i s t i l l e d w a t e r , a n d

2
a d d 18 m l . 1 N H C 1 . b ) S u s p e n d 0 . 2 2 g. s o r b i c a c i d i n a p p r o x . 2 0 m l . d i s t i l l e d w a t e r , a n d
a p p r o x i m a t e l y n e u t r a l i z e w i t h 2 m l . 1 N K O H . c ) D i s s o l v e 3 . 0 g. A T P - N a H 2 2 i n 10 m l .
d i s t i l l e d w a t e r , a n d n e u t r a l i z e w i t h a p p r o x . 3 m l . 1 N K O H . M i x s o l u t i o n s a, b , a n d c,
* We have h a d n o experience with c o m m e r c i a l p r e p a r a t i o n s

a d j u s t t o p H 8.2 w i t h 1 N K O H o r 1 N H C 1 ( g l a s s e l e c t r o d e ) , a n d m a k e u p t o 100 m l .
II. A c e t y l c o e n z y m e A , A c - C o A ( 1 5 m M ) :
Adjust solution prepared according t o 8
to p H 4 - 5 with HC1 or K H C 0 . 3 Determine
content according to p. 1988. Or dissolve 26.4 mg. o f A c - C o A (commercial preparation)
in 2 ml. o f water.
III. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E D T A ( 5 0 m M ) :
D i s s o l v e 0 . 4 6 g. E D T A - N a H - 2 H 0 in 2 0 m l . distilled w a t e r , a d j u s t t o p H 8 w i t h 1 N
2 2 2
K O H (glass electrode), a n d m a k e u p t o 25 ml. with water.

I V . P h o s p h a t e buffer ( 0 . 5 M ; p H 7 . 5 ) :
D i s s o l v e 6.8 g. K H P 0 2 4 in a p p r o x . 7 0 m l . o f w a t e r , a d j u s t t o p H 7.5 w i t h a p p r o x . 2 . 2 m l .
1 N K O H , a n d m a k e u p to 100 ml. with distilled water.
V. Thiokinase, T K (5mg. protein/ml.):
S o l u t i o n in 2 0 m M K H C 0 3 according to 2 1 0
V I . C a r n i t i n e a c e t y l t r a n s f e r a s e , A C T (1 m g . p r o t e i n / m l . ) :
D i s s o l v e 1 m l . s u s p e n s i o n in 4 m l . p o t a s s i u m p h o s p h a t e buffer ( I V ) .
VII. Perchloric acid (approx. 50 m M ) :
Dilute 4.2 ml. 7 0 % H C 1 0 4 to 100 ml. with distilled water.
Solutions I, II, and V keep for several m o n t h s when frozen at - 1 5 ° C ; solutions III, IV, VI, and VII can
be kept for a similar time at 4 °C.
Procedure
Collection of sample and deproteinization:
R a p i d p o s t - m o r t e m c h a n g e s o c c u r in t h e a c y l a t i o n s t a t u s o f c a r n i t i n e . To k e e p t h e s e c h a n g e s
2
s m a l l c o l l e c t t i s s u e s a m p l e s w i t h " q u i c k - f r e e z e " t o n g s (see p . 4 0 0 ) , o r i n t r o d u c e i n t o a c e t o n e -

dry ice m i x t u r e i m m e d i a t e l y after t h e d e a t h o f t h e a n i m a l . P o w d e r t h e f r o z e n t i s s u e in a steel
m o r t a r , q u i c k l y w e i g h a n d d e p r o t e i n i z e : triturate in a c o o l e d m o r t a r w i t h 2 t o 3 m l . i c e - c o l d per
c h l o r i c a c i d ( V I I ) p e r g r a m o f t i s s u e ; c e n t r i f u g e ; a d d 0 . 0 2 5 m l . p h o s p h a t e buffer ( I V ) p e r m l . o f
e x t r a c t t o a n a l i q u o t o f t h e s u p e r n a t a n t fluid, a n d c a r e f u l l y adjust t o p H 6.5 t o 7.0 w i t h 1 N K O H .
( T h e p H m u s t n o t in a n y c i r c u m s t a n c e s e x c e e d 8.5, s i n c e s h o r t - c h a i n a c y l c a r n i t i n e s are o t h e r
wise hydrolysed.) Separate K C 1 0 4 in t h e c o l d , a n d a n a l y s e t h e s u p e r n a t a n t fluid for c a r n i t i n e
a n d esterified c a r n i t i n e s .
C a r n i t i n e a n d a c y l c a r n i t i n e s are s t a b l e for s e v e r a l d a y s in t h e n e u t r a l i z e d extract at —15 ° C .

Assay System
W a v e l e n g t h : 3 0 0 n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 0 m l . ; 25 ° C , m a i n t a i n t e m p e r a t u r e
strictly. R e a d a g a i n s t air o r w a t e r .
Pipette into cuvettes: C o n c e n t r a t i o n in

assay mixture
Sample (deproteinized, neutralized) u p t o 1.3 m l . 1.5 t o 2 5 fiM carnitine

Tris buffer (I) 0.50 ml. 0.1 M tris, 5 m M e a c h
of sorbate, M g C l , A T P
2
A c - C o A solution (II) 0.02 ml. 0.15 m M

E D T A solution (III) 0.05 ml. 1.25 m M
Water t o 1.965 m l .
M i x , adjust temperature t o 25 °C.
T K solution (V) 0.025 ml. approx. 3 5 - 4 5 m U / m l .
M i x ; C o A in t h e s a m p l e l e a d s t o a r a p i d i n c r e a s e in
extinction. After 2 to 5 min., the reaction "creeps"
b e c a u s e o f l o w a c e t y l - C o A h y d r o l a s e a c t i v i t y in t h e
TK. Read E x and immediately pipette:
A C T solution (VI) 0.01 m l . 5 //g./ml. = 600 m U / m l .
M i x ; after 6 t o 15 m i n . r e a d e x t i n c t i o n s e v e r a l t i m e s ,
determine E 2 by extrapolation to the time o f addition
of ACT. E 2 — E j = AE is u s e d in t h e c a l c u l a t i o n s .
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o a d d i t i o n o f A C T s o l u t i o n ( V I ) b y a d d i n g a further
0.01 m l . o f s o l u t i o n ( V I ) at t h e e n d o f t h e r e a c t i o n . S u b t r a c t c h a n g e i n e x t i n c t i o n f r o m AE.
Calculations
T h e extinction coefficient 2
of sorbyl-CoA at 300 n m is 23.5 + 0.5 c m . / / i m o l e . According to eqn. (2) on2
p. 312, therefore, with an assay v o l u m e of 2 ml. a n d v ml. of sample, the c o n c e n t r a t i o n is:
2000 X ZlE r i y i n
C =
23.5 x v r n m o l e
/ m L
l
Dilution of the sample during p r e t r e a t m e n t m u s t also be taken into account.
Values of 30 + 0.46 n m o l e ( + 1.5%) were found with ( - )-carnitine s t a n d a r d solutions.
N o r m a l Values
Typical values for free carnitine in tissues of rats (fed) are, e. g. 302 + 46 n m o l e per g. of frozen cardiac
muscle a n d 173 + 24 n m o l e per g. of frozen liver (means + S.D). 2
A serious source of error is inconstancy of the measuring t e m p e r a t u r e . W a r m i n g during the m e a s u r e m e n t

leads to an increase in the extinction at 300 n m ; the p H of the tris buffer decreases, a n d m o r e undissociated
sorbic acid is formed. Very exact t e m p e r a t u r e control of the cuvette h o l d e r is necessary.
Carnitine acetyltransferase is highly specific for ( —)-carnitine. T h e only k n o w n analogue t h a t would also be
determined by this m e t h o d is n o r c a r n i t i n e 11
(3-hydroxy-4-dimethylaminobutyric acid); however, this
substance has n o t yet been found in biological material. ( + ) - C a r n i t i n e has a weak competitive inhibiting
action.
(—)-Carnitine by the DTNB* Method

Principle
(1) A c - C o A + Carnitine t
c a r m t i n e
* Acetylcarnitine + C o A S H
acetyltransferase
(2) C o A S H + S-(( )>-N0 2 S-CoA / 2
SH^JKNO 2 s^y>-No 2
co 2
_
C0 "
2
C o A S H formed in the enzymatic reaction (1) reacts non-enzymatically with D T N B to form the yellow
5-thio-2-nitrobenzoate anion, which absorbs strongly at 412 n m .
This m e t h o d is only slightly influenced by p H changes. T h e p H m u s t n o t exceed 8.5, since the carnitine
acetyltransferase is otherwise inactivated; the p H should n o t fall below 7.0, since insufficient dissociation of
the dye 5-thio-2-nitrobenzoate would then lead to excessively low results. E D T A m u s t be a d d e d for
m e a s u r e m e n t s in tissue extracts (see p . 1759).
Equipment
S p e c t r o p h o t o m e t e r , s p e c t r u m - l i n e p h o t o m e t e r , o r c o l o r i m e t e r s u i t a b l e for a c c u r a t e m e a s u r e
m e n t s at 4 1 2 n m ; p r e f e r a b l y w i t h c o u p l e d r e c o r d e r .
Reagents
1. 5 , 5 ' - D i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) , D T N B
e.g. from Aldrich Chemical Co. Inc., Milwaukee, Wis., U S A .
T h e o t h e r r e a g e n t s r e q u i r e d for t h e p r e p a r a t i o n o f s o l u t i o n s II t o V I are l i s t e d o n p . 1 7 6 0 .
* D T N B = 5,5'-dithiobis-(2-nitrobenzoic acid).
U s e w a t e r distilled f r o m g l a s s .
I. Tris buffer (1 M ; p H 7 . 8 ) :
D i s s o l v e 12.1 g. tris in 65 m l . 1 N H C 1 , a n d m a k e u p t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r .
II. 5 , 5 ' - D i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) , D T N B ( 1 0 m M ) :
D i s s o l v e 4 0 m g . D T N B in a p p r o x . 5 m l . 2 % K H C 0 3 solution, adjust t o p H 7 to 8 with
1 N H C 1 , a n d m a k e u p t o 10 m l . w i t h distilled w a t e r .
P r e p a r e t h e o t h e r s o l u t i o n s a s d e s c r i b e d o n p. 1 7 6 0 .
III. A c e t y l c o e n z y m e A , A c - C o A (15 m M )
IV. Ethylenediaminetetra-acetate, E D T A (50 m M )
V . C a r n i t i n e a c e t y l t r a n s f e r a s e , A C T (1 m g . p r o t e i n / m l . )
VI. Perchloric acid (approx. 50 m M )
Solutions I, V, a n d VI keep for several m o n t h s at 4 °C, a n d solutions II to IV keep for a similar time when
frozen at —15 °C.
Procedure
S a m p l e c o l l e c t i o n , t r e a t m e n t , a n d stability o f t h e s a m p l e a s d e s c r i b e d o n p . 1 7 6 0 .
Assay System
W a v e l e n g t h : 4 1 2 n m ; light p a t h : 1 c m . ; final v o l u m e : 2.0 m l . ; r o o m t e m p e r a t u r e ; m e a s u r e

a g a i n s t air.

assay mixture
Sample (deproteinized, neutralized) u p t o 1.6 m l . 3 to 40 ^ M carnitine

D T N B solution (II) 0.025 ml. 0.125 m M
A c - C o A solution (III) 0.02 ml. 0.15 m M
E D T A solution (IV) 0.05 ml. 1.25 m M
Water t o 1.99 m l . t o 1.99 m l .
Mix, read extinction E . x
A C T solution (V) 0.01 m l . 5 fig./ml = 600 m U / m l .
M i x ; after 3 - 5 m i n . r e a d e x t i n c t i o n several t i m e s . T h e
r e a c t i o n " c r e e p s " if A C T still c o n t a i n s traces o f
acetyl-CoA hydrolase. Determine E by extrapolation 2
to the time of addition o f A C T . E — E = z l E i s u s e d

2 x
in t h e c a l c u l a t i o n s .
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f A C T s o l u t i o n ( V ) b y a d d i n g a
further 0.01 m l . o f s o l u t i o n V at t h e e n d o f t h e r e a c t i o n . S u b t r a c t t h e c h a n g e in e x t i n c t i o n f r o m
AE.
Calculations
T h e extinction coefficient of 5-thio-2-nitrobenzoate a t 4 1 2 n m is 1 3 . 6 c m . / / i m o l e . A c c o r d i n g to eqn. (2) on

2 12
p. 312, therefore, with an assay v o l u m e of 2 ml. a n d v ml. of sample, the c o n c e n t r a t i o n is:
2000 x AE , , , n
c = [nmole/ml.J
13.6 x v
Dilution of the sample during p r e t r e a t m e n t m u s t also be taken into account.
Carnitine acetyltransferase is slowly inactivated by D T N B . In the m e t h o d described here, the reaction should
proceed to completion. If i m p r o b a b l y low carnitine values are obtained, a d d m o r e enzyme.
Specificity o f M e t h o d , see p . 1 7 6 2 .
Acetylcarnitine ' 3 13
This m e t h o d allows the determination of acetylcarnitine a n d acetyl coenzyme A in one o p e r a t i o n .
Principle
(1) Acetylcarnitine + C o A S H ^£L±_ Carnitine + A c - C o A
i : 1
citrate
(2) A c - C o A + Oxaloacetate s y n t h a s e > Citrate + C o A S H
t
,
(3) Malate + N A D +
Oxaloacetate -f N A D H + H +
T h e acetyl-CoA formed in reaction (1) reacts with oxaloacetate in the presence of citrate synthase to give
citrate [eqn. (2)]; C o A S H is regenerated. T h e c o n s u m p t i o n of oxaloacetate leads to disturbance of the
equilibrium of the malate d ehydro genase ( M D H ) reaction (3) with c o n s e q u e n t reduction of N A D . T h e
increase in the N A D H concentration, as measured by the extinction at 340 (334, 366) n m , is the unit of
measurement.
(Citrate synthase, Citrate oxaloacetate-lyase, E C 4.1.3.7; m a l a t e d e h y d r o g e n a s e , L - M a l a t e : N A D oxi
doreductase, E C 1.1.1.37).
Pearson 14
referred to the non-stoichiometric relation between the acetylcarnitine conversion a n d the N A D H
formed in this system (see a l s o ) . A simple correction factor m u s t be used in the calculations, see u n d e r
15
" C a l c u l a t i o n s " on p . 1767 a n d p . 112 et seq. As in the d e t e r m i n a t i o n of carnitine, E D T A should be a d d e d

for m e a s u r e m e n t s in tissue extracts to prevent the inhibition of carnitine acetyltransferase.
Equipment
S p e c t r o p h o t o m e t e r for m e a s u r e m e n t s at 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m .
Reagents
1. L - M a l i c a c i d 4. C i t r a t e s y n t h a s e , C S
2. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , N A D from pig heart, crystalline, suspension in 3.2 M
commercial p r e p a r a t i o n s , see p . 545. ammonium sulphate solution; ^ 70 U/mg.
3. C o e n z y m e A commercial p r e p a r a t i o n s , see p. 443.
commercial p r e p a r a t i o n s , see p . 528. 5. M a l a t e d e h y d r o g e n a s e , MDH
from pig heart, crystalline, suspension in 3.2 M
a m m o n i u m sulphate s o l u t i o n ; ^ 1100 U/mg.
T h e o t h e r r e a g e n t s r e q u i r e d f o r t h e p r e p a r a t i o n o f t h e s o l u t i o n s are l i s t e d o n p . 1 7 5 9 .
Purity of Reagents
All enzymes m u s t be free from acetyl-CoA hydrolase a n d N A D H oxidase. C S a n d M D H m u s t not contain

A C T , a n d M D H m u s t n o t contain C S .
U s e water distilled f r o m glass.

I. P o t a s s i u m m a l a t e ( 1 M ) :
D i s s o l v e 1 3 . 4 g. L - m a l i c a c i d in w a t e r , a d j u s t t o p H 7 t o 9 w i t h 5 N K O H , a n d m a k e u p
to 100 ml. with distilled water.
II. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( a p p r o x . 1 0 m M / ? - N A D ) :
D i s s o l v e 2 3 . 1 m g . N A D in a p p r o x . 1 m l . d i s t i l l e d w a t e r , a d d 0 . 0 4 5 m l . 1 N K O H , a n d
dilute to 3 ml. with distilled water.
III. C o e n z y m e A ( a p p r o x . 10 m M ) :
D i s s o l v e 10 m g . C o A in 1 m l . d i s t i l l e d w a t e r .
IV. M a l a t e d e h y d r o g e n a s e , M D H ( l m g . p r o t e i n / m l . ) :
Dilute stock suspension as required with 3.2 M a m m o n i u m sulphate solution ( p H %
« 6.0).
V . C i t r a t e s y n t h a s e , C S (1 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n as r e q u i r e d w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n ( p H « 6 . 0 ) .
P r e p a r e t h e o t h e r s o l u t i o n s as d e s c r i b e d o n p . 1 7 6 3 a n d 1 7 6 0 :
V I . Tris buffer (1 M ; p H 7.8)
VII. Ethylenediaminetetra-acetate (50mM)
V I I I . C a r n i t i n e a c e t y l t r a n s f e r a s e (1 m g . p r o t e i n / m l . )
IX. Perchloric acid (approx. 50 m M )
Solutions I, VI, a n d VII keep indefinitely at - 1 5 °C, solution II for o n e week at + 4 °C, a n d solution III for
a few days. T h e enzyme suspensions IV, V, a n d VIII are stable for several m o n t h s at 0 to + 4 °C.
Procedure
Collection, treatment, a n d stability o f the samples as described o n p. 1760.
Assay System
Wavelength: 340 ( H g 334, H g 365) n m ; light p a t h : 1 c m . ; assay v o l u m e : 2.0 m l . ; r o o m t e m p e

rature; measure against water.

assay mixture
Sample (deproteinized, neutralized) u p t o 1.65 m l . 1 0 - 1 0 0 fiM acetylcarnitine

Tris buffer (VI) 0.20 ml. 100 m M
L-Malate solution (I) 0.02 ml. 10 m M
E D T A solution (VII) 0.05 ml. 1.25 m M
N A D solution (II) 0.10 ml. a p p r o x . 0.5 m M
C o A solution (III) 0.025 ml. approx. 0.125 m M
M i x , read extinction E. 1
MDH suspension (IV) 0.01 m l . 5 jug./ml. = 5.5 U / m l .
M i x ; w a i t u n t i l e q u i l i b r i u m is r e a c h e d , a n d r e a d E . 2
E 2 - Et = AE . t
CS suspension (V) 0.02 ml. 10 j i g . / m l . = 0 . 7 U / m l .
Mix; acetyl-CoA in t h e s a m p l e reacts Wait until

reaction stops, read E . 3
E3 — E 2 = A E . 2
A C T solution (VIII) 0.01 m l . 5 ^g./ml. = 0.6 U / m l .
M i x ; a c e t y l c a r n i t i n e reacts. W a i t until r e a c t i o n s t o p s
( 5 - 1 5 min.). Read E 4 • E 4 - E 3 = A E . 3
Calculations
T h e increase in N A D H is smaller t h a n that c o r r e s p o n d i n g to stoichiometric reaction of acetylcarnitine a n d

a c e t y l - C o A ' . F o r constant experimental conditions, however, the correct values can be calculated from
14 15
the three measured A E values a n d the extinction coefficient of N A D H (e = 6.22 cm. //zmole at 340 n m ) . 2
C a r n i t i n e a n d Acylcarnitines 1767
F o r the deproteinized a n d neutralized sample used in the assay,
(4) Acetyl-CoA: c = a x
2 [/^mole/cuvette contents]
where a = ^ n ^ and B= ^ ^ 2
0 + 1 J E t
(5) Acetylcarnitine + A c e t y l - C o A : c = a x
^ R n i o l e / cuvette contents]
where «' = <?% + f and = i ^ i E ,
T h e value for acetylcarnitine is found from the difference between equations (5) a n d (4). If the sample contains
n o acetyl-CoA, A E 2 = O, a n d the calculation is simplified.
F o r routine determinations, it is advisable to construct a s t a n d a r d curve in which the m e a s u r e d jS values are
plotted against c o r r e s p o n d i n g a values.
N o r m a l Values
Typical values for acetylcarnitine in tissues of rats (fed) are 377 ± 62 n m o l e per g. of frozen heart muscle a n d
41 ± 9 n m o l e per g. of frozen liver (means ± S.D.).
2
N o serious error is to be expected, provided that the calculation is carried out correctly. T h e m e t h o d can be
used for p u r e solutions a n d for tissue extracts.
T h e m e t h o d is absolutely specific for the acetyl derivatives of C o A a n d carnitine. O t h e r short-chain acyl

carnitines are substrates for A C T , b u t only acetyl-CoA reacts with CS a c c o r d i n g t o e q u a t i o n (2).
Short-Chain (C -C ) Acylcarnitines
3 10
N o entirely satisfactory m e t h o d is k n o w n at present for the determination of short-chain acylcarnitines. T h e

m e t h o d described here gives an error of less t h a n + 2 0 % .
carnitine
(1) Acylcarnitine + C o A S H . a c e t y l t r a n s f e r a s e Carnitine + A c y l - C o A
Short-chain ( C — C ) acylcarnitines are substrates for A C T . T h e f o r m a t i o n of acyl-CoA, as measured by

2 1 0
the increase in extinction at 232 n m , is the unit of m e a s u r e m e n t .
T h e reversible reaction (1) has an equilibrium c o n s t a n t of 0.6. Q u a n t i t a t i v e conversion of acylcarnitine can

3
therefore be achieved only by the use of a large excess of C o A . However, the q u a n t i t y t h a t can be used is
limited in practice by the extinction of C o A at 232 n m . T h e experimental values m u s t therefore be corrected

for the calculations with the aid of the equilibrium c o n s t a n t s a n d the c o n c e n t r a t i o n s of the three species
present in equilibrium. T h e equilibrium c o n s t a n t has a constant value of 0.6 in the region of the p H o p t i m u m
( 0 . 7 - 8 . 0 ) of carnitine acyltransferase. T h e addition of E D T A is necessary in the analysis of tissue extracts.
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e f o r a c c u r a t e m e a s u r e m e n t at 2 3 2 n m .
Reagents
T h e r e a g e n t s r e q u i r e d for t h e p r e p a r a t i o n o f t h e s o l u t i o n s are listed o n p a g e s 1 7 5 9 a n d 1 7 6 5 .
Prepare the solutions as described o n pages 1760, 1763, and 1765.

I. Tris buffer (1 M ; p H 7 . 8 )
II. E t h y l e n e d i a m i n e t e t r a - a c e t a t e ( 5 0 m M )
III. C o e n z y m e A ( a p p r o x . 10 m M )
I V . C a r n i t i n e a c e t y l t r a n s f e r a s e , A C T (1 m g . p r o t e i n / m l . ) .
Procedure
Collection, treatment, and stability o f the sample as described o n p. 1760.
Assay System
W a v e l e n g t h : 2 3 2 n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 0 m l . ; r o o m t e m p e r a t u r e ; m e a s u r e
a g a i n s t air.
Sample (deproteinized, neutralized) up to

1.70 m l . 1 0 - 5 0 //mole acylcarnitine
E D T A solution (II) 0.005 ml. 0.125 m M
C o A solution (III) 0.05 ml. approx. 0.25 m M
M i x ; read extinction E j .
A C T solution (IV) 0.01 m l . 5 / i g . / m l . = 0.6 U / m l .
M i x ; w a i t u n t i l e q u i l i b r i u m is r e a c h e d (5 t o 10 m i n . ) .
R e a d e x t i n c t i o n E . E -E
2 2 1 = AE.
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f t h e A C T s o l u t i o n ( I V ) b y a d d i t i o n o f
a further 0.01 m l . o f s o l u t i o n I V . S u b t r a c t t h e c h a n g e in e x t i n c t i o n f r o m A E .
Calculations
T h e extinction coefficient of a c y l - C o A at 232 n m is e = 4.5 c m . / / m i o l e . T h e a m o u n t of acylcarnitine

2 8
reacting is therefore given by
c = z
^ ^ n
= 0.445 AE [/^mole/cuvette contents]
To o b t a i n the total a m o u n t of short-chain acylcarnitine, the a m o u n t of acylcarnitine remaining in the equi

librium must be a d d e d . It is calculated from the equilibrium c o n s t a n t a n d the c o n c e n t r a t i o n s of acyl-CoA,
carnitine, a n d C o A .
W i t h s q u a r e brackets t o indicate initial c o n c e n t r a t i o n s in t h e assay system in //mole, we o b t a i n
(2) c = 0.445 AE + ° 6
V^L^l^^ +
°*
M AE)
^ m o l e / c u v e t t e contents]
[CoASH]—0.445 A E
Subtraction of the acetylcarnitine value (determined according to p . 1764) from this value gives the a m o u n t
of short-chain ( C - C ) acylcarnitines.
3 1 0
In practice, the c o n c e n t r a t i o n s of C o A S H a n d acyl-CoA in tissue extracts are so low that they can be
neglected. Only the C o A c o n c e n t r a t i o n used in the assay a n d the carnitine c o n c e n t r a t i o n of the tissue ex
tract determined according t o p . 1758 need therefore be considered in e q u a t i o n (2).
The error of the m e t h o d is increased by any inaccuracy of the equilibrium c o n s t a n t of reaction (1) a n d by the
methodological errors in the d e t e r m i n a t i o n s of carnitine, acyl-CoA, a n d C o A S H . E r r o r s of u p to a b o u t 50%
of the acylcarnitine remaining in the system after equilibration should be expected. In practice, this corres
p o n d s to ± 2 0 % of the total acylcarnitine value.
N o r m a l Values
Pearson a n d Tubbs detected significant quantities (up to 700 nmole/g. fresh wt.) of short-chain acylcarni
2
tines other t h a n acetylcarnitine in rat hearts after perfusion with solutions containing short-chain fatty
acids.
T h e application of the m e t h o d t o tissue extracts is limited by its relative insensitivity a n d the high light
a b s o r p t i o n of extracts at 232 n m .
A C T specifically transfers acyl g r o u p s containing 2 to 10 C a t o m s to carnitine. Acetylcarnitine can be

specifically determined, while the o t h e r acylcarnitines c a n n o t be differentiated.
Long-Chain (C > 12) Acylcarnitines

Principle
Long-chain acylcarnitines are insoluble in dilute perchloric acid, a n d can therefore be separated from all
other carnitine derivatives o n acidic deproteinization of tissues. They are d e t e r m i n e d as described o n p . 1758
as free carnitine after alkaline hydrolysis of the acid-insoluble fraction of the tissue.
Procedure
G r i n d f r o z e n t i s s u e w i t h p e r c h l o r i c a c i d as d e s c r i b e d o n p . 1 7 6 0 a n d c e n t r i f u g e . W a s h p r e
c i p i t a t e three t i m e s w i t h 2 % ( w / v ) p e r c h l o r i c a c i d a n d t a k e u p in K O H s o l u t i o n , s o t h a t t h e
final v o l u m e is 3 m l . p e r g. o f f r o z e n t i s s u e a n d t h e final c o n c e n t r a t i o n o f K O H is 0.2 N .
I n c u b a t e this s u s p e n s i o n for 2 h. at 55 ° C ; t h e n c o o l t o 0 ° C , a c i d i f y w i t h 7 0 % ( w / w ) H C 1 0 , 4
a n d c e n t r i f u g e . Buffer s u p e r n a t a n t fluid w i t h a f e w d r o p s 0.5 M K H P 0 2 4 (solution IV o n

p. 1 7 6 0 ) , a n d a d j u s t t o p H 7 - 8 w i t h 6 N KOH.
Assay System
D e t e r m i n e carnitine as described o n p. 1758.
Normal Values
Typical values for acid-insoluble carnitine in tissues from rats (fed) are 52 + 23 n m o l e per g. of frozen heart
muscle a n d 11 + 1 n m o l e per g. of frozen liver (means ± S.D.). These values increase considerably on
starvation . 2
Specificity
Acid insoluble carnitine is e q u a t e d t o long-chain acylcarnitine for the following reasons. Synthetic pal-
mitoylcarnitine is also insoluble; w h e n a d d e d to tissue extracts, it is recovered quantitatively. Carnitine is
liberated from b o t h derivatives by alkaline hydrolysis at similar rates. This hydrolysis naturally c a n n o t
p r o v e whether long-chain acylcarnitines are present in the acid-insoluble fraction. However, o t h e r w o r k e r s 16
have found long-chain acylcarnitines, consisting mainly of palmitoylcarnitine, stearoylcarnitine, a n d

oleoylcarnitine, o n c h r o m a t o g r a p h y of tissue lipid extracts.
Other Methods
Very sensitive carnitine d e t e r m i n a t i o n s can be carried o u t with 2-oxoglutarate dehydrogenase as an indi

c a t o r enzyme. T h e C o A S H formed in reaction (1) with A C T reacts further as follows:
C o A S H + 2-Oxoglutarate + N A D +
C0 2 + Succinyl-CoA + N A D H + H +
Very small quantities of N A D H c a n also be m e a s u r e d fluorimetrically.

T h e sensitivity of the acetylcarnitine d e t e r m i n a t i o n on p . 1764 can also be increased by fluorimetric mea
surement of N A D H .
Bohmer, Norum, a n d Bremer 16

have described a m e t h o d for the d e t e r m i n a t i o n of the concentrations of
carnitine derivatives in tissues. Injected H - y - b u t y l b e t a i n e is converted into carnitine in vivo, with selective
3
labelling of carnitine a n d uniform distribution of H a m o n g s t t h e carnitine derivatives. These a u t h o r s

3
describe c h r o m a t o g r a p h i c separation systems for carnitine, acetylcarnitine, a n d long-chain acylcarnitines;

however, this m e t h o d does n o t give a b s o l u t e c o n c e n t r a t i o n values.
C a r n i t i n e a n d Acylcarnitines 1771
References
1 G. Fraenkel & S. Friedman, Vitamins a n d H o r m o n e s 15, 73 [1957].

2 D. J. Pearson & P. K. Tubbs, Biochem. J. 105, 953 [1967].
3 /. B. Fritz, S. K. Schultz & P. A. Srere, J. biol. C h e m . 236, 2509 [1963].
4 / . F. A. Chase, Biochem. J. 104, 510 [1967].
5 K. R. Norum, Biochim. Biophys. A c t a 89, 95 [1964].
6 N. R. Marquis & /. B. Fritz, J. Lipid R e s . 5, 184 [1964].
7 S. J. Wakil & G. Hubscher, J. biol. C h e m . 235, 1554 [I960].
8 E. R. Stadtman in S. P. Colowick & N. O. Kaplan: M e t h o d s in E n z y m o l o g y , A c a d e m i c Press, N e w
York 1957, Vol. I l l , p . 9 3 1 .
9 / . F. A. Chase, D. J. Pearson & P. K. Tubbs, Biochim. Biophys. A c t a 96, 162 [1965].
10 H. R. Mahler, S. J. Wakil & R. M. Bock, J. biol. C h e m . 204, 453 [1953].
11 LB. Fritz & S. K. Schultz, J. biol. C h e m . 240, 2188 [1965].
12 G. L. Ellmann, A r c h . Biochem. Biophys. 82, 70 [1959].
13 D.J. Pearson & P. K. Tubbs, N a t u r e 202, 91 [1964].
14 D. J. Pearson, Biochem. J. 95, 23c [1965].
15 H. U. Bergmeyer & H. Mollering, Biochem. Z . 344, 167 [1966].
16 T. Bohmer, K. R. Norum & 7. Bremer, Biochim. Biophys. A c t a 125, 244 [1966].
Creatine
Erich Bernt, H a n s Ulrich Bergmeyer and H a n s Mollering
Creatine, methylguanidinoacetic acid, is found mainly in muscle of various living organisms. In addition,
it occurs in blood, in brain, in some t r a n s u d a t e s a n d in thyroid glands. Assay m e t h o d s for creatine in b o d y
fluids and in muscle have so far d e p e n d e d on direct or indirect colorimetric m e t h o d s ; these m e t h o d s
are unspecific a n d are not very sensitive.
Creatine can be converted to creatine p h o s p h a t e with A T P a n d creatine kinase ( A T P : creatine ^ - p h o s p h o
transferase, EC 2.7.3.2). This specific reaction is the basis of the following m e t h o d for the assay of creatine
in s e r u m ; it is similar to the m e t h o d of Tanzer a n d Gilvarg . 1
Application of Method: In biochemistry, clinical biochemistry a n d possibly in foodstuff chemistry.
Principle
(1) Creatine + A T P c r e a t i n e
» Creatine p h o s p h a t e + ADP
kinase ,
(2) ADP + PEP • A T P + Pyruvate

kinase* .
• Lactate + N A D +
dehydrogenase**
The decrease of N A D H , as m e a s u r e d by the change in extinction at 340 (334, 365) n m , is p r o p o r t i o n a l to

the a m o u n t of creatine present.
T h e equilibrium of reaction (1) d e p e n d s strongly on the p H ; in the alkaline range it is in favour of creatine
p h o s p h a t e . It is therefore best to carry out the assay at p H values a b o v e 9. T h e t u r n o v e r n u m b e r of the
enzyme is small (25000 mole creatine per mole enzyme per min. at 38 °C) a n d the Michaelis constant is
high for creatine ( K = 1 . 6 x 1 0 ~ M at p H 8.8). Relatively large a m o u n t s of enzyme (ca. 3 mg./assay) are
M
2
therefore required, so t h a t the reaction proceeds sufficient rapidly. This is particularly i m p o r t a n t for
measurements in serum, because certain constituents of serum inhibit the enzyme. So far it has not been
possible to eliminate these.
Equipment
S p e c t r o p h o t o m e t e r or spectrum-line p h o t o m e t e r suitable for precise m e a s u r e m e n t s at 340,

334 o r 365 n m ; b e n c h centrifuge.
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 4. M a g n e s i u m chloride, M g C l • 6 H 0 , A . R .
2 2
2. P o t a s s i u m c a r b o n a t e , A . R . 5. P e r c h l o r i c acid, A. R., 70% (w/w), sp.

3. S o d i u m h y d r o g e n c a r b o n a t e , 5 % ( w / v ) g r . 1.67
* A T P : pyruvate 2-<9-phosphotransferase, E C 2.7.1.40.

** L-Lactate: N A D oxidoreductase, E C 1.1.1.27.
Creatine 1773
6. P h o s p h o e n o l p y r u v a t e , P E P 9. C r e a t i n e k i n a s e , C K
tricyclohexylammonium salt, commercial prep from rabbit muscle, lyophilized; ^ 2 5 U/mg.
aration, see p. 548. (25 °C); commercial preparation, see p. 444.
7. A d e n o s i n e t r i p h o s p h a t e , A T P 10. L a c t a t e d e h y d r o g e n a s e , L D H
disodium salt, A T P - N a H - 3 H 0 ; commercial
2 2 2 from rabbit muscle, crystalline suspension in
preparation, see p. 527. 3.2 M ammonium sulphate solution; ^550
8. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o U/mg. (25 °C); commercial preparation, see
tide, N A D H p. 4 8 1 .
disodium salt, N A D H - N a ; commercial prep
2 11. Pyruvate kinase, P K
aration, see p. 545. from rabbit muscle, crystalline suspension in
3.2 M ammonium sulphate solution; ^200
U/mg. (25 °C) commercial preparation, see
p. 509.
Purity of Reagents
L D H and PK must be free from creatine kinase. All three enzymes must contain less than 0.001% ATPase
and myokinase (related to the activity of CK). A T P must be completely free from A D P , and PEP must be
free from pyruvate.
P r e p a r e all s o l u t i o n s w i t h freshly p r e p a r e d , d o u b l y d i s t i l l e d w a t e r . T o a v o i d t h e g r o w t h o f
m i c r o - o r g a n i s m s sterilize t h e flasks.
I. T r i e t h a n o l a m i n e / K C 0 2 3 ( 0 . 2 1 M t r i e t h a n o l a m i n e ; 1.16 M K C0 ):
2 3
D i s s o l v e 1.0 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e a n d 4 . 0 g. K C 0 2 3 in d i s t i l l e d w a t e r a n d
m a k e u p t o 25 m l .
II. P h o s p h o e n o l p y r u v a t e / m a g n e s i u m c h l o r i d e ( 1 0 m M P E P ; 0 . 4 M M g C l ) : 2
D i s s o l v e 1 4 m g . P E P a n d 2 5 0 m g . M g C l - 6 H 0 in 3 m l . d i s t i l l e d w a t e r .
2 2
III. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e / a d e n o s i n e t r i p h o s p h a t e ( 1 0 m M / ? - N A D H ;
25 m M A T P ) :
D i s s o l v e 16 m g . N A D H - N a 2 and 30 mg. A T P - N a H - 3 H 0 2 2 2 in 2 m l . 5% NaHC0 3
solution.
IV. L a c t a t e d e h y d r o g e n a s e / p y r u v a t e k i n a s e , L D H / P K ( e a c h 1 m g . p r o t e i n / m l . ) :
D i l u t e the s t o c k s u s p e n s i o n s a c c o r d i n g l y w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n a n d
mix.
V. Creatine kinase, C K (120 m g . p r o t e i n / m l . ) :
D i s s o l v e 6 0 m g . p r o t e i n w i t h 0.5 m l . 5% N a H C 0 3 s o l u t i o n ; if n e c e s s a r y , r e m o v e a n y t u r b
idity b y c e n t r i f u g a t i o n . P r e p a r e t h e s o l u t i o n freshly e a c h d a y .
VI. Perchloric acid (0.6 N ) :
D i l u t e 5.2 m l . 7 0 % p e r c h l o r i c a c i d t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r .
Store all solutions and suspensions, stoppered, in a refrigerator at 0 - 4 °C. Prepare the PEP/magnesium
chloride solution and the N A D H / A T P solution freshly each week. Solutions I and VI are stable in
definitely; the enzyme suspension IV keeps for ca. 1 year.
Procedure
Collect b l o o d without venestasis. A d d i t i o n o f 1 m g . oxalate/ml., 1 m g . citrate/ml., 2 m g .

f l u o r i d e / m l . 0 . 2 m g . h e p a r i n / m l . o r 1 m g . E D T A / m l . d o e s n o t interfere. P r e f e r a b l y c o l l e c t
tissues o f laboratory animals w i t h freeze-stop c l a m p s (see "Cell a n d Tissue Disintegration",
p. 4 0 0 .
Deproteinization:
W h o l e b l o o d : Pipette into a centrifuge tube 6 ml. ice-cold perchloric acid solution (VI) a n d
3 m l . w h o l e b l o o d . M i x t h o r o u g h l y w i t h a t h i n g l a s s r o d a n d c e n t r i f u g e f o r 15 m i n . at 3 0 0 0 r p m .
A d d 1 ml. t r i e t h a n o l a m i n e / K C 0 2 3 s o l u t i o n (I) t o 3 m l . s u p e r n a t a n t fluid a n d after a l l o w i n g t o
s t a n d f o r 15 m i n . in i c e w a t e r filter off t h e p e r c h l o r a t e p r e c i p i t a t e . A f t e r e q u i l i b r a t i o n t o 25 ° C
a d d 2 . 0 0 m l . o f this s o l u t i o n buffered a t p H 8 - 9 t o t h e a s s a y .
S e r u m : Deproteinize 4 ml. serum or p l a s m a with 4 ml. perchloric acid solution (VI) a n d centri
fuge. Treat 3 m l . o f t h e s u p e r n a t a n t fluid a s d e s c r i b e d f o r w h o l e b l o o d . T a k e i n t o a c c o u n t t h e
c h a n g e in t h e v o l u m e s o n d e p r o t e i n i z a t i o n i n t h e c a l c u l a t i o n s .
T i s s u e s : W i t h h o m o g e n a t e s c a r e m u s t b e t a k e n t o e n s u r e t h a t t h e filtrate after d e p r o t e i n i z a t i o n
is free f r o m p e r c h l o r a t e a n d is a d j u s t e d t o p H 8 - 9 . H o m o g e n a t e s of, f o r e x a m p l e , s k e l e t a l
m u s c l e o f t h e m o u s e c o n t a i n s o m u c h c r e a t i n e (ca. 0.6%) t h a t o n l y a c a . 0 . 5 % h o m o g e n a t e n e e d
be analysed. In this case the a m o u n t o f t r i e t h a n o l a m i n e / K C 0 2 3 s o l u t i o n (I) r e c o m m e n d e d
for w h o l e b l o o d is n o t sufficient t o a d j u s t t h e p H t o 9 ; u s e m o r e o f s o l u t i o n I a n d t a k e t h i s i n t o
a c c o u n t in t h e c a l c u l a t i o n s . O t h e r w i s e treat a s f o r w h o l e b l o o d .
U r i n e : M i x filtered u r i n e w i t h e q u a l v o l u m e s 0 . 2 5 M glycine buffer, p H 9 , a n d a d d 2 m l . o f
this m i x t u r e t o t h e a s s a y . D e p r o t e i n i z a t i o n is n o r m a l l y n o t n e c e s s a r y . T a k e i n t o a c c o u n t t h e
dilution in the calculations.
In b l o o d t h e c r e a t i n e c o n t e n t d e c r e a s e s 2 4 hr. after c o l l e c t i o n b y 2 0 % a t r o o m t e m p e r a t u r e ,
b y 0 % at 4 ° C . I n a c i d e x t r a c t s after d e p r o t e i n i z a t i o n o r i n n e u t r a l e x t r a c t s c r e a t i n e is s t a b l e
for at least 2 4 hr. at 4 ° C o r e v e n 2 5 ° C . T h e c r e a t i n e c o n t e n t o f t i s s u e s is l i k e l y t o i n c r e a s e d u e
t o release f r o m c r e a t i n e p h o s p h a t e . T i s s u e h o m o g e n a t e s o r a c i d e x t r a c t s s h o u l d b e s t o r e d f r o z e n
if t h e a s s a y is n o t carried o u t i m m e d i a t e l y .
Creatine 1775
Assay System
a t u r e ; r e a d a g a i n s t air.
T h e i n c r e a s e in e x t i n c t i o n o n a d d i t i o n o f C K s o l u t i o n ( V ) is o b t a i n e d b y a d d i n g a further
0 . 0 2 m l . s o l u t i o n V at t h e e n d o f t h e r e a c t i o n . S u b t r a c t t h e e x t i n c t i o n c h a n g e f r o m E .
2
Pipette into cuvettes : Concentration in assay mixture
Sample (deproteinized, neutralized,

buffered) 2.00 ml. u p t o c a . 1 5 0 JUM c r e a t i n e
0.15 ml. 0.65 m M P E P
PEP/MgCl -solution2 (II) 26 m M M g C l 2
0.10 ml. 0.4 m M NADH;

N A D H / A T P solution (III) 1.1 m M A T P
0.05 ml. 12ULDH/ml.;
L D H / P K suspension (IV) 4 U PK/ml.
M i x and follow extinction c h a n g e s until constant

( 1 0 - 1 5 min.). T h e n read extinction E . t
C K solution (V) 0.02 ml. 25 U / m l .
M i x . A f t e r 15, 2 0 a n d 35 m i n . r e a d e x t i n c t i o n s ; b y
extrapolation of these values to the time o f C K addition
(see p . 3 0 8 ) d e t e r m i n e e x t i n c t i o n E . E
2 l — E is u s e d
2
for t h e c a l c u l a t i o n s .
Calculations
U n d e r the above conditions the reaction proceeds stoichiometrically, a n d therefore the calculation formula
(2) on p . 312 applies. T h e results are o b t a i n e d in /rniole creatine/ml. s a m p l e This value m u s t be multi
plied by a factor if the s a m p l e h a s been deproteinized, neutralized o r diluted in a n y way. In the case of
whole b l o o d the specific gravity (ca. 1.06) a n d t h e water c o n t e n t (ca. 80%) m u s t b e t a k e n into a c c o u n t .
Therefore a factor of 3.8 for whole b l o o d is o b t a i n e d in this m e t h o d after correction for these values a n d
for the dilution 1 + 2 in the deproteinization a n d 3 + 1 in the p e r c h l o r a t e precipitation. T h e following
relationships hold for the calculation of the creatine c o n c e n t r a t i o n of b l o o d :
c = AE x 0.723 AE x 0.709 AE x 1.30 [/miole/ml.]

c = AE x 94.76 AE x 92.93 AE x 170 [//g./ml.]
W i t h a m e a n value (n = 10) of 0.35 g. creatine per 100 g. r a b b i t skeletal muscle a s t a n d a r d deviation of

0.02 g. creatine was found. T h e coefficient of variation is 5.7%.
N o r m a l Values
T h e creatine content of muscles of different organisms is 0 . 3 - 0 . 5 % of the fresh weight. W h o l e b l o o d

contains 3 to 5 mg. creatine per 100 ml., s e r u m a n d p l a s m a contain 0 . 3 - 0 . 7 mg. per 100 ml.
Effects of drugs and other therapeutic measures: None known
Interference in the assay technique: Insufficient purity of the reagents (see p . 1773), especially of the enzymes,
results in t o o high creatine values. If the P E P contains pyruvate a n d the A T P is c o n t a m i n a t e d with A D P ,
t o o m u c h N A D H will be oxidized before t h e actual reaction of creatine. In this case m o r e N A D H must
be added before the C K . Too low C K activity results in lower creatine v a l u e s ; with 1.5 mg. of enzyme per
assay we found only 80% of the a d d e d creatine. C K is inactivated in solutions of p H less t h a n 7; highly
dilute solutions in distilled water are therefore to be avoided. T h e removal of p y r u v a t e from the sample
with ion exchange resin before the enzymatic r e a c t i o n is n o t necessary in o u r experience; it can easily
1
result in loss of creatine.
Creatine kinase is specific for creatine. Creatinine, arginine, citrulline, ureidosuccinate, canavarine a n d
glycocyamidine have n o effect o n the d e t e r m i n a t i o n . Glycocyamine reacts with creatine kinase, but only
a b o u t l/40th as rapidly as creatine. N e i t h e r adenosine-5'-diphosphate n o r inosine-5'-triphosphate can
act as p h o s p h a t e d o n o r .
References
1 M. L. Tanzer & C. Gilvarg, J. biol. C h e m . 234, 3201 [1959].

Creatine Phosphate
Walther L a m p r e c h t , Philipp Stein, Fritz Heinz, a n d H e r w i g Weisser
The need for a specific a n d reliable m e t h o d for the d e t e r m i n a t i o n of creatine p h o s p h a t e in cell a n d tissue
extracts is met by the enzymatic assay with creatine kinase, C K ( A T P - c r e a t i n e ^ - p h o s p h o t r a n s f e r a s e ,
E C 2.7.3.2).Two assay m e t h o d s based o n this reaction m a y be used.
Application of Method: In biochemistry.
Determination with Creatine Kinase, Hexokinase,

and Glucose-6-phosphate Dehydrogenase
T h e enzyme creatine kinase catalyses the transfer of p h o s p h a t e from creatine p h o s p h a t e (CP) to adenosine
d i p h o s p h a t e ( A D P ) . In the presence of hexokinase, H K ( A T P : D-hexose 6-phosphotransferase, EC
2.7.1.1), the A T P formed p h o s p h o r y l a t e s glucose to glucose-6-phosphate ( G - 6 - P ) with regeneration of
A D P . T h e N A D P - d e p e n d e n t oxidation of G - 6 - P by glucose-6-phosphate d e h y d r o g e n a s e , G 6 P - D H (D-G1U-
cose-6-phosphate: N A D P 1-oxidoreductase, E C 1.1.1.49) is used as the indicator reaction.
Principle
(1) CP + ADP — — • Creatine + A T P
|
(2) A T P + Glucose — — • G-6-P + ADP
(3) G-6-P + N A D P +
+ H 0
2
G 6 P
~ P H
> 6-Phosphogluconic acid + N A D P H + H +
O n e mole of N A D P H is formed per m o l e of creatine p h o s p h a t e . T h e increase in extinction at 334, 340, o r

365 n m is m e a s u r e d .
The reaction sequence s h o w n a b o v e allows the d e t e r m i n a t i o n n o t only of C P b u t also of G-6-P a n d A T P
in the same assay system.
C K from muscle is specific for A D P a n d creatine p h o s p h a t e or A T P a n d creatine. It requires M g 2 +

or
Mn 2 +
for the reaction. C a 2 +
gives only half of the o p t i m u m activity, B a 2 +
is inactive, a n d Z n 2 +
and C u 2 +
inhibit the reaction. A D P , A M P , a n d N A D also have an inhibitory effect. p - C h l o r o m e r c u r i b e n z o a t e

inhibits the reaction by blockage of thiol g r o u p s . T h e o p t i m u m p H for the C P f o r m a t i o n (forward reaction)
is 9.0, and that for the A T P formation (reverse reaction) is 6 . 0 - 7 . 0 . The Michaelis c o n s t a n t s at the o p t i m u m
1
p H a n d 38 °C are K M = 5 m M for creatine p h o s p h a t e a n d K M = 1 m M for A D P . O n e mole of enzyme

(molecular weight 81000) forms 150000 mole of A T P in 1 min. T h e AF for the forward reaction at p H
7.5 is 2
ca. 3 kcal.
T h e equilibria of t h e h e x o k i n a s e a n d glucose-6-phosphate d e h y d r o g e n a s e reactions lie completely t o t h e
right when they are coupled together. W i t h a suitable excess of A D P , therefore, t h e three coupled enzyme
reactions proceed quantitatively.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e o f m e a s u r e m e n t s at 3 4 0 ( 3 3 4 or 3 6 5 )
n m ; p H meter; laboratory centrifuge.
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 9. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ,
2. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e G6P-DH
phosphate, N A D P from yeast, suspension in 3.2 M a m m o n i u m
disodium salt, N A D P - N a H . C o m m e r c i a l p r e p
2 sulphate s o l u t i o n ; ^ 140 U / m g . (25 °C). C o m
aration, see p . 546. mercial p r e p a r a t i o n , see p. 458.
3. M a g n e s i u m c h l o r i d e , A . R., 10. H e x o k i n a s e , H K
MgCl -6H 0 2 2 from yeast, crystalline suspension in 3.2 M
4. Glucose a m m o n i u m sulphate solution; ^140 U/mg.
5. P e r c h l o r i c a c i d , A . R., s p . gr. 1.67 ( c a . (25 C). C o m m e r c i a l p r e p a r a t i o n , see p . 473.
7 0 % w / w ) o r s p . gr. 1.54 ( c a . 6 0 % w / w ) 11. Creatine kinase, C K
6. P o t a s s i u m c a r b o n a t e , A . R . , K C 0 2 3 from r a b b i t muscle, salt-free lyophilized p r e p
7. M e t h y l o r a n g e o r m e t h y l red a r a t i o n ; ^ 1 8 U / m g . protein (25 °C). C o m
(indicator) mercial p r e p a r a t i o n , see p . 444.
8. A d e n o s i n e d i p h o s p h a t e , A D P
disodium salt, A D P - N a . C o m m e r c i a l p r e p a r a t
2
ion, see p . 525.
Purity of Reagents
Creatine k i n a s e : T h e p r e p a r a t i o n should contain less t h a n 0 . 0 1 % (based on the specific activity of the C K )

of A T P a s e , glutathione reductase, adenylate kinase, glucose-6-phosphatase, N A D P H oxidase, a n d
p h o s p h o g l u c o n a t e dehydrogenase.
H e x o k i n a s e : C o n t a m i n a t i n g activities, based on the specific activity of H K , as for C K .
Glucose-6-phosphate d e h y d r o g e n a s e : If G-6-P a n d A T P in tissue extracts are determined in the same
system, the hexokinase activity of the G 6 P - D H p r e p a r a t i o n must be 0 . 0 1 % based on the specific activity
of the G 6 P - D H . These conditions are satisfied by crystalline G 6 P - D H (Type I, Boehringer M a n n h e i m ) .
If the biological material contains n o G-6-P or if it is n o t desired to determine A T P a n d G-6-P, the contami
nating hexokinase activity is of n o i m p o r t a n c e . T h e specific activity should be a b o u t 140 U / m g . , or a b o u t
300 U / m g . in the case of crystalline p r e p a r a t i o n s .
The other c o n t a m i n a t i n g activities (based on the specific activity of the G 6 P - D H ) should be less t h a n
0 . 0 1 % of A T P a s e , glutathione reductase, 6-phosphogluconate dehydrogenase, adenylate kinase, p h o s p h o -
glucomutase, a n d p h o s p h o g l u c o s e isomerase. If one does not wish to d e t e r m i n e G-6-P, c o n t a m i n a t i o n by
these last enzymes is of not i m p o r t a n c e .
U s e o n l y fresh d o u b l y distilled w a t e r .
I. T r i e t h a n o l a m i n e buffer ( 5 0 m M ; p H 7 . 5 - 7 . 6 ) :
D i s s o l v e 4 . 6 5 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in 2 0 0 m l . d i s t i l l e d w a t e r , a d d 11 m l . 1 N
N a O H a n d d i l u t e t o 5 0 0 m l . w i t h distilled w a t e r ( c h e c k w i t h p H m e t e r ) .
Creatine P h o s p h a t e 1779
II. N A D P (ca. 7 m M 0 - N A D P ) :
D i s s o l v e 7.5 m g . N A D P - N a H in distilled w a t e r a n d m a k e u p t o 1.5 m l .
2
III. M a g n e s i u m c h l o r i d e (0.1 M ) :
D i s s o l v e 2 . 0 3 3 g. M g C l * 6 H 0 in distilled w a t e r a n d m a k e u p t o 100 m l .
2 2
IV. G l u c o s e (0.5 M ) :
D i s s o l v e 9.91 g. g l u c o s e ( C H 6 1 2 0 6 • H 0 ) in distilled w a t e r a n d m a k e u p t o 100 m l .
2
V . P e r c h l o r i c a c i d (6 % w / w ) :
D i l u t e 7.8 m l . 7 0 % H C 1 0 4 o r 9.7 m l . 6 0 % H C 1 0 4 t o 150 m l . w i t h d i s t i l l e d w a t e r .
VI. P o t a s s i u m c a r b o n a t e (ca. 5 M ) :
D i s s o l v e 7 2 g. K C 0 2 3 in distilled water a n d m a k e u p t o 100 ml.
VII. Methyl orange indicator or methyl red:
D i s s o l v e 50 m g . indicator in 100 ml. distilled water.
VIII. A d e n o s i n e diphosphate, A D P (ca. 22 m M ) :
Dissolve 24 mg. A D P - N a 2 in 2 m l . d i s t i l l e d w a t e r .
I X . G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (1 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n a s r e q u i r e d w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
X . H e x o k i n a s e (2 m g . p r o t e i n / m l . ) :
X I . C r e a t i n e k i n a s e (5 m g . p r o t e i n / m l . ) :
D i s s o l v e 5 m g . l y o p h i l i z e d e n z y m e in 1 m l . t r i e t h a n o l m i n e buffer.
Store all solutions, stoppered, at 1-4 °C. P r e p a r e fresh C K solution (XI) daily from dry powder, a n d
p r e p a r e fresh N A D P , A D P , a n d glucose solutions weekly.
Procedure
U s e t h e q u i c k - f r e e z e m e t h o d ( s e e p . 4 0 0 ) for c o l l e c t i o n o f t i s s u e s a m p l e s . F o r d e p r o t e i n i z a t i o n ,
see A T P d e t e r m i n a t i o n ( p . 2 0 9 9 ) .
Assay System
T h e v o l u m e o f s a m p l e ( V ) u s e d for t h e a s s a y is s u c h t h a t t h e c h a n g e in e x t i n c t i o n for C P at
5
H g 3 6 5 n m d o e s n o t e x c e e d 0 . 2 0 0 a n d t h e r e a c t i o n is c o m p l e t e in 30 m i n .
W a v e l e n g t h : 3 4 0 , H g 3 3 4 , o r H g 3 6 5 n m ; light p a t h : 1 c m . ; final v o l u m e : 3 . 0 0 m l . ; ca. 25 ° C ;
m e a s u r e a g a i n s t air. T h e q u a n t i t y o f e x t r a c t r e q u i r e d c a n b e d e c r e a s e d b y t h e u s e o f s e m i m i c r o
cuvettes. Immediately before use, prepare reagent mixture:
Buffer ( s o l u t i o n I) 1.5 v o l .
N A D P s o l u t i o n (II) 0.1 v o l .
MgCl 2 s o l u t i o n (II) 0.1 v o l .
A D P solution (VIII) 0.02 vol.
G 6 P - D H suspension (IX) 0.005 vol.
Glucose solution (IV) 0.100 vol.
H K suspension (X) 0.005 vol.
Water t o 1.99 v o l .
Reagent mixture 1.99 m l . 2 5 m M buffer, 0 . 2 3 m M

N A D P , 3.3 m M M g C l 2
0 . 1 5 m M A D P , 16.7 m M
glucose
1.7 fig. G 6 P - D H / m l . ^
240 m U / m l .
3.3 fig. H K / m l . ^ 4 7 0 m U / m l .
Sample + water 1.00 m l .
M i x . F o l l o w r e a c t i o n w i t h a r e c o r d e r if p o s s i b l e ,
o t h e r w i s e f o l l o w c h a n g e in e x t i n c t i o n for c a . 25 m i n .
ATP and G-6-P in t h e sample react. Wait until
r e a c t i o n s t o p s o r r e c o r d slight " c r e e p " . D e t e r m i n e E j .
C K solution (XI) 0.01 m l . 17 fig. C K / m l . ^ 3 0 6 m U / m l .
M i x , read e x t i n c t i o n E 2 after a b o u t 15 m i n .
E 2 — E l = A E.
D e t e r m i n e the c h a n g e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f t h e C K s o l u t i o n by a d d i t i o n o f a
further 0.01 m l . o f C K s o l u t i o n ( X I ) .
C P , A T P , a n d G - 6 - P c a n b e d e t e r m i n e d t o g e t h e r in t h e s a m e c u v e t t e . T h i s is d o n e by p i p e t t i n g
the r e a g e n t s o l u t i o n s s i n g l y in t h e a b o v e o r d e r a n d d e t e r m i n i n g t h e A E v a l u e s c o r r e s p o n d i n g
to the c o n c e n t r a t i o n o f G - 6 - P , A T P , a n d C P after a d d i t i o n o f G 6 P - D H , H K , a n d C K r e s p e c t i
vely.
Calculations
With organ extracts, the changes in extinction often do not come to a stop even after a long time. It is
then necessary to extrapolate to the starting times of the individual reactions.
T h e reactions proceed stoichiometrically; formula (2) o n p . 312 is therefore valid. T h e C P concentration

per ml. sample diluted with distilled water is t h u s :

c = 0.492 x AE 0.482 x AE 0.870 x AE [/miole/ml.]
F o r biological samples, the fluid c o n t e n t of the tissue a n d the dilution during the p r e t r e a t m e n t of the sample
m u s t be taken into account.
If
V x = weight (g.) or v o l u m e (ml.) of the tissue sample
V 2 = Vj + g- or ml. perchloric acid used for deproteinization
V 3 = volume of the perchloric acid extract before neutralization
V 4 = V 3 + ml. K C 0
2 3 required for neutralization
V 5 = volume of the deproteinized sample in the cuvette (ml.),
V x V
the dilution factor is F = — - —.
V, x V 3
A water content of 7 5 % is assumed for tissue (liver, muscle, heart). A c c o r d i n g to p . 2107, therefore, 3.25 ml.
perchloric acid solution (V) are required to deproteinize 1 g. tissue. If it is wished to express the results in
/zmole/g. tissue, then it is necessary to insert V in g. a n d V in g. tissue + 1.035 x ml. perchloric acid
x 2
(1.035 = specific gravity of 6% perchloric acid) in the calculations.

If it is wished to express the results as /rniole C P / m l . tissue, then the weight of the tissue sample V x (g.)
is divided by the specific gravity of the tissue a n d the volume (ml.) of perchloric acid is a d d e d in V . To 2
obtain the /ig. CP/g. tissue, the /imole C P / g . tissue must be multiplied by the molecular weight of C P
(211.08). F o r corrections for the blood content of the tissue a n d for the intercellular space see " D e t e r
mination of A T P with Hex okinase a n d Glucose-6-phosphate D e h y d r o g e n a s e " , p . 2107.
In the presence of large a m o u n t s of P O j " (e. g. in the analysis of deproteinized i n c u b a t i o n mixtures which
contain Krebs-Ringer p h o s p h a t e saline) turbidity occurs in the cuvette, often only d u r i n g the assay,
owing to the precipitation of fine crystals of m a g n e s i u m a m m o n i u m p h o s p h a t e (simulating a n increase in
extinction).
If there is d o u b t whether the reaction is proceeding, a d d crystalline C P a n d check t h a t the system functions
correctly.
Determination with Creatine Kinase, Phosphoglycerate

Kinase, and Glyceraldehyde Phosphate Dehydrogenase 3
C r e a t i n e k i n a s e c a t a l y s e s t h e transfer o f p h o s p h a t e f r o m c r e a t i n e p h o s p h a t e t o a d e n o s i n e
d i p h o s p h a t e . T h e r e s u l t i n g A T P , in t h e p r e s e n c e o f p h o s p h o g l y c e r a t e k i n a s e , P G K (ATP:
3-phospho-D-glycerate 1-phosphotransferase, E C 2.7.2.3), phosphorylates D-glycerate-3-phos-
phate to 1,3-diphosphoglycerate, w h i c h undergoes N A D H - d e p e n d e n t reduction with glyceral-
dehyde-3-phosphate dehydrogenase, G A D P H (D-Glyceraldehyde-3-phosphate: N A D oxido
reductase, phosphorylating, E C 1.2.1.12) to D - g l y c e r a l d e h y d e - 3 - p h o s p h a t e (indicator reaction).
Principle
(1) CP + ADP — > Creatine + A T P
i 1
(2) A T P + Glycerate-3-P P G K
> 1,3-Diphosphoglycerate + ADP
(3) 1,3-Diphosphoglycerate + N A D H + H + G A P D H
> Glyceraldehyde-3-P -f N A D +
+ V {
1 mole of N A D H is oxidized per mole of creatine p h o s p h a t e . T h e decrease in extinction at 334, 340, or

365 n m is measured.
U n d e r the conditions indicated, the equilibrium of the overall reaction lies on the right, so that creatine
p h o s p h a t e can be determined together with A T P . T h e m e t h o d can also be c o m b i n e d with the d e t e r m i n a t i o n
of pyruvate a n d of dihydroxyacetone p h o s p h a t e .
Equipment, see p . 1778.
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 8. P o t a s s i u m c a r b o n a t e , A . R .
2. R e d u c e d nicotinamide-adenine 9. M e t h y l o r a n g e o r m e t h y l r e d
dinucleotide, NADH 10. C r e a t i n e k i n a s e , C K
d i s o d i u m salt, N A D H - N a ; commercial p r e p
2 from rabbit muscle, salt-free lyophilized p r e p
a r a t i o n , see p . 545. aration; ^18 U / m g . protein (25 ° C ) ; c o m
3. M a g n e s i u m s u l p h a t e , M g S 0 - 7 H 0 4 2 mercial p r e p a r a t i o n , see p . 444.
4. D-Glycerate-3-phosphate 11. P h o s p h o g l y c e r a t e kinase, P G K
crystalline t r i c y c l o h e x y l a m m o n i u m salt ( 3 H 0 ) ; 2 from yeast, crystalline suspension in 3.2 M a m
commercial p r e p a r a t i o n , see p . 540. m o n i u m sulphate solution (1 m M EDTA);
5. A d e n o s i n e d i p h o s p h a t e , A D P ^ 4 0 0 U / m g . (25 ° C ) ; commercial p r e p a r a t i o n ,
d i s o d i u m salt, A D P - N a ; 2 commercial prep s e e p . 502.
a r a t i o n , see p . 525. 12. Glyceraldehyde-3-phosphate
6. G l u t a t h i o n e , r e d u c e d dehydrogenase, GAPDH
C o m m e r c i a l p r e p a r a t i o n , see p . 538. from rabbit muscle, crystalline suspension in
7. P e r c h l o r i c a c i d , A . R . , s p . g r . 1.67; 3.2 M a m m o n i u m sulphate solution (0.1 m M
c a . 7 0 % ( w / w ) o r s p . g r . 1.54; c a . 6 0 % E D T A ) ; ^ 36 U / m g . (25 °C); commercial prep
(w/w) a r a t i o n , see p. 466.
Creatine k i n a s e : see p . 1778. Phosphoglycerate kinase a n d glyceraldehyde-3-phosphate d e h y d r o g e n a s e :

based o n the specific activities, the c o n t a m i n a t i n g activities of the following enzymes should be < 0 . 0 1 % :
adenylate kinase, A T P a s e , glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, L-gly-
cerol-3-phosphate dehydrogenase, aldolase, a n d t r i o s e p h o s p h a t e isomerase.
U s e only fresh d o u b l y distilled w a t e r .

I. T r i e t h a n o l a m i n e b u f f e r ( 5 0 m M ; p H = 7.5):
D i s s o l v e 4 . 6 5 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in 2 0 0 m l . d i s t i l l e d w a t e r , a d d 11 m l .
1 N N a O H , a n d m a k e u p t o 500 ml. with distilled w a t e r ; c h e c k the p H with a p H m e t e r .
II. M a g n e s i u m s u l p h a t e ( 0 . 5 M ) :
D i s s o l v e 1 2 . 3 2 g. M g S 0 - 7 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
4 2
III. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( c a . 10 m M ) :
D i s s o l v e 6.8 m g . N A D H - N a 2 in 1 m l . d i s t i l l e d w a t e r .
I V . G l y c e r a t e - 3 - p h o s p h a t e ( c a . 0.1 M ) :
D i s s o l v e 5 3 . 8 m g . o f t h e t r i c y c l o h e x y l a m m o n i u m salt in 1 m l . d i s t i l l e d w a t e r .
V. G l u t a t h i o n e , G S H (ca. 50 m M ) :
D i s s o l v e 1 5 . 4 m g . g l u t a t h i o n e in 1 m l . d i s t i l l e d w a t e r .
V I . A d e n o s i n e d i p h o s p h a t e ( c a . 10 m M ) :
D i s s o l v e 5.4 m g . A D P - N a 2 in 1 m l . d i s t i l l e d w a t e r .
VII. Phosphoglycerate kinase, P G K (10 mg. protein/ml.):
U s e stock suspension undiluted.
VIII. Glyceraldehyde-3-phosphate dehydrogenase, G A P D H (10 mg. protein/ml.):
U s e stock suspension undiluted.
I X . C r e a t i n e k i n a s e , C K (5 m g . p r o t e i n / m l . ) :
D i s s o l v e 5 m g . l y o p h i l i z e d p r e p a r a t i o n in 1 m l . d i s t i l l e d w a t e r .
X. Perchloric acid (6% w / w ) :
D i l u t e 7.8 m l . 7 0 % p e r c h l o r i c a c i d o r 9.7 m l . 6 0 % a c i d t o 1 5 0 m l . w i t h d i s t i l l e d w a t e r .
X I . P o t a s s i u m carbonate (ca. 5 M ) :
D i s s o l v e 7 2 g. K C 0 2 3 in 100 m l . d i s t i l l e d w a t e r .
XII. Methyl orange or methyl red:
D i s s o l v e 5 0 m g . i n d i c a t o r in 1 0 0 m l . d i s t i l l e d w a t e r .
K e e p all solutions in stoppered containers at 1 - 4 °C. P r e p a r e fresh C K solution (IX) daily from dry powder,
and fresh N A D H , A D P , a n d glycerate-3-phosphate solutions weekly.
Procedure
F o r t r e a t m e n t o f t h e m a t e r i a l u n d e r i n v e s t i g a t i o n , see p . 1 7 7 9 , 2 0 9 9 .
Assay System
T h e v o l u m e o f s a m p l e ( V ) u s e d for the a s s a y s h o u l d b e s u c h that t h e c h a n g e in e x t i n c t i o n d o e s

5
not exceed 0.300.

W a v e l e n g t h : 3 4 0 , H g 3 3 4 , o r H g 3 6 5 n m ; light p a t h : 1 c m . ; final v o l u m e ; 3 . 0 0 m l . ; ca. 25 ° C ;
m e a s u r e a g a i n s t air.
T h e v o l u m e o f t h e a s s a y m i x t u r e c a n b e d e c r e a s e d t o 2 m l . b y d e c r e a s e o f t h e q u a n t i t y o f buffer
a n d o m i s s i o n o f the w a t e r . If o n l y a s m a l l q u a n t i t y o f extract is a v a i l a b l e , t h e v o l u m e p i p e t t e d
c a n b e d e c r e a s e d b y t h e u s e o f s e m i m i c r o c u v e t t e s a n d b y a p p r o p r i a t e m o d i f i c a t i o n o f the a s s a y
system.
Prepare the following reagent mixture immediately before use:
Buffer s o l u t i o n (I) 1.50 v o l .

MgS0 4 s o l u t i o n (II) 0.04 vol.
N A D H s o l u t i o n (III) 0.10 vol.
Glycerate-3-phosphate solution (IV) 0.20 vol.
G S H solution (V) 0.10 vol.
A D P solution (VI) 0.03 vol.
P G K suspension (VII) 0.010 vol.
G A P D H suspension (VIII) 0.010 vol.
Reagent mixture 1.99 m l . 25 m M buffer, 6.7 m M

MgSO ,0.33 mM
4 NADH
6.7 m M g l y c e r a t e - 3 - p h o s -
phate
1.7 m M G S H , 0.1 m M
ADP
33 fig. P G K / m l . = 13 U / m l .
33 fig. G A P D H / m l . =
1.2 U / m l .
S a m p l e -j- w a t e r 1.00 m l .
M i x ; f o l l o w r e a c t i o n w i t h a r e c o r d e r if p o s s i b l e ,
o t h e r w i s e f o l l o w c h a n g e in e x t i n c t i o n for ca. 15 m i n .
A T P in t h e s a m p l e r e a c t s . Wait u n t i l r e a c t i o n s t o p s o r
r e c o r d slight " c r e e p " . D e t e r m i n e e x t i n c t i o n E ^
C K solution (IX) 0.01 m l . 16.7 fig. C K / m l . = 3 0 0 m U / m l .
M i x ; after 15 m i n . , d e t e r m i n e e x t i n c t i o n E . 2
E t - E 2 = A E.
T h e e x t i n c t i o n d u e t o t h e C K itself m u s t b e d e t e r m i n e d at the e n d o f t h e a s s a y . T h i s d e t e r m i n a
t i o n m u s t be carried o u t at least o n c e d a i l y o r w h e n e v e r n e w e n z y m e s o l u t i o n s o r s u s p e n s i o n s
are u s e d .
C P a n d A T P c a n b e d e t e r m i n e d t o g e t h e r in t h e s a m e c u v e t t e . T h i s is d o n e b y p i p e t t i n g t h e
r e a g e n t s o l u t i o n s s i n g l y in t h e a b o v e o r d e r a n d d e t e r m i n i n g the A E v a l u e s c o r r e s p o n d i n g t o
the c o n c e n t r a t i o n s o f A T P a n d C P s e p a r a t e l y after a d d i t i o n o f G A P D H a n d o f C K r e s p e c t i v e l y .
Calculations, Sources of Error, see p . 1780, 1781.
O t h e r M e t h o d s for the D e t e r m i n a t i o n o f C r e a t i n e P h o s p h a t e
N o other m e t h o d s have so far been described for the enzymatic d e t e r m i n a t i o n of C P . T h e m e t h o d s described

here have proved useful in investigations on heart metabolism. Enzymatic s p e c t r o p h o t o m e t r i c determina
tions of energy-rich p h o s p h a t e s have been p r o p o s e d by Slater 4
a n d Kratzing et a l . . 5
K n o w n C P analyses are based mainly on the determination of the p h o s p h a t e c o m p o n e n t of the C P . T h e

most c o m m o n m e t h o d is the colour test with reduced p h o s p h o m o l y b d i c a c i d 6 - 1 0
.
C P analyses based o n the detection of the creatine c o m p o n e n t use either the Jaffe r e a c t i o n 1 1 , 1 2
or the colour
reaction with d i a c e t y l - a - n a p h t h o l 1 3 - 1 5
. In m a n y cases, C P is separated by c h r o m a t o g r a p h y or electro
phoresis before such d e t e r m i n a t i o n s 1 6 - 2 1
.
References
1 L. Noda, S. A. Kuby & H. Lardy in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c

Press, N e w Y o r k , Vol. II, p . 605.
2 S. A. Kuby c.s., J. biol C h e m . 209, 191 [1954]; 2 / 0 , 65, 83 [1954].
3 E. N. Fawaz, G. Fawaz & K. von Dahl, P r o c . Soc. Exp. Biol. M e d . 709, 38 [1962].
4 E. C Slater, Biochem. J. 50, vii [1951]
5 C. C. Kratzing & A. Narayanaswami, Biochem. J. 54, 317 [1953].
6 C. H. Fiske & J. Subbarow, J. biol. C h e m . 81, 629 [1929].
7 O. H. Lowry & J. A. Lopez, J. biol. C h e m . 162, 421 [1945].
8 B. E. Wahler & A. Wollenberger, Biochem. Z. 329, 508 [1958].
9 J. Berenblum & E. Chain, Biochem. J. 32, 295 [1938].
10 J. B. Martin & D. M. Doty, Analytic. C h e m . 21, 965 [1949].
11 H.H. Taussky, J. biol. C h e m . 208, 835 [1954].
12 H. Mcllwain, H. L. Buchel & /. D. Ceshire, Biochem. J. 48, 12 [1951].
13 D. Eggleton, S. R. Elsden & N. Gough, Biochem. J. 37, 526 [1943].
14 A. E. Ennor & H. Stocken, Biochem. J. 42, 557 [1948].
15 A. H. Ennor & H. Rosenberg, Biochem. J. 51, 606 [1952].
16 A. Fleckenstein & E. Gerlach, N a u n y n - S c h m i e d e b e r g s A r c h . exp. P a t h o l . P h a r m a c o l . 2 1 9 , 531 [1953].
17 A. Fleckenstein & J. Jahnke, Pfliigers Arch. ges. Physiol. Menschen, Tiere 258, 111 [1953].
18 A. Martonosi, Biochem, biophys. Res. C o m m u n . 2, 12 [I960].
19 P. C Caldwell, Biochem, J. 55, 458 [1955].
20 W. Thorn, W. Isselhard & K. Irmsher, Biochem. Z. 330, 385 [1958].
21 E. Gerlach & J. Jahnke, Biochem. Z. 330, 565 [1958].
Creatinine
August Wilhelm Wahlefeld, Gunter H o l z and H a n s Ulrich Bergmeyer
Creatinine occurs in muscle cells of vertebrates as the only intermediate p r o d u c t of creatine metabolism.
It is formed from creatine by t h e formation of a cyclic amide a n d the removal of water. This reaction occurs
spontaneously in muscle, b u t h a s been shown to be enzymically catalysed in micro-organisms.
Creatinine is of clinical i m p o r t a n c e because of its very constant renal clearance. D e t e r m i n a t i o n of the
creatinine clearance is a valuable p a r a m e t e r as a measure of the glomerular filtration of the kidney, because
creatinine is n o t excreted or reabsorbed by the tubules.
The assay m e t h o d s so far described for the determination of creatinine in serum a n d urine, as well as in
muscle are unspecific colour tests. They include colour reactions with 3,5-dinitrobenzoic a c i d , with the 1
potassium salts of mercury t h i o c y a n a t e a n d l , 4 - n a p h t h o q u i n o n e - 2 - s u l p h o n a t e . T h e most c o m m o n is the

2 3
so-called Jaffe r e a c t i o n , in which creatinine a n d picric acid form an orange-red colour in alkaline conditions.
4
This reaction is relatively unspecific; t h e specificity can be increased by a b s o r p t i o n of creatinine on Lloyd's

r e a g e n t (aluminium silicate, kaolin). Enzymatic assay m e t h o d s
6 7 , 8
were n o t practicable because the necessary
enzymes were n o t readily available. Recently, this p r o b l e m h a s been o v e r c o m e 9 , 1 0
. T h e enzymatic m e t h o d
described h e r e 11
depends o n the specific, quantitative conversion of creatinine t o creatine with creatinine
a m i d o h y d r o l a s e a n d the subsequent specific determination of the creatine f o r m e d . 12
Application of Method: In biochemistry, clinical biochemistry a n d foodstuff chemistry.
Principle
(1) Creatinine + H 0 „ 2
c r e a t i n i n a s e
* , Creatine
(2) Creatine + A T P l r a t i n e kinase** , C r e a t i n e phosphate + ADP
I
(3) ADP + PEP pyruvate k i n a s e * * ^ ^ j p + p y r u v a t e
, LDH**** „ L A C T A T E + N A D +
The oxidation of N A D H , as measured by the decrease of extinction at 340 (334, 365) n m , is directly
p r o p o r t i o n a l t o the a m o u n t of creatinine present.
T h e e q u i l i b r i u m c o n s t a n t f o r r e a c t i o n ( 1 ) is K H O = 1 . 2 7 at p H 8 . 0 in 0 . 1 M g l y c y l g l y c i n e buffer
2
a n d 3 7 ° C . S i n c e t h e Michaelis c o n s t a n t o f c r e a t i n i n a s e for c r e a t i n i n e is v e r y large ( 1 0 " 2

M )a
s l o w r e a c t i o n is t o b e e x p e c t e d in t h e c o u p l e d a s s a y ; i n c r e a s e d rates c a n b e o b t a i n e d o n l y b y
a d d i t i o n o f large a m o u n t s o f c r e a t i n i n a s e w i t h sufficient a c t i v i t y o f a u x i l i a r y a n d i n d i c a t o r
enzymes.
* Creatinine a m i d o h y d r o l a s e ( n o systematic n a m e yet assigned by t h e E n z y m e Commission).

** A T P : creatine ^ - p h o s p h o t r a n s f e r a s e , E C 2.7.3.2.
*** A T P - p y r u v a t e 2 - 0 - p h o s p h o t r a n s f e r a s e , E C 2.7.1.40.
**** L-Lactate: N A D oxidoreductase, E C 1.1.1.27.
Creatinine 1787
T h e p H profile o f t h e c o u p l e d a s s a y s h o w s a b r o a d o p t i m u m b e t w e e n p H 7.5 a n d 8.5 if 0.1 M

g l y c i n e o r 0.1 M g l y c y l g l y c i n e buffer is u s e d . T r i e t h a n o l a m i n e b u f f e r (0.1 M ) d i s p l a c e s t h e
o p t i m u m t o t h e a c i d r a n g e ; tris, d i e t h a n o l a m i n e , a m e d i o l a n d 2-amino-2-methylpropanol
buffers h a v e a s i m i l a r effect. In a d d i t i o n , this series o f buffers i n h i b i t t h e o v e r a l l r e a c t i o n ; t h e
i n h i b i t i o n is c o m p l e t e w i t h 2 - a m i n o - 2 - m e t h y l p r o p a n o l buffers a b o v e p H 7.5. S i m i l a r l y , buffers
o f h i g h i o n i c s t r e n g t h i n h i b i t t h e o v e r a l l r e a c t i o n . In t h e m e t h o d for s e r u m d e s c r i b e d h e r e
d e p r o t e i n i z a t i o n is o m i t t e d . N o r m a l s e r u m c o n t a i n s r e l a t i v e l y little c r e a t i n i n e ( c a . 10 ~ 4
M),
therefore a comparatively large a m o u n t o f sample must be taken. To avoid the occurrence o f
t u r b i d i t y in t h e a s s a y m i x t u r e d e t e r g e n t s are a d d e d (0.1 t o 0 . 2 % v / v ) . U l t r a v o n ® or T r i t o n - X -
100® h a v e p r o v e d s u i t a b l e . T h e t e m p e r a t u r e c h o s e n for t h e a s s a y ( 3 0 ° C ) is a c o m p r o m i s e
b e t w e e n a r a p i d r e a c t i o n a n d t e m p e r a t u r e - d e p e n d e n t " c r e e p " . B y a d d i t i o n o f 0.1 M N a H P 0 2 4
t o t h e buffer ( p H 8) t h i s s e c o n d a r y r e a c t i o n m a y b e r e d u c e d b y half. P h o s p h a t e c a n h o w e v e r
o n l y b e u s e d in t h e test m i x t u r e in t h e a b s e n c e o f a m m o n i u m i o n s ( u s e e n z y m e s o l u t i o n s in
glycerol).
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e for p r e c i s e m e a s u r e m e n t s at 3 4 0 , 3 3 4
o r 365 n m ; p r e f e r a b l y a r e c o r d e r b u t t h i s is n o t a b s o l u t e l y e s s e n t i a l .
Reagents*
1. G l y c i n e , A . R . 10. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o
2. D i s o d i u m h y d r o g e n p h o s p h a t e , tide, N A D H
Na HP0 12H 0
2 4 2 disodium salt, N A D H - N a ; commercial p r e p
2
3. H y d r o c h l o r i c a c i d , H C 1 , A . R . aration, see p . 545.

4 . S o d i u m h y d r o x i d e , N a O H , A . R. I N 11. C r e a t i n e k i n a s e , C K
5. U l t r a v o n J F N W , C i b a A G , W e h r ( B a d e n ) , from rabbit muscle, lyophilized preparation;
West G e r m a n y ^ 18 U / m g . (25 °C); commercial p r e p a r a t i o n ,
6. M a g n e s i u m c h l o r i d e , M g C l • 6 H 0 , A . R.
2 2 see p . 4 4 4 .
7. S o d i u m h y d r o g e n c a r b o n a t e , NaHC0 , 3 12. Lactate d e h y d r o g e n a s e , L D H
A.R. from skeletal muscle, solution in 50% glycerol,
8. P h o s p h o e n o l p y r u v a t e , P E P p H ca. 7; ^ 5 5 0 U / m g . (25 °C); commercial
tricyclohexylammonium salt; commercial p r e p p r e p a r a t i o n , see p . 4 8 1 .
aration, see p . 548. 13. Pyruvate kinase, P K
9. A d e n o s i n e t r i p h o s p h a t e , A T P from rabbit muscle, solution in 5 0 % glycerol,
d i s o d i u m salt, A T P - N a H • 3 H 0 ; commercial
2 2 2 p H ca. 6; ^ 200 U / m g . (25 °C); commercial
p r e p a r a t i o n , see p . 527. p r e p a r a t i o n , see p . 509.
14. C r e a t i n i n e a m i d o h y d r o l a s e (creatininase)
from micro-organisms, solution in 5 0 % glycerol;
^ 50 U / m g . (25 °C); commercial p r e p a r a t i o n ,
see p . 445.
Purity of Reagents
A T P m u s t b e c o m p l e t e l y free f r o m A D P , a n d P E P free f r o m p y r u v a t e . A l l f o u r e n z y m e s m u s t
n o t c o n t a i n m o r e t h a n 0.01 % A T P a s e , m y o k i n a s e , h e x o k i n a s e o r o t h e r k i n a s e s (relative t o
their r e s p e c t i v e a c t i v i t i e s ) .
* Complete reagent kits are available commercially, see p . 558.

P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y distilled w a t e r .

I. G l y c i n e / p h o s p h a t e buffer (0.1 M g l y c i n e ; 0.1 M N a H P 0 ; 0 . 1 % U l t r a v o n ; p H 8 . 0 ) :
2 4
D i s s o l v e 7.51 g. g l y c i n e a n d 3 5 . 8 g. N a H P 0 2 4 • 1 2 H 0 in 8 0 0 ml. distilled w a t e r , a d d 1 m l .

2
U l t r a v o n , adjust t o p H 8.0 w i t h d i l u t e h y d r o c h l o r i c a c i d a n d d i l u t e t o 1 0 0 0 m l . w i t h
distilled w a t e r .
II. M a g n e s i u m c h l o r i d e s o l u t i o n (1 M ) :
D i s s o l v e 2 . 0 3 g. M g C l - 6 H 0 w i t h distilled w a t e r a n d m a k e u p t o 10 m l .
2 2
III. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e / a d e n o s i n e triphosphate/phosphoenolpyru-
v a t e (2 m M £ - N A D H , 5.5 m M A T P , 6 m M P E P , 0.1 M g l y c y l g l y c i n e buffer, p H 8 . 0 ) :
D i s s o l v e 132 m g . g l y c y l g l y c i n e in 8.0 m l . distilled w a t e r , a d j u s t t o p H 8.0 w i t h 1 N N a O H .
A d d 16.6 m g . / ? - N A D H - N a , 3 4 m g . A T P - N a H - 3 H 0 a n d 2 8 . 4 m g .
2 2 2 2 PEP(CHA) , 3
c h e c k t h e p H a n d d i l u t e t o 10 m l . w i t h distilled w a t e r .
IV. Lactate d e h y d r o g e n a s e / p y r u v a t e kinase, L D H / P K (700 U / m l . ; 300 U / m l . , respectively):
D i l u t e the s t o c k s u s p e n s i o n s a c c o r d i n g l y w i t h 5 0 % g l y c e r o l a n d m i x .
V. S o d i u m b i c a r b o n a t e s o l u t i o n ( 0 . 5 % N a H C 0 ) : 3
D i s s o l v e 0.5 g. N a H C 0 3 in d i s t i l l e d w a t e r a n d d i l u t e t o 100 m l .
VI. Creatine kinase, C K (1500 U / m l . ) :
D i s s o l v e 75 m g . p r o t e i n in 1.0 m l . 0 . 5 % N a H C 0 3 s o l u t i o n ( V ) if n e c e s s a r y , c e n t r i f u g e
off a n y p r e c i p i t a t e .
VII. Creatinine amidohydrolase, creatininase (500 U / m l . ) :
Dilute stock solution with 5 0 % glycerol accordingly.
S t o r e all s o l u t i o n s a n d s u s p e n s i o n s , s t o p p e r e d , in a refrigerator at 0 - 4 ° C . S o l u t i o n s I, II, I V ,

V a n d V I I are s t a b l e f o r c a . 1 y e a r , s o l u t i o n III m u s t b e p r e p a r e d f r e s h l y e v e r y f o r t n i g h t a n d
solution VI every day.
Procedure
Collection of sample :
C o l l e c t b l o o d w i t h o u t v e n e s t a s i s a n d a v o i d h a e m o l y s i s . A h a e m o g l o b i n c o n t e n t o f o v e r 100 m g . /
100 m l . g i v e s c o n s i d e r a b l y l o w e r v a l u e s for c r e a t i n i n e . A d d i t i o n o f 1 m g . o x a l a t e / m l . , 1 m g . E D T A /
ml. a n d 0.2 or 2 . 0 m g . h e p a r i n / m l . d o e s n o t interfere w i t h t h e a s s a y . A d d i t i o n o f citrate o r
fluoride should be avoided.
U s e s e r u m o r p l a s m a directly. D i l u t e u r i n e 1 -f 4 9 w i t h d o u b l y d i s t i l l e d w a t e r .
In the f r o z e n state s a m p l e s are s t a b l e indefinitely, w h i l e at 4 ° C t h e y k e e p for at least 2 d a y s .
Creatinine 1789
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 3 . 2 2 m l . ; t e m p e r a t u r e :
30 ° C ; read a g a i n s t air.
G l y c i n e / P h o s p h a t e buffer (I) 2.00 ml. 62.5 m M glycine,

62.5 m M p h o s p h a t e
0.06% Ultravon
M g C l solution
2 (II) 0.05 ml. 15 m M MgCl 2
N A D H / A T P / P E P solution (HI) 0.50 ml. 0.3 m M NADH

0.85 m M A T P
0.93 m M P E P
L D H / P K suspension (IV) 0.05 m l . c a . 10 U L D H / m l .
c a . 4.5 U P K / m l .
C K solution (VI) 0.10 ml. ca 45 U C K / m l .
Sample 0.50 ml. u p t o 0.11 m M
M i x a n d i n c u b a t e for 1 0 - 1 5 m i n . at 30 ° C .
Creatininase solution (VII) 0.02 ml. ca. 3 U / m l .
M i x a n d i m m e d i a t e l y r e a d e x t i n c t i o n E . I n c u b a t e for
x
e x a c t l y 30 m i n . at 30 ° C a n d r e a d E . F o r e x t r a p o l a t i o n
2
o f the c r e e p , d e t e r m i n e E 3 after a further 10 m i n .
Calculations
U n d e r the above conditions the reaction proceeds stoichiometrically. By m e a s u r e m e n t of the creep a n d

correction for this by extrapolation all possible interfering side reactions are eliminated. T h e extinction
difference representing the creatinine content is:
AE = ( E - E ) - 3 x
x 2 (E -E )
2 3
T h e concentration of creatinine in serum is calculated as follows:
c = AE x 1.056 AE x 1.035 z l E x 1.894 [^mole/ml.]

c = AE x 11.94 AE x 11.71 AE x 21.43 [mg./lOO ml.]
In the case of urine the a p p r o p r i a t e calculation factor must be multiplied by 50 to correct for the dilution
(1 + 49).
The precision of the m e t h o d measured in series, e. g. at a wavelength of 365 n m , is 1 % at a concentration of

7 mg. creatinine/100 ml. a n d 4 . 6 % at a concentration of 0.8 m g . / l 0 0 ml.
T h e precision from day to day was found to be 3 . 3 % for 1.8 mg. creatinine/100 ml.
N o r m a l Values
Creatinine has not been measured in serum by this m e t h o d on a sufficiently large p o p u l a t i o n . T h e n o r m a l

values for the modification of the Jaffe m e t h o d with Lloyd's reagent are therefore g i v e n : 13
W o m e n 0.5 to 1.0 mg./lOO ml.
Men 0.7 to 1.2 mg./lOO ml.
Effects of drugs and other therapeutic measures on the creatinine levels: refer to Christian . 14
Interference in the assay: Care should be taken that the initial extinction is sufficiently high, e.g. at 365 n m
E x should be 0.800. Lower initial extinctions are due to old N A D H / A T P / P E P solutions or too high
pyruvate content in the sample. In this case m o r e N A D H should be a d d e d . Too low C K activity prolongs
the reaction and too little creatinine is recovered. Interference from other c o m p o u n d s has not been en
countered.
The specificity of creatinine a m i d o h y d r o l a s e has not been intensively studied, e. g. it is not k n o w n whether
glycocyamidine is hydrolysed. T h e high specificity of the m e t h o d is conferred by the creatine kinase r e a c t i o n ;
see the specificity of the creatine assay, p. 1776.
References
1 S. R. Benedict & /. A. Behre, J. biol. C h e m . 114, 115 [1936].

2 P. Stelgens, Biochem. Z. 324, 228 [1953].
3 M. X. Sullivan & F. Irreverre, J. biol. C h e m . 223, 530 [1958].
4 M. Jaffe, Z. Physiol. C h e m . 10, 391 [1886].
5 S. Narayanan & H. D. Appleton, Clin. C h e m . 18, 270 [1972].
6 R. S. Hare. Proc. Soc. Exp. Biol. M e d . 74, 148 [1950].
7 R. Dubos & B. F. Miller, J. biol. C h e m . 121, 429 [1937].
8 B. F. Miller & R. Dubos, J. biol. C h e m . 121, 457 [1937].
9 Boehringer M a n n h e i m G m b H . G e r m a n patents P2122294.6, P 2 1 2 2 2 9 8 . 0 .
10 M.H. McLean & J. Gallwas, Abstract 30.2. Scand. J. Clin. L a b . Invest. 29, Suppl. 126 [1972]; 8th
Intern. Congress Clinical Chemistry, C o p e n h a g e n .
11 A. W. Wahlefeld, G. Herz & H. U. Bergmeyer, Abstract 30.1, Scand. J. Clin. L a b . Invest. 29, Suppl. 126
[1972]; 8th Intern. Congress of Clinical Chemistry, C o p e n h a g e n .
12 M. L. Tanzer & C. Gilvarg, J. biol. C h e m . 234, 3201 [1959].
13 R. J. Henry, Clinical Chemistry, p . 303, 1964, H o e b e r Medical Division, H a r p e r & R o w , N e w York,
USA.
14 D. G. Christian, Amer. J. Clin. Path. 54, 129 [1970].
Urea
Ingeborg Gutmann and Hans Ulrich Bergmeyer
Urea, the diamide of c a r b o n i c acid, is the m o s t i m p o r t a n t d e g r a d a t i o n p r o d u c t of protein metabolism in

m a n , in other m a m m a l s a n d in certain other a n i m a l species. U r e a is formed in the liver a n d c o m p o s e s the
major fraction of the organic substances present in urine.
There are m a n y m e t h o d s for the d e t e r m i n a t i o n of urea. T h e qualitative identification is u n s p e c i f i c . 1,2
Of the quantitative m e t h o d s the following are w o r t h y of m e n t i o n :

a) d e t e r m i n a t i o n with x a n t h y d r o l , which can be carried out gravimetrically , by the d e t e r m i n a t i o n of 3
nitrogen according t o Kjeldahl*, colorimetrically , o x i d i m e t r i c a l l y 5 6,7

a n d n e p h e l o m e t r i c a l l y ; b) the colori
8
metric determination with d i a c e t y l m o n o x i m e 9,10

; c) the colorimetric d e t e r m i n a t i o n with dimethyl-
g l y o x i m e ; d) the colorimetric d e t e r m i n a t i o n with p - d i m e t h y l a m i n o b e n z a l d e h y d e
11 12,13
.
T h e enzymatic hydrolysis of u r e a with urease ( U r e a a m i d o h y d r o l a s e , E C 3.5.1.5), which results in the form
ation of a m m o n i a a n d c a r b o n dioxide, was earlier used for the d e t e r m i n a t i o n of urea in m u s c l e , in food 14
stuffs , fertilizers
15 16
a n d drinking w a t e r . T h e two reaction p r o d u c t s can serve to determine the urea
17
content of the sample. C a r b o n dioxide can be determined g a s o m e t r i c a l l y . A m m o n i a can be determined 18
d i r e c t l y , after d i s t i l l a t i o n
19 20
o r titrated after diffusion . Instead of t i t r a t i o n the a m m o n i a can also be
21
determined with Nesslers' reagent after separation by d i s t i l l a t i o n , by microdiffusion 22 23

or by ion exchange
adsorption . 24
Simpler t h a n the a b o v e - m e n t i o n e d m e t h o d s is the d e t e r m i n a t i o n with Nesslers' reagent directly in the

hydrolysis mixture after deproteinization, with inclusion of the a p p r o p r i a t e b l a n k s 2 0 , 2 2
' ' .
2 5 2 6
A m m o n i a can be determined m o r e sensitively a n d m o r e accurately with the Berthelot reaction 27

using
sodium n i t r o p r u s s i d e 28
as the c a t a l y s t ' . T h e first m e t h o d described here is based on t h a t of
29 30
Fawcett
and Scott . 31
In the second m e t h o d described here a m m o n i a is determined with the aid of g l u t a m a t e d e h y d r o g e n a s e

( L - G l u t a m a t e : N A D oxidoreductase, d e a m i n a t i n g , E C 1 . 4 . 1 . 2 ) 32,33
.
Application of Method: In biochemistry, clinical chemistry a n d in foodstuff chemistry.
Determination of Urea, Indicator Reaction with

Phenol and Hypochlorite
Principle
(1) Urea + H 0 2 2NH 3 + C0 2
, , I
I
(2) 2NH 3 + 2NaC10 • 2NH C1 + 2 NaOH
2
(3) 2 Phenol + 2 N H C 1 + 2 N a O H
2 • 2-p-Aminophenol + 2 NaCl + 2H 0 2
2 p-Aminophenol + 2 Phenol + 0 2 • 2 Indophenol + 2H 0 2
A m m o n i u m ions react with p h e n o l a n d hypochlorite to give a blue dye i n d o p h e n o l , which is p r o p o r t i o n a l 34
to urea c onc e ntra tion a n d can be m e a s u r e d between 530 a n d 650 n m .

T h e p H o p t i m u m for u r e a s e 35
is p H 7.0; it alters with the type of buffer solution used. T h e turnover n u m b e r
of the e n z y m e 36
is 4.6 x 1 0 mole urea per min. per mole at 20 °C. T h e Michaelis c o n s t a n t
5 35
is 0.4 m M . T h e
Berthelot colour reaction is complete in 2 0 - 3 0 min. at 37 ° C ; the colour remains c o n s t a n t for u p to 24 hr.
T h e m a x i m u m of the a b s o r p t i o n spectrum is at 630 n m . It is preferable to read between 530 and 580 n m
(e. g. with the filters H g 546, H g 578), a n d to carry out the hydrolysis of the u r e a at the p H of the serum or
urine.
Equipment
S p e c t r o p h o t o m e t e r , s p e c t r u m - l i n e p h o t o m e t e r o r filter p h o t o m e t e r s u i t a b l e for m e a s u r e
m e n t s at 5 3 0 n m t o 5 8 0 n m ; c o n s t a n t t e m p e r a t u r e w a t e r b a t h (37 ° C ) .
Reagents*
1. U r e a , A . R. 6. Urease
2. P h e n o l , l i q u i d from soya b e a n s ; dry p o w d e r ;
3. S o d i u m n i t r o p r u s s i d e - 2 H 0 , A . R.
2 ^ 5 U / m g . (25 °C) Commercial preparation,
4. S o d i u m h y p o c h l o r i t e s o l u t i o n see p. 517.
a b o u t 13% active chlorine 7. G l y c e r o l , A . R . , 5 0 % ( v / v )
5. S o d i u m h y d r o x i d e , A . R .
Purity of Reagents
All reagents must be A. R. quality.
P r e p a r e all s o l u t i o n s w i t h freshly p r e p a r e d , d o u b l y distilled w a t e r . T o p r e v e n t t h e g r o w t h

o f m i c r o - o r g a n i s m s , sterilize t h e c o n t a i n e r s .
I. U r e a s e ( 2 m g . / m l . ) :
G r i n d 2 4 m g . u r e a s e w i t h 2 m l . 5 0 % g l y c e r o l , a d d a further 10 m l . 5 0 % g l y c e r o l a n d
mix thoroughly.
II. U r e a s t a n d a r d s o l u t i o n (0.5 m M ) :
D i s s o l v e 3 0 . 0 m g . u r e a in 1 0 0 0 m l . distilled w a t e r .
III. P h e n o l - s o d i u m n i t r o p r u s s i d e ( 0 . 1 0 6 M p h e n o l ; 0 . 1 7 m M s o d i u m n i t r o p r u s s i d e ) :
D i s s o l v e 25 m g . s o d i u m n i t r o p r u s s i d e a n d 5 m l . l i q u i d p h e n o l in 5 0 0 m l . distilled w a t e r .
I V . H y p o c h l o r i t e (11 m M N a C I O ; 0 . 1 2 5 N N a O H ) :
D i s s o l v e 2.5 g. s o d i u m h y d r o x i d e in distilled w a t e r , a d d 2.5 m l . N a C I O s o l u t i o n a n d d i l u t e
t o 5 0 0 m l . w i t h distilled w a t e r .

Urea 1793
Store all solutions, stoppered, in a refrigerator at 0 - 4 °C. T h e p h e n o l a n d h y p o c h l o r i t e solution (III, IV)

are stable for ca. 6 m o n t h s , the s t a n d a r d solution (II) a n d the urease suspension (I) for ca. 1 year.
Procedure
O b t a i n p l a s m a o r s e r u m f r o m b l o o d w h i c h is a s fresh a s p o s s i b l e . A d d i t i o n o f 1 m g . o x a l a t e / m l . ,
1 m g . c i t r a t e / m l . o r 0 . 2 m g . h e p a r i n / m l . d o e s n o t interfere. A v o i d a d d i t i o n o f fluoride to
b l o o d b e c a u s e it i n h i b i t s u r e a s e . T h e i n h i b i t i o n c a n b e r e v e r s e d b y a d d i t i o n o f c a f f e i n e - m a g n e s
ium s a l i c y l a t e . P l a s m a or serum have the s a m e urea content as w h o l e b l o o d .
37
M i x 0.1 m l . s e r u m o r p l a s m a w i t h 0.9 m l . p h y s i o l o g i c a l N a C l . D i l u t e u r i n e 1 : 1 0 0 0 w i t h
physiological NaCl.
In s a m p l e s w h i c h are n o t a b s o l u t e l y fresh a m m o n i a is r e a d i l y f o r m e d f r o m b a s i c a m i n o a c i d s
r e s u l t i n g in t o o h i g h v a l u e s .
Assay System
W a v e l e n g t h : 5 3 0 - 5 8 0 n m ; light p a t h : 1 c m . ; final v o l u m e : 1 0 . 3 m l . ; t e m p e r a t u r e : 37 ° C .
R e a d a g a i n s t b l a n k w i t h o u t s a m p l e . O n e b l a n k a n d o n e s t a n d a r d is sufficient for e a c h series
of measurements.
C o n c e n t r a t i o n in
P i p e t t e i n t o test t u b e s : Standard Sample
assay mixture
Urease suspension (I) 0.10 ml. 0.10 ml. 0.33 mg. ^ 1.7 U / m l .
Urea standard solution (II) 0.20 ml. 0.33 m M
Sample 0.20 ml. u p t o ca. 2.2 m M
urea
M i x , s t o p p e r test t u b e s w i t h c l e a n s t o p p e r s o r P a r a f i l m ® a n d
i n c u b a t e for 15 m i n .
Phenol solution (III) 5.00 ml. 5.00 ml. 53 m M p h e n o l ;

8 0 pM N a n i t r o p r u s s i d e
Hypochlorite solution (IV) 5.00 ml. 5.00 ml. 5.5 m M s o d i u m
hypochlorite
M i x i m m e d i a t e l y , i n c u b a t e for 3 0 m i n . P o u r i n t o c u v e t t e s a n d r e a d
extinction of sample ( E ) and extinction of standard ( E
s a m p l e )
s t a n d a r d
against the blank.
W i t h v a l u e s a b o v e 2 0 0 m g . u r e a / 1 0 0 m l . s e r u m o r 2 0 g. u r e a / 1 0 0 m l . u r i n e r e p e a t t h e a s s a y
w i t h h a l f t h e s a m p l e v o l u m e ( m u l t i p l y result b y 2 ) .
Calculations
T h e extinction is p r o p o r t i o n a l t o the c o n c e n t r a t i o n u p to a content of 200 mg. urea/100 ml. serum (or 20 g.

urea/100 ml. urine). T h e calculation of the urea c o n t e n t is based o n the extinction of the u r e a s t a n d a r d .
T h e s t a n d a r d solution contains 30 ug. u r e a per ml. T h e serum was diluted 1 : 1 0 . Therefore to calculate
the urea c o n c e n t r a t i o n :
E x 30
in s e r u m : c = s a m p l e
— [mg./lOO ml.]
^standard
in u r i n e : c = E s a m p l e X 3
° [g./100ml.]
^standard
With a mean value of 31 mg. urea/100 ml. serum a s t a n d a r d deviation of 0.6 mg. urea was found. T h e
coefficient of variation is 2 % .
Normal Values
1 0 - 5 0 mg. urea/100 ml. s e r u m ; 2 0 - 3 5 g. u r e a / 2 4 hr. u r i n e .

3 8 39
Traces of a m m o n i a interfere with the m e t h o d , therefore use only fresh distilled water a n d always keep
bottles containing reagents stoppered. U r e a s e is inhibited by p o t a s s i u m a n d s o d i u m ions, whereas p h o s
p h a t e ions a c t i v a t e . T h e formation of i n d o p h e n o l is inhibited by heavy metal i o n s . Organic nitrogen
35 34
c o m p o u n d s interfere with the r e a c t i o n . 34
Urease reacts specifically with u r e a . 3 5
Determination of Urea with Glutamate Dehydrogenase as

Indicator Enzyme
Principle
(1) Urea + H 0 - ^ s s ^ 2 N H 2 3 + C0 2
i 1
(2) 2 2-Oxoglutarate + 2 N A D H + 2 N H 4
+
2 L-Glutamate + 2 N A D +
+ 2H 02
T h e decrease of N A D H , as measured by the change of extinction at 340 (334, 365) n m , is p r o p o r t i o n a l to

the a m o u n t of a m m o n i a present. Two moles of a m m o n i a are formed from 1 mole urea.
F o r p H o p t i m u m of urease, see p . 1792. T h e p H o p t i m u m of g l u t a m a t e d e h y d r o g e n a s e lies between 7.6

a n d 8.6; it depends on the buffer s o l u t i o n 4 0 , 4 1
. T h e equilibrium c o n s t a n t of the r e a c t i o n 41
is ca. 2.0 x 1 0 " 1 5
M ; the equilibrium is strongly in favour of the a m i n o acid. T h e Michaelis c o n s t a n t for a m m o n i a is depend-

Urea 1795
ent on the pH and the buffer: K M = 56 m M (phosphate buffer, pH 7 . 6 ) , K 41

M = 3.2 m M (tris buffer, pH
8 . 0 ) . The affinity of the enzyme for ammonia is not very great, therefore an excess of glutamate dehydro
42
genase must be used. The enzyme is activated and stabilized 43

by A D P .
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e for m e a s u r e m e n t s at 3 4 0 , 3 3 4 o r
365 n m .
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 5. Urease
2. 2 - O x o g l u t a r a t e from jack beans; lyophilized: ^100 U/mg.
commercial p r e p a r a t i o n , see p . 548. (25 °C). Commercial preparation, see p. 517.
3. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo 6. G l y c e r o l , A . R., 5 0 % ( v / v )
tide, N A D H 7. S o d i u m h y d r o x i d e , 1 N
2 8. S o d i u m h y d r o g e n c a r b o n a t e , A . R.
aration, see p . 545. 9. A d e n o s i n e - 5 ' - d i p h o s p h a t e , A D P
4. G l u t a m a t e d e h y d r o g e n a s e , G I D H disodium salt A D P - N a or free acid; commercial
2
from bovine liver, N H + -free, in 5 0 % glycerol; preparations see p. 525.

^ 90 U / m g . ; c o m m e r c i a l p r e p a r a t i o n , see p . 4 6 1 .
Purity of Reagents
All reagents must be A. R. or purest grade available, a n d free from a m m o n i u m ions.
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y d i s t i l l e d w a t e r . T o a v o i d t h e g r o w t h o f m i c r o - o r g a n i s m s
sterilize t h e c o n t a i n e r s .
I. T r i e t h a n o l a m i n e buffer ( 0 . 2 M ; p H 8 . 6 ) :
D i s s o l v e 7 . 4 3 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in 1 5 0 m l . w a t e r , a d j u s t t o p H 8.6 w i t h
1 N N a O H a n d dilute to 200 ml. with distilled water.
II. 2 - O x o g l u t a r a t e s o l u t i o n ( 0 . 4 M ) :
D i s s o l v e 5 8 5 m g . 2 - o x o g l u t a r i c a c i d in 7.5 m l . 1 N s o d i u m h y d r o x i d e s o l u t i o n , c h e c k
p H ( p H m u s t b e n e u t r a l ) d i l u t e t o 10 m l . w i t h d i s t i l l e d w a t e r .
III. R e d u c e d n i c o t i n a m i d e - a d e n i n e - d i n u c l e o t i d e ( c a . 7 m M / f - N A D H ) :
D i s s o l v e 6 0 m g . N A D H a n d 100 m g . N a H C 0 3 in 10 m l . d i s t i l l e d w a t e r .
IV. A d e n o s i n e - 5 ' - d i p h o s p h a t e (20 m M ) :
Dissolve 216.3 mg. A D P - N a 2 o r 176 m g . A D P free a c i d in 2 0 m l . d i s t i l l e d w a t e r .
V. G l u t a m a t e dehydrogenase (10 mg. protein/ml.):
U s e s t o c k s o l u t i o n in 5 0 % g l y c e r o l u n d i l u t e d .
VI. U r e a s e (2.5 m g . p r o t e i n / m l . ) :
D i s s o l v e 100 m g . l y o p h i l i z a t e in 10 m l . 5 0 % g l y c e r o l .
VII. Reagent mix:
2 0 m l . Buffer (I)
2 ml. A D P (IV)
1 ml. 2-oxoglutarate (II)
1 ml. N A D H (III)
6 ml. Distilled water mix thoroughly.
Store all solutions, stoppered, in a refrigerator at 0 - 4 °C. Solutions ( I ) - ( I V ) are stable for 4 weeks, solution
(VI) for 6 m o n t h s , solution (V) for 1 year a n d the reagent mix for 1 week.
Procedure
O b t a i n p l a s m a o r s e r u m f r o m t h e freshest p o s s i b l e b l o o d . A d d i t i v e s u p t o 3 0 m g . o x a l a t e / m l . ,
3 0 m g . c i t r a t e / m l . , 3 0 m g . f l u o r i d e / m l . , 2 m g . h e p a r i n / m l . a n d 10 m g . E D T A / m l . Dilute
u r i n e 1 : 2 0 0 0 w i t h d i s t i l l e d w a t e r d o n o t interfere. D i l u t e 0.1 m l . s e r u m o r p l a s m a w i t h 2 . 4 m l .
water or use 20 /d. s a m p l e undiluted for the assay.
Stability of sample: F o r 3 d a y s at 0 - 4 ° C at m a x i m u m .
Assay System
W a v e l e n g t h : 3 4 0 ( 3 3 4 , 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 6 8 m l . ; t e m p e r a t u r e : 2 0 -
25 ° C ; r e a d a g a i n s t air. P r e p a r e a b l a n k c o n t a i n i n g buffer i n s t e a d o f s a m p l e for e a c h series
of measurements.
Reagent mix (VII) 2.50 ml. 0.11 M buffer

10.9 m M 2 - O x o G
1.09 m M ADP
0.19 m M NADH
Diluted sample 0.50 ml. u p t o 8 0 pM u r e a
0.02 ml. c a . 6 6 pg. G l D H / m l . = 6 U / m l .
G I D H solution (V)
M i x a n d read e x t i n c t i o n E j after 10 m i n .
Urease solution (VI) 0.02 ml. c a . 16 pg. u r e a s e / m l . = 1.6 U / m l .
M i x a n d r e a d e x t i n c t i o n E after 15 m i n . ( E — E )
2 t 2 s a m p l e
— (Ej — E ) 2 b l a n k = A E is u s e d for c a l c u l a t i o n .
Urea 1797
Calculations
U n d e r the above conditions the reaction proceeds stoichiometrically a n d therefore the calculation formula
(2) on p . 312 applies. T h e results are obtained as //mole urea per ml. sample. This value m u s t be multiplied
by a factor if the sample has been diluted. F o r this m e t h o d with a dilution of 1:25 for serum a n d 1 : 2 0 0 0
for urine the following relationships h o l d :

Serum c = AE x 12.46 AE x 12.22 AE x 22.35 [/imole/ml.]
Serum c - AE x 74.83 AE x 73.39 AE x 134.3 [mg./lOO ml.]
Urine c = AE x 0.997 AE x 0.977 AE x 1.79 [mmole/ml.]
Urine c = AE x 5.99 AE x 5.87 AE x 10.74 [g./lOO ml.]
W i t h a m e a n value of 24.8 mg. urea/100 ml. serum a s t a n d a r d deviation of 0.75 mg. urea was found.
T h e coefficient of variation is 2.7.
N o r m a l Values
See " D e t e r m i n a t i o n of U r e a , Indicator Reaction with Phenol a n d H y p o c h l o r i t e " , p . 1791.
Effects of drags and other therapeutic measures: None known.
Interference in the assay technique: T h e a m m o n i a content of the reagents is eliminated by the blank.
Nevertheless the bottles containing the reagents should always be kept stoppered to keep the a m m o n i a
c o n t a m i n a t i o n low.
Urease reacts specifically with u r e a . G l u t a m a t e d e h y d r o g e n a s e reacts specifically with a m m o n i a , o t h e r

3 5
amines such as methylamine, ethylamine a n d diethylamine d o n o t r e a c t . 41
References
1 H. K. Barrenscheen & D. Weltmann, Biochem. Z. 131, 591 [1922].

2 W. R. Fearon, Biochem. J. 33, 902 [1939].
3 P. C. Wenger, C. Limeran & A. Maulbetsch, M i k r o c h e m i e 8, 132 [1933/34].
4 / . F. Barrett & E. B. Jones, Biochem. J. 26, 1246 [1932].
5 M. H. Lee & E. M. Widdowson, Biochem. J. 31, 2035 [1937].
6 L. Cuny & J. Robert, Bull. Soc. C h i m . biol. 12, 171 [1930]; 13, 1167 [1931].
7 F. W. Allen & / . M . Luck, J. biol. C h e m . 82, 693 [1929].
8 Z . F. Ch. Kachani, Arztl. L a b o r a t o r i u m 9, 81 & 408 [1963].
9 G. Ceriotti & L. Spandrio, Clin. chim. A c t a 8, 295 [1963].
10 T. Momose, Y. Ohkura & /. Tomita, Clin. Chemistry 11, 113 [1965].
11 H. Dudek, Clin. chim. A c t a 14, 108 [1966].
12 J. Michon & R. Arnaud, Clin. chim. A c t a 7, 739 [1962].
13 A. F. M. Roijers & M. M. Tas, Clin. chim. A c t a 9, 197 [1964].
14 / . B. Sumner, J. biol. C h e m . 27, 95 [1916].
15 / . Schormuller & H. Tdnzler, Z. Lebensmittel-Unters. u. Forsch. 109, 234 [1959].
16 J. Y. Yee & R. O. E. Danis, J. Assoc. off. agric. Chemists 20, 104 [1937].
17 C. Engelhard, Z. ges. Brauereiwesen 54, 160 [1931].
18 D. D. van Slyke, J. biol. C h e m . 73, 695 [1927].
19 L. D. Scott, Brit. J. exp. P a t h o l . 21, 93 [1940].
20 B. Reichert & H. Schwebs, P h a r m a z i e 2, 348 [1947].
21 V. E. Kinsey & P. Robison, J. biol. C h e m . 162, 325 [1946].
22 O. Folin & A. Sredberg, J. biol. C h e m . 88, 77 [1930].
23 R. Richterich, J. P. Colombo & H. Weber, Arztl. L a b o r a t o r i u m 8, 259 [1962].
24 / . Ellinghaus, Hoppe-Seylers Z. physiol. C h e m i e 150, 211 [1925].
25 W. Ohlson, A c t a physiol. scand. / , 278 [1940].
26 A. M. Roman, J. U r o l o g . 4, 531 [1921].
27 M. P. E. Berthelot, Repert. C h i m . a p p . 1, / , 284 [1959].
28 B. Lubochinsky & J. P. Zalta, Bull. Soc. C h i m . biol. 36, 1363 [1954].
29 H. Weller, D a s M e d . L a b o r a t o r i u m 15, 142 [1962].
30 R. L. Searcy, G. S. Gough, J. L. Korotzer & L. M. Berquist, A n n . J. med. Techn. 27, 255 [1961].
31 / . K. Fawcett & /. E. Scott, J. clin. P a t h , 13, 156 [I960].
32 H. Talke & G. E. Schubert, Klin. Wschr. 43, 174 [1965].
33 H. Kaltwasser & H. G. Schlegel, Analyt. Biochem. 16, 132 [1966].
34 K. Lorenz, Z. f. Klin. Chemie 5, 291 [1967].
35 / . E. Varner in P . D. Boyer, H. Lardy & K. Myrbdck: T h e Enzymes Vol. 4, p . 247, A c a d e m i c Press,
N e w Y o r k - L o n d o n , 2nd. E d n . 1960.
36 Hoppe-Seyler-Thierfelder: H a n d b u c h der Physiologisch- u n d Pathologisch-chemischen Analyse,
10th. Edn., Vol. 6, P a r t C, p . 362, Springer-Verlag, Berlin, B 1966.
37 Ch. F. M. Rose, Brit. J. exp. P a t h o l . 14, 339 [1933].
38 E. M. Mackay & L. L. Mackay, J. clin. Invest. 4, 295 [1927].
39 H. Sarre: N i e r e n k r a n k h e i t e n , G e o r g T h i e m e Verlag, Stuttgart 1959.
40 H. J. Strecker in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, Vol. 2, p . 220, A c a d e m i c
Press, N e w Y o r k 1955.
42 C. Frieden in P. D. Boyer, H. Lardy & K. Myrbdck, T h e Enzymes, Vol. 7, p . 3, Academic Press,
New Y o r k - L o n d o n , 2nd. edn. 1963.
43 C. Frieden, J. biol. C h e m . 234, 815 [1959].
Determination with Automatic Analysers

G a b o r Szasz
T h e a u t o m a t e d d e t e r m i n a t i o n of urea c o r r e s p o n d s in principle to the c o n v e n t i o n a l m a n u a l m e t h o d (see

p . 1791 a n d is based on the m e t h o d developed by Wilcox 1
for the A u t o Analyzer ®.
Equipment
S t a n d a r d A u t o A n a l y z e r ® * : s a m p l e r I I ; p r o p o r t i o n i n g p u m p I, w i t h 15 s e c . / r o t a t i o n ; d i a l y s e r
w i t h d i a l y s i s m e m b r a n e , t y p e C ; t h e r m o s t a t , a d j u s t a b l e , w i t h d o u b l e h e a t i n g c o i l s ( e a c h 12 m .
l o n g , 1.6 m m . i n t e r n a l d i a m e t e r ) ; p h o t o m e t e r w i t h 15 m m . flow-through cuvette and selenium
p h o t o c e l l , filter 5 5 0 n m ; r e c o r d e r w i t h c h a r t s p e e d o f 0 . 4 c m . / m i n .
Reagents**
1. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA 2. D i s o d i u m t a r t r a t e , A . R .
disodium salt, E D T A N a H - 2 H 0 , A . R .
2 2 2 3. S o d i u m c a r b o n a t e , A . R . , a n h y d r o u s
* Technicon G m b H , F r a n k f u r t / M . , G e r m a n y
•* Complete reagent kits are available commercially, see p . 558.
Urea 1799
4. S o d i u m a z i d e , p u r e , N a N 3 8. S o d i u m h y p o c h l o r i t e
5. Urease chlorine bleaching agent containing ca. 12%
suspension in 50% glycerol; ^ 3 U / m g . ; c o m active chlorine
mercial p r e p a r a t i o n , see p . 517. 9. S o d i u m h y d r o x i d e , A . R .
6. P h e n o l , A . R . 10. S o d i u m c h l o r i d e , A . R .
7. S o d i u m n i t r o p r u s s i d e , A . R . 11. U r e a , A . R .
[Fe ( C N ) N O ] N a - 2 H 0
5 2 2 12. Tween-20
Purity of Reagents
Urease should not contain any arginase a n d must be free from a m m o n i u m ions.
P r e p a r a t i o n o f S o l u t i o n s (for 3 0 0 - 5 0 0 a s s a y s )
I. E t h y l e n e d i a m i n e t e t r a - a c e t a t e / t a r t r a t e ( 1 3 m M E D T A ; 11 m M t a r t r a t e ) :
D i s s o l v e 17.5 g. E D T A - N a H - 2 H 0 + 8.75 g. N a - D - t a r t r a t e - 2 H 0 + 2 . 1 g. N a C 0
2 2 2 2 2 2 3 +
+ 3.5 g. N a N 3 in 3 5 0 0 m l . d i s t i l l e d w a t e r . T h e p H s h o u l d b e b e t w e e n 6 . 4 a n d 6.6. A d d
1.75 m l . T w e e n - 2 0 a n d m i x t h o r o u g h l y .
II. U r e a s e ( 3 0 /zg. p r o t e i n / m l . ) :
D i s s o l v e 10.5 m g . p r o t e i n in 3 5 0 m l . s o l u t i o n I ; if n e c e s s a r y r e m o v e a n y t u r b i d i t y b y
filtration. P r e p a r e t h e s o l u t i o n freshly e a c h d a y .
III. S o d i u m c h l o r i d e ( 1 7 m M ) :
D i s s o l v e 3.5 g. N a C l in 3 5 0 0 m l . d i s t i l l e d w a t e r , a d d 1.75 m l . T w e e n - 2 0 a n d m i x t h o r o u g h l y .
IV. Phenol/nitroprusside (88 m M p h e n o l ; 0.14 m M N a nitroprusside):
D i s s o l v e 15 g. p h e n o l + 75 m g . [ F e ( C N ) N O ] N a - 2 H 0 in 1 8 0 0 m l . d i s t i l l e d w a t e r .
5 2 2
V . H y p o c h l o r i t e (9.1 m M h y p o c h l o r i t e ; 1.05 m M NaOH):

D i s s o l v e 7.5 m l . c h l o r i n e b l e a c h i n g a g e n t -f- 7.5 g. N a O H in 1 8 0 0 m l . d i s t i l l e d w a t e r .
VI. Urea stock solution ( 1 % w / v ) :
D i s s o l v e 1.0 g. u r e a + 0.1 g. N a N 3 in 100 ml. distilled water.
VII. Urea working solutions ( 1 2 . 5 - 2 0 0 mg. % ) :
D i l u t e 1.25, 2 . 5 , 5.0, 1 0 . 0 , 1 5 . 0 , 2 0 . 0 m l . s o l u t i o n V I t o 1 0 0 m l . w i t h 0 . 1 % N a N 3 solution.
T h e s e s o l u t i o n s c o r r e s p o n d t o u r e a c o n c e n t r a t i o n s o f 1 2 . 5 , 2 5 , 5 0 , 1 0 0 , 1 5 0 a n d 2 0 0 m g . %.
Store all solutions, stoppered, at 0 - 4 °C. Store solutions IV a n d V in b r o w n bottles. P r e p a r e solution II

freshly each day. Solutions I, III, IV a n d V are stable for several m o n t h s , solutions VI a n d VII are stable
for a year.
Procedure
A s for t h e m a n u a l m e t h o d ( s e e p . 1 7 9 3 ) .
Assay System
Set u p t h e t u b i n g s y s t e m a c c o r d i n g t o t h e flow s c h e m e s h o w n in F i g . 1. R i n s e t h e w h o l e
t u b i n g s y s t e m for 15 m i n . , first w i t h d o u b l y d i s t i l l e d w a t e r a n d t h e n w i t h t h e c o r r e s p o n d
i n g r e a g e n t s . T a k e u p 4 0 s a m p l e s p e r h r . ; p r o b e 3 0 sec. in t h e s a m p l e a n d 6 0 sec. in t h e w a s h
fluid. A t t h e e n d w a s h t h e t u b i n g f o r 15 m i n . w i t h d o u b l y d i s t i l l e d w a t e r .
Waste
Waste
Proportioning
pump Tubing
lower upper ml/min
- - • l e v e l 'Sample 0.15
B u f f e r / E n z y m e (soln.II) 3M
Air 2.0
NaCKsoln.m) 3.U
Air 2.0
Hypochlorite Isoln.V) 2.0
Phenol (soln. IV) 2.0
H02
1.2
Return 2.9
Pulse suppressor
a = 2 inch l o n g ,
0.010inch int.diam.
Colorimeter Recorder b = 1 . 5 inch l o n g ,
550 n m 0.005inch int.diam.
Fig. 1. F l o w scheme for the a u t o m a t e d d e t e r m i n a t i o n of u r e a .
Calculations
The extinctions (transmissions) of the samples are converted t o m g . % u r e a by c o m p a r i n g the samples to

the s t a n d a r d s by m e a n s of t h e c h a r t reader*.
Accuracy of Method
T h e urea concentrations of 496 sera have been determined with b o t h the a u t o m a t e d and m a n u a l m e t h o d s
(see p . 1791). The urea c o n c e n t r a t i o n of these sera varied between 12 a n d 600 mg.%. Sera with a urea
concentration over 200 m g . % were diluted 1 : 10. W i t h a m e a n value of 63.0 m g . % urea ( a u t o m a t e d
m e t h o d ) a coefficient of correlation of 0.981 was calculated.
Precision of M e t h o d
Ten assays were carried out on each of 2 sera. T h e s t a n d a r d deviation with a m e a n of 45.0 a n d 99.1 m g . %
respectively was 0.67 a n d 0.74 m g . % respectively, a n d the c o r r e s p o n d i n g coefficients of variation were
1.48 a n d 0.74%.
* Available from Technicon.

Urea 1801
W i t h a s t a n d a r d solution of 200 m g . % urea the extinction should be between 0.5 a n d 0.7. Deviation from
this range can be corrected by change of the wavelength, the sampler tubing a n d the incubation t e m p e r a t u r e
( 4 0 - 5 0 °C). Alteration of the ratio of sample/wash (1 : 2) in favour of the sample increases the carry over
from sample to sample. Increasing the rate of the assays has an unfavourable effect on the reproducibility.
0.6 200
0.5
150
100
0.3
50
Q
25
0.1
12.5
Fig. 2. S t a n d a r d curve in the range 1 2 . 5 - 2 0 0 m g . % u r e a .
References
1 A. A. Wilcox, W. E. Caroll, R. E. Sterling, H. A. Davis & A. G. Ware, Clin. C h e m . 72, 151 [1966].
Ammonia
Ernest K u n * and E d n a B. K e a r n e y
The enzymatic determination of a m m o n i a ~ is suitable for the analysis of tissue extracts or enzymatic
1 4
incubation mixtures; it can also be coupled with all enzymatic systems in which a m m o n i a is formed
(e. g. A M P - d e a m i n a s e ) .
5
Principle
(1) NADH + NH 4
+
+ 2-Oxoglutarate-°^H> L-Glutamate. + N A D +
+ H 0
2
T h e reductive a m i n a t i o n of 2-oxoglutarate is catalysed by the enzyme g l u t a m a t e dehydrogenase, G I D H

( L - G l u t a m a t e : N A D ( P ) oxidoreductase, d e a m i n a t i n g , E C 1.4.1.3). T h e equilibrium of the reaction lies
far to the right. T h e decrease of N A D H , as measured by the change of extinction at 340, 334 or 365 n m ,
is a measure of the reaction. At p H 8.0 the reaction is stoichiometric.
The o p t i m u m p H is 8.0 with excess N A D H , 2-oxoglutarate a n d g l u t a m a t e dehydrogenase. This p H

should be maintained exactly at t e m p e r a t u r e s between 20 a n d 25 °C.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r for a c c u r a t e m e a s u r e m e n t s at 3 4 0 , 3 3 4 o r
365 n m ; refrigerated c e n t r i f u g e ( 0 - 4 ° C ) .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 5. P o t a s s i u m hydrogen carbonate, A. R.,

2. 2-Oxoglutarate l%(w/v)
free acid, crystalline, commercial p r e p a r a t i o n , 6. A m m o n i u m c h l o r i d e , A . R .
see p. 548. 7. G l u t a m a t e d e h y d r o g e n a s e , GIDH
3. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo from liver, crystalline, solution in 5 0 % (v/v)
tide, N A D H glycerol, N H ^ - f r e e : ^ 9 0 U / m g . ; commercial
disodium salt, N A D H - N a 2 ; commercial p r e p p r e p a r a t i o n see p . 4 6 1 .
aration, see p . 545. 8. H y d r o c h l o r i c a c i d , A . R., 3 N
4. T r i c h l o r o a c e t i c a c i d , A . R. 9. S o d i u m h y d r o x i d e , A . R . , 1 N
* Research Career A w a r d of the U . S . Public Health Service. This w o r k was s u p p o r t e d by grants from
U S P H S ( U S P H S R O 1 H D 01239-11, U S P H S R O 1 C A 07955-3) from Life I n s u r a n c e Medical Research
F u n d (G-67-30) and from the American Cancer Society (E-493).
Ammonia 1803
Purity of Reagents
Use only A. R. c o m p o u n d s . Special care should be t a k e n t h a t n o c o n t a m i n a t i o n of the reagents occurs

from a m m o n i a in the air. Enzymes crystallized from a m m o n i u m sulphate m u s t be completely free from
ammonia.
T o a v o i d t r a c e s o f a m m o n i a u s e fresh d i s t i l l e d w a t e r a n d k e e p t h e c o n t a i n e r s w e l l - s t o p p e r e d .
I. Tris buffer ( 0 . 5 M ; p H 8 . 0 ) :
D i s s o l v e 3 0 . 2 5 g. tris in c a . 4 0 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 8.0 at r o o m t e m p e r a t u r e
with 3 N HC1 a n d dilute to 500 ml. with distilled water.
II. 2 - O x o g l u t a r a t e (0.1 M ; p H 7 . 4 ) :
A d d 4 0 ml. distilled water to 730.5 m g . 2-oxoglutaric acid a n d dissolve the acid by
stirring w h i l e a d j u s t i n g t h e p H t o 7 . 4 w i t h 1 N N a O H . D i l u t e t o 5 0 m l . w i t h d i s t i l l e d
water.
III. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( c a . 8 m M /f-NADH):
Dissolve 70 mg. N A D H - N a 2 in 10 m l . 1 % K H C 0 3 solution.
IV. Trichloroacetic acid ( 1 0 % w / v ) :
D i s s o l v e 10 g. t r i c h l o r o a c e t i c a c i d in 1 0 0 m l . d i s t i l l e d w a t e r .
V . P o t a s s i u m h y d r o g e n c a r b o n a t e (2 M ) :
D i s s o l v e 2 0 g. K H C 0 3 in 1 0 0 m l . d i s t i l l e d w a t e r .
V I . A m m o n i u m c h l o r i d e (0.1 M ) :
D i s s o l v e 5 3 5 m g . N H C 1 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
4
V I I . G l u t a m a t e d e h y d r o g e n a s e , G I D H ( c a . 10 m g . p r o t e i n / m l . ) :
U s e c o m m e r c i a l p r e p a r a t i o n s o f this c o n c e n t r a t i o n .
Stability of Solution
Store tris buffer (solution I), trichloroacetic acid solution (IV) a n d the enzyme solution (VII) at 0 - 6 °C.
Store the N A D H solution (II) a n d the oxoglutarate solution (III) frozen in small p o r t i o n s at - 1 5 °C,
t h a w out the daily requirement. Store solutions (II), (III), (IV) a n d (VII) in an ice b a t h d u r i n g the day.
Solution (V) can be stored at r o o m t e m p e r a t u r e , b u t it should be m a d e u p freshly when the a m o u n t
required to neutralize the trichloroacetic acid is t o o large.
Procedure
Collection, Treatment and Stability of Samples

C a r r y o u t all o p e r a t i o n s at 0 - 4 ° C t o r e d u c e t h e l i b e r a t i o n o f a m m o n i a f r o m a c i d - l a b i l e
c o m p o u n d s (e. g. g l u t a m i n e ) .
C o o l b i o l o g i c a l fluids a n d s a m p l e s in a n ice b a t h a n d d e p r o t e i n i z e w i t h a n e q u a l v o l u m e o f
t r i c h l o r o a c e t i c a c i d s o l u t i o n ( I V ) . In t h e c a s e o f i n c u b a t i o n m i x t u r e s o f e n z y m a t i c r e a c t i o n s ,
treat a m i x t u r e o f t h e r e a g e n t s u s e d a s a b l a n k . A f t e r 5 t o 10 m i n . c e n t r i f u g e off t h e p r e c i p i t a t e d
p r o t e i n a n d n e u t r a l i z e t h e s u p e r n a t a n t fluid w i t h K H C 0 3 solution (V). Preferably determine
the r e q u i r e d a m o u n t o f K H C 0 3 in a s e p a r a t e e x p e r i m e n t : d i l u t e t h e t r i c h l o r o a c e t i c a c i d
s o l u t i o n ( I V ) t o 5 % a n d titrate t o p H 7 t o 7.5 ( p H m e t e r ) u n t i l C 0 2 production ceases. K H C 0 3
is u s e d i n s t e a d o f K C 0 2 3 o r K O H t o a v o i d t h e p o s s i b i l i t y o f t h e s a m p l e b e c o m i n g t o o al
kaline.
C o l l e c t t i s s u e s a m p l e s w i t h f r e e z e - c l a m p s (see p . 4 0 0 ) . w e i g h , p o w d e r a n d h o m o g e n i z e in
t r i c h l o r o a c e t i c a c i d s o l u t i o n ( I V ) at 0 ° C (ice b a t h ) . T i s s u e s a m p l e s c a n a l s o b e t r e a t e d at
0 ° C directly w i t h t r i c h l o r o a c e t i c a c i d ( s o l u t i o n I V ) in a m i c r o h o m o g e n i z e r (e. g. S o r v a l l
O m n i m i x e r ) . T h e r a t i o o f t i s s u e w e i g h t t o t r i c h l o r o a c e t i c a c i d s o l u t i o n s h o u l d b e 1 : 10.
T h e p r o t e i n c a n b e d e t e r m i n e d in t h e s e d i m e n t b y t h e B i u r e t m e t h o d ; t h e p r e c i p i t a t e d p r o t e i n
6
is d i s s o l v e d in 1 0 % K O H .
A c i d s a m p l e s are s t a b l e indefinitely. H o w e v e r , a c i d a m i d e s in a c i d s o l u t i o n l i b e r a t e a m m o n i a
at r o o m t e m p e r a t u r e w i t h i n / t o 1 hr. C o n s e q u e n t l y , it is n e c e s s a r y t o c a r r y o u t t h e d e p r o t e i n
l
2
i z a t i o n a n d n e u t r a l i z a t i o n r a p i d l y in t h e c o l d . S a m p l e s c a n b e s t o r e d at —15 ° C for l o n g
periods.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 o r H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 1.0 m l . ; r o o m

t e m p e r a t u r e ; read a g a i n s t w a t e r . P r e p a r e a b l a n k for e a c h series o f e s t i m a t i o n s w i t h n e u t r a l i z e d
trichloroacetic acid.
assay mixture
S a m p l e (neutral e x t r a c t ) 0.50 ml. 10 t o 8 0 pM NH4

Tris buffer s o l u t i o n (I) 0.20 ml. 100 m M
2-Oxoglutarate solution (II) 0.10 ml. 10 m M
M i x and read extinction E j .
G I D H solution (VII) 0.02 ml. 2 0 0 pg./ml = 9 U/ml.
M i x . After ca. 9 0 m i n . r e a d e x t i n c t i o n E 2 a n d after a

further 5 m i n . read e x t i n c t i o n E 2 again to check
whether the reaction has stopped.
E x — E 2 = A E is u s e d for t h e c a l c u l a t i o n s .
E x t i n c t i o n E at 3 4 0 n m is u s u a l l y b e t w e e n 1.5 a n d 1.7. If t h e a c c u r a c y o f t h e s p e c t r o p h o t o m e t e r
x
is n o t sufficient in this r a n g e r e a d at 365 n m .

It is p r e f e r a b l e t o o c c a s i o n a l l y i n c l u d e N H 4 standards to check that the sample contains
n o c o m p o u n d s w h i c h interfere w i t h t h e s p e c t r o p h o t o m e t r i c a s s a y . In t h i s c a s e a d d N H C 1 4
s o l u t i o n ( V I ) t o t h e s a m p l e . G e n e r a l l y a b o u t 9 8 % o f t h e a m m o n i a is r e c o v e r e d .
Ammonia 1805
Calculations
(2) on p . 312 applies. T h e results are obtained in /zmole N H 4 /ml. sample. This value m u s t be multiplied
by a factor if the sample has been diluted by deproteinization, neutralization or in any other way. U n d e r
these conditions the N H 4 c o n c e n t r a t i o n of the deproteinized and neutralized sample is:

1 1
AE x- AE x- [^mole/ml.]
6.22 x v 3.4x v
v = volume of sample t a k e n for assay.
Parallel determinations (at least 5) on the same sample deviate from the m e a n by 5 % (mainly d u e to
pipetting errors). T h e values obtained in the enzymatic assay over the range 10-80 N H 4 (0.2 — 1.4 ug.
N H / m l . assay mixture) agree within 1 - 2 % of the theoretical values.
3
A m m o n i a in the reagents can interfere. If the reagent blank at 340 n m gives a > 0.100 p r e p a r e new solutions
with fresh, doubly distilled water. All c o m p o u n d s which react with N A D at p H 8 to form an a d d u c t
interfere in the assay, because the resulting a b s o r p t i o n a r o u n d 340 n m simulates N A D H (examples
are acetone, d i h y d r o x y a c e t o n e ' ) . Monofluoro-oxaloacetate, an inhibitor of m a l a t e d e h y d r o g e n a s e
7 8 9 - 1 1
,
reacts at p H > 8 with N A D to form an adduct which has the same s p e c t r o p h o t o m e t r i c a n d fluorimetric
properties as N A D H . 1 2
M o s t of these c o m p o u n d s can be destroyed by heating for 1 0 - 2 0 min. at 100 °C a n d p H 1, but that is

not possible here (see above). As the rate of the G I D H reaction at p H values nearer 7.5 is m o r e rapid
t h a n that of the a d d u c t formation, this type of interference can be o v e r c o m e by the addition of several-
fold m o r e G I D H t h a n stated.
G l u t a m a t e dehydrogenase reacts specifically with a m m o n i a .
Other Methods
A m m o n i a can also be determined fluorimetrically by the same p r i n c i p l e .

13
References
1 G. Fawaz & K. v. Dahl, Lebanese M e d . J. 16, 169 [1963].

C h e m . Abstr. 61, 2177 [1964].
2 E. Kirsten, C. Gerez & R. Kirsten, Biochem. Z. 337, 312 [1963].
3 A. Mondzac, G. E. Ehrlich & J. E. Seegmiller, J. L a b o r a t . Clin. Med. 66, 526 [1965].
4 E. Manoukian & G. Fawaz, Z. klin. C h e m . u n d klin. Biochem. 7, 32 [1969].

5 E. Kun, H. H. Loh & S. B. I. El-Fiky, M o l . P h a r m a c o l . 2, 481 [1966].
6 G. Beisenherz, H. J. Baltze, Th. Bucher, R. Czok, K. H. Garbade, E. Meyer-Arendt & G. Pfleiderer,
Z. Naturforsch. 8b, 555 [1953].
7 N. O. Kaplan, M. M. Ciotti & F. E. Stolzenbach, J. biol. C h e m . 221, 833 [1956].
8 R. M. Burton, A. San Pietro & N. O. Kaplan, Arch. Biochem. Biophys. 70, 87 [1957].
9 E. Kun, D. R. Grassetti, D. W. Fanshier & R. M. Featherstone, Biochem. P h a r m a c o l . / , 207 [1958].
10 E. Kun, L. K. Gottwald, D. W. Fanshier & J. E. Ayling, J. biol. C h e m . 238, 1456 [1963].
11 D. Dupourque & E. Kun, Eur. J. Biochem. 7, 247 [1969].
12 E. Kun & H. G. Williams-Ashman, N a t u r e 194, 316 [1962].
13 M. Rubin & L. Knott, Clin. C h e m . 13, 341 [1967].
Polyunsaturated Fatty Acids
H. Whitney Wharton
T h e m e t h o d described h e r e determines p o l y u n s a t u r a t e d fatty acids which contain at least o n e pair of cis-

1
d o u b l e b o n d s separated by a methylene g r o u p . T h e methylene g r o u p m u s t be located at the a> 8 C - a t o m . 2
T h e enzyme lipoxygenase ( L i n o l e a t e : oxygen oxidoreductase, E C 1.13.11.12) catalyses the oxidation of these

pairs of double b o n d s by a t m o s p h e r i c oxygen. T h e reaction p r o d u c t s are conjugated diene h y d r o p e r o x i d e s .
T h e m e c h a n i s m of oxidation has been elucidated by Hamburg a n d Samuelsson . 2
Application of Method: In foodstuff chemistry, the oil a n d fat industry, biochemistry a n d clinical chemistry.
Typical experimental materials are free fatty acids, fatty acid esters, oils, fats, h y d r o g e n a t e d plant oils, b l o o d
p l a s m a a n d serum, seeds a n d micro-organisms. T h e m e t h o d allows the d e t e r m i n a t i o n of as little as 5 fig.
(0.018 /rniole) linoleic acid.
Principle
lipoxygenase
(1) x cis, m - 9 , 1 2 - O c t a d e c a d i e n o a t e + x 0 2 •
13-Hydroperoxy-cw,/ra«s-9,11 - o c t a d e c a d i e n o a t e (92 % ) 2
+ 9-Hydroperoxy-/raAw,cw-10,12-octadecadienoate ( 8 % ) 2
T h e a b s o r p t i o n of the conjugated diene h y d r o p e r o x i d e at 234 n m . is a m e a s u r e of the total a m o u n t of

p o l y u n s a t u r a t e d fatty acids containing a s - d o u b l e b o n d s separated by methylene g r o u p s . T h e a b s o r p t i o n
obeys Beer's law between 5 a n d 25 pg. substrate.
T h e m e t h o d described here is o p t i m u m if all the necessary p r e c a u t i o n s are observed.
Equipment
A l u m i n i u m o r g l a s s d e s i c c a t o r , c o v e r e d w i t h a l u m i n i u m foil o r p a i n t e d b l a c k . S p e c t r o p h o t o
m e t e r . S e m i - m i c r o b a l a n c e . M i c r o - b e a k e r s : 4 0 - 4 5 m m . h i g h , i n t e r n a l d i a m e t e r 13 m m . , w i t h
a l i p . 7.5 m l . S t o p p e r e d flasks.
Reagents
1. B o r i c a c i d , H B 0 , A . R .
3 3 5. Lipoxygenase
2. P o t a s s i u m h y d r o x i d e , A . R . lyophilizate, ^ 500 U / m g . (25 ° C ) ; commercial
3. H y d r o c h l o r i c a c i d , A . R . , c o n e p r e p a r a t i o n , see p . 483.
4. E t h a n o l , b e n z e n e - f r e e
T h e activity of each enzyme p r e p a r a t i o n should be checked before use. Lipoxygenase (20 pg.) should oxidize
the free p o l y u n s a t u r a t e d fatty acids from 30 pg. c o t t o n seeds or maize oil. A s s a y : see u n d e r "Oils, fats a n d
fatty acid ester", p . 1808 a n d " A s s a y S y s t e m " , p . 1810. Plot the m e a s u r e d E values against time.
1808 M e t a b o l i t e s : F a t t y Acid Metabolism, etc.
I. P o t a s s i u m h y d r o x i d e ( 1 0 N ) :
D i s s o l v e 6 6 g. K O H p e l l e t s ( 8 5 % K O H ) o r 56 g. a n h y d r o u s K O H in a b o u t 8 0 m l .
distilled w a t e r , c o o l a n d d i l u t e t o 100 m l . w i t h distilled w a t e r .
II. A l c o h o l i c p o t a s s i u m h y d r o x i d e (0.5 N ) :
D i l u t e 5 m l . 10 N K O H ( s o l u t i o n I) t o 100 m l . w i t h e t h a n o l . P r e p a r e freshly e a c h d a y .
III. A l c o h o l i c p o t a s s i u m h y d r o x i d e ( 1 . 5 N ) :
D i l u t e 15 m l . 10 N K O H ( s o l u t i o n I) t o 100 m l . w i t h e t h a n o l . P r e p a r e freshly e a c h d a y .
I V . H y d r o c h l o r i c a c i d (0.5 N ) :
D i l u t e 5 0 m l . c o n e . H C 1 t o 1 0 0 0 m l . w i t h distilled w a t e r .
V . P o t a s s i u m b o r a t e buffer (1 M ; p H 9 . 0 ) :
D i s s o l v e 6 3 g. b o r i c a c i d a n d 2 5 . 0 g. K O H pellqts ( 8 5 % K O H ) in a b o u t 8 0 0 m l . distilled
w a t e r , w i t h stirring a n d w a r m i n g o n a s t e a m b a t h . C o o l t h e c l e a r s o l u t i o n t o r o o m
t e m p e r a t u r e , adjust t o p H 9.0 w i t h 1 N H C 1 o r 1 N K O H a n d d i l u t e w i t h distilled w a t e r
to 1000 ml.
VJ. P o t a s s i u m b o r a t e buffer ( 0 . 2 M ; p H 9 . 0 ) :
D i l u t e 2 0 0 m l . s o l u t i o n V t o 1 0 0 0 m l . w i t h distilled w a t e r .
V I I . L i p o x y g e n a s e s t o c k s o l u t i o n (1 m g . p r o t e i n / m l . ) :
D i s s o l v e 10 m g . l i p o x y g e n a s e in 10 m l . i c e - c o l d buffer ( s o l u t i o n V I ) .
VIII. Lipoxygenase, w o r k i n g solution (0.2 m g . protein/ml.):
M i x 2.0 m l . s o l u t i o n V I I w i t h 8.0 m l . i c e - c o l d buffer ( s o l u t i o n V I ) .
IX. Lipoxygenase, inactivated w o r k i n g solution (0.2 mg. protein/ml.):
P i p e t t e 5 m l . s o l u t i o n V I I I i n t o a test t u b e in s u c h a w a y t h a t t h e w a l l s a b o v e the surface
o f the l i q u i d r e m a i n dry. P l a c e t h e test t u b e in a b o i l i n g w a t e r b a t h for 5 m i n . , s o that
u p t o a b o u t 1 c m . a b o v e t h e s u r f a c e o f t h e s o l u t i o n is h e a t e d . U n d e r t h e s e c o n d i t i o n s
t h e e n z y m e is c o m p l e t e l y i n a c t i v a t e d .
T h e enzyme solutions can be stored frozen for several m o n t h s without loss of activity. They m a y be thawed
by placing in water at r o o m t e m p e r a t u r e , but after thawing completely, they should be mixed well and placed
immediately in an ice-water b a t h . Repeated freezing a n d thawing does not affect the enzyme activity. T h e
alcoholic K O H solutions should be prepared freshly each day.
Procedure
F a t t y a c i d s o r g l y c e r i d e s s h o u l d b e c o n v e r t e d at r o o m t e m p e r a t u r e t o their p o t a s s i u m s o a p s .
Preliminary treatment of samples

O i l s , fats a n d fatty a c i d s : b e t w e e n 5 a n d 25 pg. s h o u l d b e u s e d for t h e a s s a y . In g e n e r a l :
weight of sample (mg.) = — — - - — —

% free p o l y u n s a t u r a t e d fatty a c i d s
P o l y u n s a t u r a t e d F a t t y Acids 1809
Transfer t h e s a m p l e w i t h a g l a s s r o d t o t h e b o t t o m o f a m i c r o b e a k e r w i t h o u t t o u c h i n g t h e sides.
D i s c a r d t h e s a m p l e if it d o e s t o u c h . W e i g h a n d a d d 1 m l . a l c o h o l i c K O H ( s o l u t i o n II). A l l o w
t h e s a m p l e t o m e l t in a h o t w a t e r b a t h a n d m i x t h o r o u g h l y w i t h t h e K O H . A l l o w t o s t a n d
for 4 hr. i n t h e d a r k at r o o m t e m p e r a t u r e t o s a p o n i f y . A d d 1 m l . 0.5 N H C 1 ( s o l u t i o n I V ) .
Transfer t h e c o n t e n t s o f t h e b e a k e r t o a 100 m l . v o l u m e t r i c flask, rinse t h e b e a k e r w i t h 2 0 m l .
1 M b o r a t e buffer ( s o l u t i o n V ) , d i l u t e t o m a r k w i t h distilled w a t e r a n d m i x . O c c a s i o n a l l y h a r d
s o a p s s o l i d i f y a n d t h e s o l u t i o n s b e c o m e t u r b i d . G e n t l y w a r m i n g results in r e - s o l u b i l i z a t i o n .
F a t t y a c i d s : C o l l e c t s a m p l e a s d e s c r i b e d a b o v e . Transfer t o a 100 m l . m e a s u r i n g c y l i n d e r w i t h
2 0 m l . 1 M b o r a t e buffer ( s o l u t i o n V ) a n d treat a s d e s c r i b e d a b o v e .
B l o o d p l a s m a : C o l l e c t s a m p l e a n d s a p o n i f y as d e s c r i b e d for o i l s a n d fats.
Transfer t h e s a p o n i f i e d s a m p l e t o a 25 m l . v o l u m e t r i c flask w i t h 5 m l . 1 M b o r a t e buffer a n d
d i l u t e t o m a r k w i t h d i s t i l l e d w a t e r . B e c a u s e o f t h e h i g h a b s o r p t i o n o f t h e c o n t r o l c u v e t t e at
2 3 4 n m , the e x t i n c t i o n is m e a s u r e d at 2 4 5 rtm a n d t h e f a c t o r 5 6 8 0 i n s t e a d o f 4 1 2 3 is u s e d for
the calculations.
M i c r o - o r g a n i s m s : ( e x a m p l e : Penicillium). A l l o w 100 m g . o f t h e d r i e d m y c e l i a t o s t a n d for
2 4 hr. at r o o m t e m p e r a t u r e w i t h 2 m l . 1.5 N a l c o h o l i c K O H ( s o l u t i o n III) in a 1 0 0 m l . v o l u
m e t r i c flask. T h e n a d d 2 0 m l . 1.0 M buffer ( s o l u t i o n V ) a n d 6 m l . 0.5 N H C 1 ( s o l u t i o n I V )
a n d d i l u t e t o 1 0 0 m l . d i s t i l l e d w a t e r . F i l t e r t h r o u g h a dry filter p a p e r a n d d i l u t e a p o r t i o n o f
t h e filtrate t e n - f o l d w i t h 0 . 2 M buffer ( s o l u t i o n I V ) .
P l a n t s e e d s : ( e x a m p l e : w h e a t k e r n e l s ) . W e i g h five soft w h e a t k e r n e l s a n d slice e a c h i n t o five
t r a n s v e r s e s e c t i o n s . G e n t l y w a r m w i t h 4 m l . 1.5 N a l c o h o l i c K O H ( s o l u t i o n III) in a 2 0 0 m l .
v o l u m e t r i c flask for 2 hr. o n a s t e a m b a t h a n d t h e n a l l o w t o s t a n d for 2 4 hr. in t h e d a r k . A d d
4 0 m l . 1.0 M buffer ( s o l u t i o n V ) a n d 6 m l . 0.5 N H C 1 ( s o l u t i o n I V ) a n d d i l u t e w i t h distilled
w a t e r t o 2 0 0 m l . a n d filter t h r o u g h a d r y filter p a p e r .
The extinction o f the solution m a y also be t o o high to permit accurate m e a s u r e m e n t o f the
a b s o r p t i o n at 2 3 4 n m w i t h s o m e s p e c t r o p h o t o m e t e r s . In this c a s e , r e a d t h e a b s o r p t i o n a g a i n s t
a reference c u v e t t e (see " A s s a y S y s t e m " , c o n t r o l ) . To c a l c u l a t e t h e p o l y u n s a t u r a t e d fatty a c i d
content of the sample use the conversion factor 4123.
Stability of samples
A l l s o l u t i o n s w h i c h c o n t a i n p o l y u n s a t u r a t e d fatty a c i d s m u s t b e p r o t e c t e d a g a i n s t a t m o s p h e r i c
o x y g e n , e x c e p t d u r i n g t h e e n z y m a t i c r e a c t i o n w h e r e o x y g e n is n e c e s s a r y . S t o r a g e in o x y g e n -
free c o n d i t i o n s is a c c o m p l i s h e d b y d i s p l a c i n g t h e air a b o v e t h e s o l u t i o n s w i t h n i t r o g e n a n d
then stoppering the vessels.
1810 M e t a b o l i t e s : F a t t y Acid M e t a b o l i s m , etc.
Assay System
W a v e l e n g t h : 2 3 4 n m ; silica c u v e t t e s , light p a t h : 1 c m . ; final v o l u m e : 3.1 m l .
P i p e t t e i n t o 7.5 m l . flasks: Control Experimental
assay mixture
S a m p l e (buffered) 3.00 ml. 3.00 ml. 1 . 7 - 8 jug. free fatty

acids/ml.
0 . 2 M b o r a t e buffer
Lipoxygenase
inactive (IX) 0.10 ml. — —
Lipoxygenase
working solution (VIII) — 0.10 ml. ^ 1 0 0 U/ml.
Stopper the tubes a n d mix by inversion. R e m o v e stoppers a n d

a l l o w t o s t a n d f o r 6 0 m i n . at r o o m t e m p e r a t u r e . P o u r t h e c o n
tents into cuvettes, adjust the s p e c t r o p h o t o m e t e r t o zero with
the control cuvette a n d read the extinction E o f the experimental
cuvette.
Calculations
In principle the calculation formula (1) on p . 312 applies.

F r o m t h e extinction E calculate the percentage p o l y u n s a t u r a t e d fatty acids in t h e sample according t o the
formula:
% p o l y u n s a t u r a t e d fatty acids
E x 4123
W
Divide by 0.956 t o calculate on a fatty acid basis where W = ug. sample in 3.0 m l . solution
the factor 4123 is obtained as follows:
4l23=Mxl«»x 100x3
31
= dilution factor (3 ml. of sample + 0.1 m l . enzyme solution)
75.2 = extinction coefficient after reaction of 1 g. p o l y u n s a t u r a t e d fatty acid/litre with a 1 cm. light p a t h at
234 n m .
1000 = conversion from g./litre t o ug./m\.
100 = conversion t o %
3 = volume of sample (ml.)
T h e extinction coefficient used above has the dimension g . " x cm. ~ . Its absolute numerical value depends
1 1
to a large extent o n experimental factors (technique of m e a s u r e m e n t s , adjustment of spectrophotometer,

" r o o m t e m p e r a t u r e " , etc.). It should b e determined in the particular l a b o r a t o r y (see below). MacGee gave a
value of 78.2 in t h e first edition of this b o o k a n d earlier a value of 79.9. Zmachinski et a l . used a value of
1 3
82.6 a n d with this achieved a high degree of agreement between the results with the lipoxygenase m e t h o d and
other m e t h o d s . We found o n analysis of 14 samples of three types of refined a n d bleached plant oils a m e a n
value of 75.2 (19 determinations). A variation of 3 units in this value m e a n s a b o u t 1 % absolute difference in
the % of u n s a t u r a t e d fatty acids with values of 2 5 % u n s a t u r a t e d fatty acids.
P o l y u n s a t u r a t e d F a t t y Acids 1811
D e t e r m i n a t i o n of the extinction coefficient:

Determine the total c o n t e n t of p o l y u n s a t u r a t e d fatty acids in n o n h y d r o g e n a t e d plant oil by the s t a n d a r d
m e t h o d for alkaline isomerization. S u m the values for dienoic, trienoic a n d tetraenoic fatty acids. As this
4
value is expressed as fatty acids (as in the A O C S * m e t h o d ) , multiply by 0.956 to convert to a glyceride basis.
Analyse the same sample with the lipoxygenase m e t h o d described above.
T h e extinction coefficient is calculated as follows:
extinction coefficient =
C x Q 3.0
where
C = c o n c e n t r a t i o n (g./litre) of the saponified sample a d d e d
C x = a m o u n t of the sample which h a s been determined to u n d e r g o isomerization in alkali by a s t a n d a r d
m e t h o d (e.g. 0.60 o r 6 0 % ) .
4
We found a m e a n value of 3 0 % p o l y u n s a t u r a t e d free fatty acids with a s t a n d a r d deviation of 1 % in 24

determinations over 3 m o n t h s ; the coefficient of variation is 3.1 %. Beare et a l . found s t a n d a r d deviations
5
of 11 % over the range 0 - 2 0 % p o l y u n s a t u r a t e d fatty acids a n d 8 . 5 % over the r a n g e 2 0 - 3 0 % .
C o n t a m i n a t i o n of the c o n t r o l or solution IX with active enzyme results in low values for E.

Extremes of p H are to be a v o i d e d ; neutralize the samples before the last dilution. O r g a n i c solvents which
have been used for the p r e p a r a t i o n of the samples should be removed by w a r m i n g in a s t r e a m of nitrogen.
E t h a n o l at a c o n c e n t r a t i o n of 3 % does n o t interfere, b u t at a concentration of 5 % it causes a 50 % inhibition
of the enzyme a n d consequently p r o l o n g s the time for the reaction. According to Kock et a l . fatty acid 6
peroxides d o n o t inhibit lipoxygenase. T h e enzyme is n o t inhibited by t r a n s - u n s a t u r a t e d fatty acids like

elaidic or linelaidic acid in c o n c e n t r a t i o n u p to 40 % . 7
Conjugated dienes o b t a i n e d after alkaline isomerization of soya bean oil d o n o t inhibit when a d d e d in
concentrations of u p to 1 0 % . Naturally-occurring dienes are automatically c o m p e n s a t e d for in the m e t h o d
7
described here. N o n e of these substances (such as elaidic, linelaidic acid or conjugated dienes) give false
positives when they are tested as substrates.
Specificity
All free p o l y u n s a t u r a t e d fatty acids which have at least one pair of cis, d s - d o u b l e b o n d s separated by a
methylene in ca 8 position react quantitatively. Therefore, linoleic, linolenic a n d a r a c h i d o n i c acids, the acids
which are m o s t i m p o r t a n t b i o l o g i c a l l y 8,9
are substrates for the enzyme. 8,14-eicosadienoic, 5,8,11-eico-
satrienoic, 8,11,14-docosatrienoic acids a n d 9,12,15-tricosatrienoic acids d o n o t react with lipoxygenase.
Esterified p o l y u n s a t u r a t e d fatty acids react, but only extremely slowly.
T h e m o l a r extinction coefficient at 234 n m , as far as it has been studied, is the s a m e for all p o l y u n s a t u r a t e d
fatty acids.
As only 1 m o l e diene h y d r o p e r o x i d e is formed enzymatically for each mole of fatty acid, it is n o t possible to
differentiate between the individual fatty acids. Therefore, linolenic acid (cis, cis, cis-A ' ' ) 9 12 15
a n d arachidonic
acid (cis, cis, cis, cis-A ' ' ' )
9 12 15 18
are determined as linoleic acid (cis, cis-A ' ). 9 12
T h e s t a n d a r d m e t h o d s (spectro
p h o t o m e t r y after alkaline i s o m e r i z a t i o n or gas c h r o m a t o g r a p h y ) can be used to distinguish these acids.
4 10
* A O C S = A m e r i c a n Oil Chemists Society.

Note
Dolev et a l . 1 1
have elucidated the mechanism of the lipoxygenase reaction a n d have shown the origin of the
oxygen incorporated in linoleic hydroperoxide. According to Koch 12
lipoxygenase h a s an absolute require
ment tor C a 2 +
for activity. H 0
2 2 or cysteine can inactivate the e n z y m e . 13
References
1 X MacGee, A n a l . C h e m . 31, 2 9 8 - 3 0 2 [1959].

2 M. Hamberg & B. Samuelsson, J. biol. C h e m . 242, 5 3 2 9 - 5 3 3 5 [1967].
3 77. Zmachinski, A. Waltking & J. D. Miller, J. A m e r . Oil C h e m . Soc. 43, 4 2 5 - 4 2 8 [1966].
4 A m e r . Oil C h e m . Soc. Official M e t h o d Cd 7 - 5 8 , revised 1959, i n : Official a n d Tentative M e t h o d s of
the A m e r i c a n Oil Chemist's Society, Chicago.
5 / . L. Beare, C. Heroux & T. K. Murray, C a n a d i a n M e d . Assn. J. 96, 1 5 7 5 - 1 576 [1967].
6 R. B. Kock, J. W. Smull, A. S. Henick & D. H. Calloway, J. A m e r . Oil C h e m . Soc. 36, 2 0 5 - 2 0 8
[1959].
7 W. J. Brabbs, P. F. Vornheder & H. W. Wharton, unpublished results.
8 F. H. Mattson, J. N u t r i t i o n 71, 366 [I960].
9 N u t r i t i o n Reviews 19, 62 [1961].
10 A m e r Oil C h e m . Soc. Official M e t h o d . Ce2-66 a n d Tentative M e t h o d Cel-62, revised 1964,
i n : Official a n d Tentative M e t h o d s of the A m e r i c a n Oil Chemist's Society, C h i c a g o .
11 A. Dolev, W. K Rohwedder, T. L. Mounts & H. J. Dutton, Lipids 2, 442 [1967].
12 R. B. Koch, Arch. Biochem. Biophysics 125, 303-307 [1968].
13 H. Mitsuda, K Yasumoto & A. Yamamoto, Agric. Biol. C h e m . 31, 8 5 3 - 8 6 0 [1967].
Lecithin
H a n s Mdllering and H a n s Ulrich Bergmeyer
M e t h o d s for the d e t e r m i n a t i o n of phospholipids, especially lecithin, have so far been r a t h e r unsatisfactory.

T h e determination of lipid c o m p o n e n t s in egg yolk by m e a n s of thin layer c h r o m a t o g r a p h y (separation
of fatty acids, cholesterol a n d phospholipids) using special elution techniques with organic solvent mixtures
has been described . F o r b l o o d Tesoro a n d Birchwooct
1 2
give a chemical semimicro m e t h o d ( p h o s p h a t e
determination after extraction of the total lipids). In c o m p a r i s o n to the a c i d 3 , 4
or alkaline h y d r o l y s i s '5 6
the enzymatic d e t e r m i n a t i o n with p h o s p h o l i p a s e (lecithinase), D , P L - D (Phosphatidylcholine p h o s p h a t i d o -

hydrolase, E C 3 . 1 . 4 . 4 ) 7 - 1 0
is preferable because of the greater specificity (see e . g . ) . 11
Application of Method: In foodstuff chemistry, e. g. as a m e a s u r e of the egg content. Possibly also in b o t a n y

and in the p h a r m a c e u t i c a l a n d cosmetic industries.
Principle
(1) Lecithin + H 0 2
P
^ ° D > P h o s p h a t i d e acid + Choline
The reaction goes to completion from left to right. Lecithin a n d p h o s p h a t i d e acid are very soluble in ether,
while choline is not. After the enzymatic reaction the p r o d u c t s can be separated by shaking with ether a n d
the choline precipitated from the a q u e o u s phase as the reineckate. T h e red-violet choline reineckate is
soluble in acetone a n d its c o n c e n t r a t i o n is measured at 520 n m .
The m e a s u r e m e n t s 10
are m a d e at the o p t i m u m p H 5 . 5 - 6 . 0 . The reaction is activated by C a 2 +
and e t h e r ;
the o p t i m u m a m o u n t of calcium is between 0.01 a n d 0.05 M ; concentrations above 0.1 M inhibit. Relatively
large a m o u n t s of enzyme (ca. 20 mg./assay) are required to obtain quantitative hydrolysis within a reason
able time because of the low specific activity of phospholipase D (0.5 U / m g . ) . T h e o p t i m u m t e m p e r a t u r e
for the enzymatic h y d r o l y s i s 11
is between 25 °C a n d 35 °C.
Equipment
P h o t o m e t e r for precise m e a s u r e m e n t s at 520 n m ; b e n c h centrifuge; s h a k e r ; glass e x t r a c t i o n

a p p a r a t u s (e. g. s e p a r a t i n g f u n n e l ) a n d d e p e n d i n g o n t h e t y p e o f s a m p l e , a r o t a r y e v a p o r a t o r .
Reagents
1. E t h e r , A . R . 7. R e i n e c k e s a l t , A . R .
2. M e t h a n o l , A . R . (Cr(NH ) (SCN) )NH H 0
3 2 4 4 2
3. A c e t o n e , A . R . 8. T r i c h l o r o a c e t i c a c i d , A . R .
4. C h l o r o f o r m , A . R. 9. S o d i u m a c e t a t e , A . R . , a n h y d r o u s
5. A c e t i c a c i d , A . R . 10. C a l c i u m c h l o r i d e , A . R . , CaCl -2H 0
2 2
should not react with c h r o m i c acid. 11. Phospholipase D (lecithinase D )

6. C h o l i n e c h l o r i d e , A . R . from white cabbage, lyophilized; ^ 0 . 5 U / m g .
(25 °C); commercial p r e p a r a t i o n , see p . 504.
Purity of Reagents
Phospholipase D isolated from white cabbage by the m e t h o d of Casson a n d Griffin is a relatively c r u d e 11
enzyme p r e p a r a t i o n , but is does not contain any enzymes which destroy choline. Before use the ether
must be treated with ferrous sulphate to remove peroxides.
P r e p a r e all s o l u t i o n s w i t h freshly p r e p a r e d , d o u b l y d i s t i l l e d w a t e r . T o p r e v e n t t h e g r o w t h o f
m i c r o - o r g a n i s m s , sterilize t h e c o n t a i n e r s .
I. A c e t a t e buffer (0.1 M ; p H 5 . 6 ; 10 m M C a C l ) : 2
M i x 9.6 m l . 0.1 M a c e t i c a c i d (5.8 m l . a c e t i c a c i d d i l u t e d w i t h d i s t i l l e d w a t e r t o 1 0 0 0 m l . )

a n d 9 0 . 4 m l . 0.1 M s o d i u m a c e t a t e s o l u t i o n ( 8 . 2 g. s o d i u m a c e t a t e d i s s o l v e d i n 1 0 0 0 m l .
distilled w a t e r ) . A d d 147 m g . C a C l - 2 H 0 . 2 2
II. R e i n e c k a t e s o l u t i o n (85 m M ) :
D i s s o l v e 6 0 0 m g . r e i n e c k e salt in m e t h a n o l a n d m a k e u p t o 2 0 m l .
III. C h o l i n e c h l o r i d e s t a n d a r d s o l u t i o n ( 2 % w / v ) :
D i s s o l v e 2 g. c h o l i n e c h l o r i d e in distilled w a t e r a n d m a k e u p t o 1 0 0 ' m l . D e t e r m i n e t h e
c o n t e n t o f t h e s o l u t i o n b y t i t r a t i o n w i t h silver n i t r a t e s o l u t i o n .
I V . T r i c h l o r o a c e t i c a c i d (3 M ) :
D i s s o l v e 4 9 g. t r i c h l o r o a c e t i c a c i d in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
V . P h o s p h o l i p a s e D (ca. 2 0 m g . / m l . ) :
S u s p e n d 4 0 0 m g . p h o s p h o l i p a s e D w i t h 2 0 m l . a c e t a t e buffer ( s o l u t i o n I). U s e the t u r b i d
enzyme suspension.
Prepare the enzyme suspension freshly each day a n d store at 0 ° C ; it rapidly loses activity. Also p r e p a r e
the reineckate solution freshly each day. All other solutions are stable at r o o m t e m p e r a t u r e .
Procedure
Collection and treatment of sample
F o o d s t u f f s w h i c h c o n t a i n large a m o u n t s o f l e c i t h i n (e. g. e g g s , e g g l i q u o r , m i l k p o w d e r , s o y a
beans, mayonnaise), drugs and cosmetics and also pure lecithins, can generally be analysed
w i t h o u t p r i o r e x t r a c t i o n . It is u s u a l l y sufficient t o d i s s o l v e t h e s a m p l e in e t h e r o r t o a d d a
finely-powdered or well-suspended, w e i g h e d a m o u n t (equivalent to 5 - 5 0 m g . lecithin) to
the assay s y s t e m . 1 2
F r e e p h o s p h o l i p i d s , w i t h t h e e x c e p t i o n o f s p h i n g o m y e l i n , are s o l u b l e in ether, but n o t in

acetone or methyl acetate.
B o u n d p h o s p h o l i p i d s are o n l y e x t r a c t a b l e after p r e l i m i n a r y t r e a t m e n t w i t h e t h a n o l o r e t h a n o l /
chloroform or ethanol/benzene 1 3
" . T h e ease with w h i c h lecitihin can be completely extracted
1 6
Lecithin 1815
f r o m tissues d e p e n d s o n t h e e x t e n t o f the b i n d i n g t o p r o t e i n . By t r e a t m e n t w i t h a l c o h o l i c s o l v e n t s
the p r o t e i n is d e n a t u r e d a n d t h e l e c i t h i n is r e l e a s e d .
D e f a t a n i m a l t i s s u e s , b l o o d , p l a s m a a n d s e r u m w i t h 3 v o l u m e s o f a c e t o n e at l o w t e m p e r a t u r e
(the p h o s p h o l i p i d s are p r e c i p i t a t e d ) . T a k e u p the filter c a k e w i t h e i t h e r a m i x t u r e o f e t h a n o l /
ether (3 : 1) o r p e t r o l e u m e t h e r / c h l o r o f o r m ( 2 : 1) at r o o m t e m p e r a t u r e . H o m o g e n i z e a n d ,
after c e n t r i f u g a t i o n , e x t r a c t t h e p r e c i p i t a t e in a s i m i l a r m a n n e r . C o m b i n e t h e e x t r a c t s , e v a p o r a t e
off the s o l v e n t at 4 0 ° C u n d e r n i t r o g e n a n d t a k e u p t h e r e s i d u e in e t h e r 1 7 1 8
.
D i s s o l v e o i l s a n d fats o r o t h e r s a m p l e s c o n t a i n i n g large a m o u n t s o f fat in e t h e r o r m a k e a fine
s u s p e n s i o n a n d p r e c i p i t a t e o u t t h e l e c i t h i n w i t h 4 t o 5 v o l u m e s i c e - c o l d a c e t o n e (in w h i c h a
s p a t u l a tip o f c a l c i u m c h l o r i d e o r m a g n e s i u m c h l o r i d e is d i s s o l v e d ) . D i s s o l v e t h e p r e c i p i t a t e
in ether a n d a n a l y s e 1 4 1 9
. Alternatively, extract directly 3 - 4 times with a c e t o n e and a d d e d salts
(this results in c o n s i d e r a b l e s e p a r a t i o n o f t h e fat), t a k e u p t h e i n s o l u b l e r e s i d u e in e t h e r a n d
t a k e a p o r t i o n for a n a l y s i s .
Stability of sample
D u r i n g t h e e x t r a c t i o n g r e a t c a r e s h o u l d b e t a k e n . P r o d u c t s w h i c h h a v e b e e n s t o r e d for l o n g
p e r i o d s in t h e air l o s e a p o r t i o n o f their p h o s p h a t i d e c o n t e n t b y o x i d a t i o n . T o a v o i d h i g h
temperatures and the action o f light and o x y g e n , the extracts s h o u l d be concentrated under
n i t r o g e n . S a m p l e s p r e p a r e d in t h i s w a y s h o u l d b e a n a l y s e d as s o o n as p o s s i b l e .
Assay System
Enzymatic hydrolysis: P r e p a r e a b l a n k w i t h o u t e n z y m e for e a c h a s s a y ; this g i v e s the c h o l i n e

c o n t e n t o f t h e s a m p l e , the test g i v e s t h e c h o l i n e + lecithin.
Incubation volume: 11 ml. a q u e o u s p h a s e + 10 m l . ether or e t h e r e x t r a c t ; r o o m t e m p e r a t u r e .
P i p e t t e i n t o 100 m l . assay
Test Blank Standard
volumetric flasks: (related to aqueous
phase)
Ether extract or solid

sample + ether 10.0 ml. 10.0 ml. 5 - 5 0 m g . lecithin
Ether 10.0 ml.
Acetate/CaCl 2 buffer (I) 10.0 m l . 10.0 m l . 10.0 ml. 90 m M N a acetate
9 m M CaCl 2
Phospholipase D
suspension (V) 1.0 m l . 1.8 m g . / m l .
Choline standard solution (III) 0 . 1 - 0 . 4 ml. 2, 4, 6 and 8 mg.
choline chloride
Distilled water 1.0 m l . 0 . 9 - 0 . 6 ml.
S t o p p e r flasks w e l l a n d s h a k e v i g o r o u s l y for 3 hr. o n a s h a k e r ( e x

cept standards).
Trichloroacetic acid (IV) 1.0 m l . 1.0 m l . 1.0 m l . 0.25 M
M i x . S h a k e t w i c e w i t h 5 0 ml. p e r o x i d e - f r e e ether, r u n off e t h e r p h a s e

a n d d i s c a r d . Filter a q u e o u s p h a s e . T a k e 2 m l . filtrate for c o l o r i m e t r i c
measurements.
Colorimetric measurements. W a v e l e n g t h : 5 2 0 n m ; light p a t h : 1 c m . ; final v o l u m e : 3 . 0 0 m l . ;

r o o m t e m p e r a t u r e ; read a g a i n s t b l a n k .
P i p e t t e i n t o 10 m l . c o n i c a l Test&
Blank C o n c e n t r a t i o n in a s s a y
centrifuge t u b e s : Standards
Filtrate from enzymatic

reaction 2.00 ml. 2.00 ml. 0 . 8 - 8 m g . lecithin
Reineckate solution (II) 1.00 m l . 1.00 m l . 28 m M
P l a c e for 3 hr. in ice b a t h , c e n t r i f u g e off c h o l i n e r e i n e c k a t e in

c o l d . W a s h p r e c i p i t a t e t w i c e w i t h 3 m l . distilled w a t e r at 0 ° C .
D i s s o l v e s e d i m e n t in
Acetone 3.00 ml. 3.00 ml.
(the c h o l i n e r e i n e c k a t e s h o u l d g o c o m p l e t e l y i n t o s o l u t i o n ) .
C e n t r i f u g e off a n y i n s o l u b l e m a t e r i a l . P o u r clear s u p e r n a t a n t
fluids i n t o c u v e t t e s a n d read t h e e x t i n c t i o n E a g a i n s t b l a n k . E is
u s e d for t h e c a l c u l a t i o n s .
Lecithin 1817
Calculations
To pre pa re a s t a n d a r d curve plot the extinctions E of the s t a n d a r d s (ordinate) against the mg. choline/
assay (abscissa). T h e s t a n d a r d curve cuts the abscissa at a b o u t 0.1 mg. due to the i n c o m p l e t e precipitation
of choline reineckate a n d the washing with water. Nevertheless, the m e t h o d is quite reproducible. We
find an extinction of 0.280 for 1 mg. choline/assay.
As the blank gives the free choline of the sample a n d the test the free + esterified choline, the m e t h o d
described here only determines the choline liberated enzymatically from lecithin.
Read off the a m o u n t s of choline c o r r e s p o n d i n g to the measured E values from the s t a n d a r d curve.
With a mean molecular weight of 785 for l e c i t h i n 14
a n d of 121.18 for choline, 1 m g . choline c o r r e s p o n d s
to 6.5 mg. lecithin ( 1 5 . 5 % ) ; from o u r s t a n d a r d s multiplication of the value for mg. choline/assay by
6 x 6.5 = 39 gives the mg. lecithin in the sample.
With 10 determinations of lecithin in egg p o w d e r we found a m e a n value of 15.21 mg. lecithin per 200 m g .
dry powder. T h e assays gave a precision (2 s) of 1.97 m g . T h e coefficient of variation is 6 . 5 % .
N o r m a l Values
The lecithin content of dried egg is ca. 1 0 % , in fresh egg yolk ca. 1 4 . 5 % , in milk p o w d e r ca. 0 . 6 5 % a n d
in butter ca. 0 . 0 6 5 % of the dry w e i g h t .11
Insufficient purity of the reagents (see p . 1814), especially the p h o s p h o l i p a s e D , results in t o o low choline
values; therefore the enzyme must be completely free from enzymes which destroy choline a n d also m u s t
not contain choline.
Triethanolamine gives the same precipitate with reineckate as choline. E t h a n o l a m i n e a n d serine d o n o t

react u n d e r the above conditions. Cephalin is therefore n o t determined. P h o s p h o l i p a s e D hydrolyses
the b o n d between the nitrogen base a n d p h o s p h o r i c a c i d . T h e enzyme reacts with the following l e c i t h i n s ;
7 10
egg lecithin, h y d r a t e d egg lecithin, dipalmityl-L-a-lecithin, lysolecithin, dimyristyl-L-a-lecithin, dipalmityl-

DL-a-lecithin a n d distearyl-DL-a-lecithin. Distearyl-/?-lecithin a n d sphingomyelin react slowly. Alkenyl-
acyl glycerol-3-phosphorylcholine is only hydrolysed at a slow rate by p h o s p h o l i p a s e D 2 0
.
References
1 E. Marchetti & G. Bergesi, Rass. Clin. Terap. Sci. Affini 64, 403 [1965].
2 A. Tesoro & B. Birchwood, Clin. Biochem. 5, 121 [1972].
3 C. G. Daubney & G. E. W. Sexton, Analyst 75, 305 [1950].
4 V. E. Munsey, J. Ass. off. agric. Chemists 36, 766 [1953].
5 R. W. Engel, J. N u t r i t . 25, 441 [1943].
6 H. Salwin, M. D. Devine & H. Mitchell, J. agric. F o o d . C h e m . 6, 475 [1958].
7 D. J. Hanahan & /. L. Chaikoff, J. biol. C h e m . 169, 699 [1947].
8 M. Kates, C a n a d . J. Biochem. P h y s i o U J , 575 [1955].
9 E. Einset & W. L. Clark, J. biol. C h e m . 231, 703 [1958].
10 F. M. Davidson & C Long, Biochem. J. 69, 458 [1958].
11 C. B. Casson & F. J. Griffin, Analyst 84, 281 [1959]; 86, 544 [1961].
12 E. Condrea, 1. Fabian & A. de Vries, Experentia 20, 557 [1964].
13 R. Kunze: Lecithin. Verlag Rosenmeier & D r . Saenger, Berlin [1941].
14 W. Halden in A. Juckenack, B. Barnes, W. Bleyer & 7 . Grossfeld: H a n d b u c h der Lebensmittelchemie,
Vol. IV, p. 708, Springer Verlag, Berlin [1939].
15 A. Beythien & W. Diemair: L a b o r a t o r i u m s b u c h fur den Lebensmittelchemiker, p. 158. Th. Stein-
kopff Verlag, D r e s d e n u n d Leipzig [1957].
16 / . Schormueller: L e h r b u c h der Lebensmittelchemie, p . 53, Springer-Verlag, Berlin-Gottingen-Heidel-
berg [1961].
17 M. H. Hack, J. biol. C h e m . 169, 137 [1947].
18 R. G. Sinclair, J. biol. C h e m . 174, 343 [1948].
19 7 . Nerking, Biochem. Z. 23. 262 [1910].
20 W. E. M. Lands & P. Hart, Biochim. biophys. A c t a 98, 532 [1965].
Acetylcholine and Choline
John G. Hildebrand*
Acetylcholine is found mainly in n e r v o u s a n d n e u r o m u s c u l a r tissues of a wide r a n g e of animals. Choline,

the biosynthetic precursor of acetylcholine a n d m a n y lipids, occurs widely. T h e c u r r e n t m e t h o d s for the
determination of acetylcholine are the classical h y d r o x a m a t e d e t e r m i n a t i o n (colour test) for pmole
quantities, a n d biological m e t h o d s a n d pyrolysis g a s - c h r o m a t o g r a p h i c techniques for the d e t e r m i n a t i o n
of p m o l e and n m o l e quantities. T h e latter m e t h o d s are very sensitive, b u t inconvenient, technically difficult
to carry out, a n d possibly n o t very specific. In m o d e r n neurobiological investigations there is a great need
for a highly sensitive, specific m e t h o d for the d e t e r m i n a t i o n of acetylcholine in small tissue samples. These
requirements are satisfied by the m e t h o d described here. It c o r r e s p o n d s in principle a n d in m a n y of its
details to the m e t h o d s published by Goldberg a n d McCaman 1
a n d Reid et a l . .
2
Application of Method: In neurochemistry, biochemistry, a n d clinical biochemistry.
Principle
(1) Acetylcholine O H
~ > Acetate+Choline
(2) Choline+ATP-y- P 3 2 c
™™ > ADP+ P-Phosphorylcholine
3 2
Choline is p h o s p h o r y l a t e d t o p h o s p h o r y l c h o l i n e with the aid of A T P a n d choline kinase ( A T P x h o l i n e

phosphotransferase, E C 2.7.1.32). T h e formation of P - p h o s p h o r y l c h o l i n e , which is m e a s u r e d with a
32
liquid scintillation spectrometer, is p r o p o r t i o n a l to the q u a n t i t y of choline. Acetylcholine is converted

into choline by alkaline hydrolysis.
Choline kinase from brewer's yeast exhibits o p t i m u m activity in reaction (2) at p H 8 . 5 - 9 . 5 a n d 37 °C.
T h e total quantity of choline o r acetylcholine should be between 10 a n d 2 0 0 0 p m o l e , since the formation
of phosphorylcholine follows a linear course in this range.
Equipment
Liquid scintillation spectrometer (manufacturers, see p. 286), paper electrophoresis apparatus

( B e c k m a n - D u r r u m cell), microcentrifuge ( M i s c o or B e c k m a n microcentrifuge); water bath,
ice b a t h , B e c k m a n m i c r o c e n t r i f u g e t u b e s , P a s t e u r p i p e t t e s .
Reagents
1. G l a c i a l a c e t i c a c i d , A . R . 5. A c e t o n e , A . R .
2. F o r m i c a c i d , A . R . , 2 3 M 6. M e t h a n o l , A . R .
3. T e t r a e t h y l a m m o n i u m b r o m i d e 7. C o n c e n t r a t e d a m m o n i a s o l u t i o n , NH ,
3
4. Iodine, I , A . R .
2 A . R . ; 7.2 M
* W i t h the s u p p o r t of U S P H S G r a n t s 5 R O l NS-07848 a n d 5 P O l NS-02253 a n d of the A m e r i c a n H e a r t

Association Established Investigatorship.
8. G l y c y l g l y c i n e , free b a s e 12. C h o l i n e k i n a s e
9. M a g n e s i u m c h l o r i d e , M g C l 2 6 H 0 , A . R.
2
from brewer's yeast, partly purified after Mc
10. A d e n o s i n e t r i p h o s p h a t e , A T P Caman et a l . ; solution in 10 m M glycylglycine
3
d i s o d i u m salt A T P - N a H - 3 H 0 . C o m m e r c i a l
2 2 2 buffer, p H 7.2, containing 1 0 % glycerol and
p r e p a r a t i o n s see p . 527. 0.1 m M E D T A ; ^ 1 0 0 m U / m g . (37 °C).
11. A d e n o s i n e triphosphate ( y - P ) , 3 2
13. D o w e x - 1 - X 8 ( 2 0 0 - 4 0 0 mesh)
ATP-y-[ P] 3 2 formate form, a n i o n exchange resin, A. R.
tetra (trimethylammonium) salt; 15-25 Ci/ 14. A c e t y l c h o l i n e c h l o r i d e
m m o l e (e.g. from New England Nuclear C o r p . , 15. C h o l i n e chloride
Boston, Mass., U S A ) . 16. B a r i u m c h l o r i d e , B a C l - 2 H 0 , A . R .
2 2
Purity of Reagents
As far as possible, the enzyme must be free from A T P a s e , p h o s p h a t a s e , a n d myokinase. A T P - y - P should 32
be freshly prepared, a n d should c o n t a i n not m o r e t h a n 0 . 0 2 % of radioactive impurities that are not retained
by the Dowex-1 (formate) c o l u m n . All other chemicals are of A. R. quality. Store choline chloride, acetyl
choline chloride, a n d A T P dry at — 20 °C.
M a k e u p all s o l u t i o n s w i t h f r e s h l y p r e p a r e d d o u b l y d i s t i l l e d w a t e r .
I. F o r m a t e - a c e t o n e t i s s u e e x t r a c t i o n s o l u t i o n ( 0 . 1 5 M f o r m i c a c i d / a c e t o n e , 3 : 1 7 , v / v ) :
4
M i x 0 . 6 5 m l . 2 3 M f o r m i c a c i d w i t h 1 4 . 3 5 m l . w a t e r a n d 85 m l . a c e t o n e .
II. T e t r a e t h y l a m m o n i u m b r o m i d e s o l u t i o n (3 m g . / m l . ) :
D i s s o l v e 3 0 m g . t e t r a e t h y l a m m o n i u m b r o m i d e in w a t e r a n d m a k e u p t o 10 m l . K e e p
in a s t o p p e r e d v e s s e l at r o o m t e m p e r a t u r e .
III. E l e c t r o p h o r e s i s buffer ( 1 . 4 M a c e t i c a c i d ; 0.5 M f o r m i c a c i d ; p H 1.9):
M i x 8 0 m l . g l a c i a l a c e t i c a c i d ( 1 7 . 4 M ) a n d 25 m l . 2 3 M f o r m i c a c i d , a n d m a k e u p t o
1 0 0 0 m l . w i t h w a t e r . K e e p in a s t o p p e r e d vessel at r o o m t e m p e r a t u r e .
I V . E l e c t r o p h e r o g r a m e l u t i o n s o l u t i o n (2 M f o r m i c a c i d / m e t h a n o l , 1 : 1 0 , v / v ) :
M i x 10 m l . m e t h a n o l w i t h 1 m l . 2 3 M f o r m i c a c i d .
V. M e t h a n o l i c a m m o n i a solution (0.9 M N H 3 in w a t e r / m e t h a n o l , 1 : 7 , v / v ) :
M i x 7 ml. m e t h a n o l with 1 ml. concentrated a m m o n i a (7.2 M ) :
V I . G l y c y l g l y c i n e buffer ( 0 . 3 5 M g l y c y l g l y c i n e 12 m M M g C l ; p H 9 . 2 ) : 2
D i s s o l v e 0 . 4 6 g. g l y c y l g l y c i n e a n d 2 4 . 4 m g . M g C l - 6 H 0 in 9 m l . w a t e r , adjust p H
2 2
to 9.2 with N a O H , and m a k e u p to 10.0 ml. with water.

V I I . A T P s o l u t i o n (0.1 M A T P ) :
D i s s o l v e 6 0 . 5 m g . A T P - N a H - 3 H 0 in 0.5 m l . w a t e r , a d j u s t p H t o 7.0, a n d m a k e u p
2 2 2
t o 1.0 m l . w i t h w a t e r . S t o r e in p o r t i o n s at — 2 0 ° C .
V I I I . S t a n d a r d c h o l i n e a n d a c e t y l c h o l i n e s o l u t i o n s ( 1 0 pM a c e t y l c h o l i n e c h l o r i d e , 10 / / M
choline chloride):
D i s s o l v e 0 . 1 4 0 g. c h o l i n e c h l o r i d e a n d 0 . 1 8 2 g. a c e t y l c h o l i n e c h l o r i d e in w a t e r a n d
m a k e u p t o 1 0 0 m l . B e f o r e u s e , d i l u t e 0 . 1 0 ml. o f this s o l u t i o n t o 100 ml. with i c e - c o l d
w a t e r . 10 /d. o f t h i s s t a n d a r d m i x t u r e c o n t a i n s 10 1 0
m o l e = 0.1 n m o l e o f e a c h s u b s t a n c e .
IX. Barium chloride solution (50 m M B a C l ) : 2
D i s s o l v e 122 m g . B a C l - 2 H 0 in w a t e r a n d m a k e u p t o 10 m l .
2 2
Acetylcholine a n d Choline 1821
X. Phosphorylcholine solution (50 m M phosphorylcholine):

D i s s o l v e 0 . 1 1 g. p h o s p h o r y l c h o l i n e i n w a t e r a n d m a k e u p t o 1 0 m l .
X I . A m m o n i u m f o r m a t e buffer ( 1 0 m M a m m o n i u m f o r m a t e ; p H 4 ) :
Dilute 0.44 ml. 23 M formic acid to 9 5 0 ml. with water, adjust p H to 4.0 with a m m o n i a ,
and m a k e u p t o 1 0 0 0 ml. with water. K e e p in a stoppered vessel at r o o m temperature
for n o t m o r e t h a n 2 - 3 d a y s .
XII. Choline kinase (10 mg. protein/ml.):
D i l u t e s t o c k s o l u t i o n t o 10 m g . p r o t e i n / m l . ( ^ 1 U / m l . ) w i t h 10 m M g l y c y l g l y c i n e buffer
p H 7 . 2 ( V I ) c o n t a i n i n g 1 0 % g l y c e r o l a n d 0.1 m M E D T A . C e n t r i f u g e t o e l i m i n a t e
t u r b i d i t y if n e c e s s a r y a n d s t o r e a t - 2 0 ° C .
Unless otherwise indicated, keep all solutions in stoppered vessels in a refrigerator at 0 t o 4 °C. Solutions
I - V , I X , a n d X a r e stable for a long time u n d e r these conditions. W h e n kept frozen at - 20 °C, choline
kinase (solution X I I ) is stable for a n u m b e r of m o n t h s , even after repeated freezing a n d thawing.
Procedure
Collection and preliminary treatment of sample: C u t u p tissue s a m p l e s ( u p to a b o u t 100 m g .

w e t w e i g h t , d e p e n d i n g o n t h e s o u r c e o f t h e t i s s u e ) i n p h y s i o l o g i c a l salt s o l u t i o n a n d transfer,
w i t h a s little a d h e r i n g l i q u i d a s p o s s i b l e , i n t o a g l a s s m i c r o h o m o g e n i s e r c o n t a i n i n g 5 0 - 1 0 0 p\.
of formate-acetone extraction solution (I). A d d a cholinesterase inhibitor, such as eserine,
t o t h e s o l u t i o n d u r i n g t h e c u t t i n g o f t h e t i s s u e . Treat larger t i s s u e s a m p l e s i n a s i m i l a r m a n n e r
with suitable modification o f the technique. H o m o g e n i z e the sample. Place the h o m o g e n a t e
a n d t h e h o m o g e n i z e r w a s h fluid ( 5 0 - 1 0 0 p\.) in a m i c r o c e n t r i f u g e t u b e a n d c e n t r i f u g e f o r
5 m i n . in a m i c r o c e n t r i f u g e at 5 0 0 0 - 1 0 0 0 0 r p m . W a s h p r e c i p i t a t e w i t h a little e x t r a c t i o n
s o l u t i o n (I) a n d c e n t r i f u g e a g a i n . C o n c e n t r a t e t h e c o m b i n e d s u p e r n a t a n t a n d w a s h fluid t o
a b o u t 10 p\. at r o o m t e m p e r a t u r e in a s t r e a m o f n i t r o g e n . T o d e t e r m i n e t h e w e i g h t o f p r o t e i n
or the dry weight o f the sample, keep the centrifuge precipitate.
Blanks contain only formate-acetone solution, a n d standard solutions contain formate-
a c e t o n e s o l u t i o n w i t h 5 , 1 0 , a n d 1 0 0 pi. s t a n d a r d c h o l i n e - a c e t y l c h o l i n e m i x t u r e ( V I I I ) . F u r t h e r
t r e a t m e n t o f t h e s t a n d a r d s is a s f o r t h e s a m p l e s .
Electrophoresis: A d d 5 p\. t e t r a e t h y l a m m o n i u m b r o m i d e s o l u t i o n ( I I ) t o 1 0 pi. sample,

mix, apply at t h e starting line 5 c m . f r o m t h e e d g e o f a 3 x 30 c m . strip o f electrophoresis paper
( B e c k m a n n o . 3 2 0 0 4 6 ) , a n d a l l o w t o d r y . P l a c e t h e strip o n t h e f r a m e o f a B e c k m a n - D u r r u m
cell a n d m o i s t e n w i t h sufficient e l e c t r o p h o r e s i s buffer ( I I I ) t o r e a c h 0 . 5 c m . o n e i t h e r s i d e o f
t h e s t a r t i n g line, s o t h a t t h e s a m p l e is " c h r o m a t o g r a p h i c a l l y " c o n c e n t r a t e d b y t h e b u f f e r
f r o n t s f l o w i n g t o g e t h e r . F i x t h e f r a m e i n t h e B e c k m a n - D u r r u m cell ( s t a r t i n g l i n e a t t h e a n o d e ) ,
a n d a p p l y 4 5 0 v o l t s f o r 2 h o u r s a t r o o m t e m p e r a t u r e . R e m o v e t h e strip, d r y u n t i l it is o n l y
slightly m o i s t , a n d e x p o s e t o i o d i n e v a p o u r in a l a r g e d i s h w i t h s o l i d i o d i n e o n t h e b o t t o m .
The tetraethylammonium band (which corresponds exactly to the acetylcholine) becomes
v i s i b l e . M a r k t h e b a n d w i t h a p e n c i l . T h e n d r y t h e strip a t 2 0 - 2 2 ° C u n t i l t h e i o d i n e h a s
evaporated from the paper.
Elution of acetylcholine and choline: C u t t h e a c e t y l c h o l i n e z o n e ( 2 . 3 c m . in t h e d i r e c t i o n o f

t h e start f r o m t h e e d g e o f t h e c a t h o d i c t e t r a e t h y l a m m o n i u m b a n d ) o u t o f t h e strip o f p a p e r ;
cut o u t the choline z o n e in the s a m e w a y (the next 2.3cm. towards the cathode). Extract samples
o f t h e s e p i e c e s o f p a p e r w i t h e l e c t r o p h e r o g r a m e l u t i o n s o l u t i o n ( I V ) after Feigenson a n d
Saelens . 5
P r e p a r e B e c k m a n m i c r o c e n t r i f u g e t u b e s f o r s a m p l e s a n d s t a n d a r d s . C u t a ring o u t o f t h e c a p
o f a tube with a scalpel a n d centrifuge into the centrifuge tube t o a distance o f a b o u t 2 c m . from
t h e r i m . F o l d t h e e l e c t r o p h o r e s i s p a p e r 3 t i m e s l e n g t h w i s e a n d p l a c e i n t h e t u b e in s u c h a
w a y t h a t t h e o u t e r e d g e o f t h e c o r r e s p o n d i n g b a n d lies o n t h e ring. M o i s t e n t h e strip o f p a p e r
w i t h 5 0 pi. o f e l u t i o n s o l u t i o n ( V I ) a n d c e n t r i f u g e . T h e e l u a t e c o l l e c t s at t h e b o t t o m o f t h e t u b e .
M o i s t e n a g a i n w i t h a further 1 0 0 pi o f solvent a n d recentrifuge. T h e n cut each tube with a
r a z o r b l a d e j u s t a b o v e t h e s u r f a c e o f t h e e l u a t e a n d h e a t t h e t u b e i n a w a t e r b a t h for 15 m i n .
at 7 0 ° C t h e n f o r 15 m i n . a t 9 0 ° C s o t h a t all t h e l i q u i d e v a p o r a t e s .
Hydrolysis of acetylcholine: A d d 5 0 pi methanolic a m m o n i a solution (V) to each tube and

heat at 70 °C until the liquid h a s completely evaporated. C h o l i n e a n d acetylcholine samples
are t r e a t e d in t h e s a m e w a y , s o t h a t all s a m p l e s h a v e t h e s a m e b l a n k .
Stability of sample: T h e d r i e d s a m p l e s k e e p i n d e f i n i t e l y in s t o p p e r e d v e s s e l s at 4 ° C . D u r i n g
the preparation o f the samples, acetylcholine should n o t be e x p o s e d t o alkaline p H values or
s t o r e d f o r a l o n g t i m e , s i n c e it is n o t v e r y s t a b l e u n d e r t h e s e c o n d i t i o n s . Carry o u t all s t e p s
in t h e p r e p a r a t i o n o f t h e s a m p l e s a s q u i c k l y a s p o s s i b l e .
Preparation of Reagent Mixture
F o r g r o u p s o f 2 0 s a m p l e s , i n c l u d i n g b l a n k s a n d s t a n d a r d s , p r e p a r e a m i x t u r e o f 4 0 pi. buffer
s o l u t i o n ( V I ) , 5 pi A T P s o l u t i o n ( V I I ) , a n d 4 0 pi. c h o l i n e k i n a s e ( X I I ) . I n c u b a t e this s o l u t i o n
for 3 0 m i n . at 37 ° C a n d t h e n c o o l t o 0 ° C in a n i c e b a t h . A d d 25 pi A T P - y - [ P ] s o l u t i o n ( 2 0
3 2
pCi) shortly before use.
Assay System
Enzymatic Reaction and Radiochemical Determination of Phosphorylcholine
D i s s o l v e t h e d r i e d s a m p l e s in 5 pi p o r t i o n s o f w a t e r in p l a s t i c t u b e s in a n ice b a t h w i t h t h e a i d
o f a d r a w n - o u t g l a s s r o d . A d d 5 pi. r e a g e n t m i x t u r e , m i x , a n d i n c u b a t e f o r 6 0 m i n . in a 3 7 ° C
w a t e r b a t h . C o o l in a n i c e b a t h a n d a d d 10 pi BaCl 2 s o l u t i o n ( I X ) a n d 5 pi phosphoryl
c h o l i n e s o l u t i o n ( X ) w i t h m i x i n g . C e n t r i f u g e off t h e p r e c i p i t a t e d B a s a l t s o f A T P a n d p h o s p h a t e
at 5 0 0 0 r p m ( r o o m t e m p e r a t u r e ) . U s e t h e s u p e r n a t a n t . P a c k s m a l l c h r o m a t o g r a p h y c o l u m n s
(cut-off Pasteur pipettes, 0 . 5 x 2 . 5 c m ) with D o w e x - l - X 8 ( 2 0 0 - 4 0 0 mesh) a n d equilibrate
w i t h a m m o n i u m f o r m a t e buffer ( X I ) . I n t r o d u c e 2 0 pi. p o r t i o n s o f s u p e r n a t a n t (samples)
s e p a r a t e l y i n t o t h e c o l u m n s . E l u t e e a c h c o l u m n w i t h 2 m l . buffer i n t o a s c i n t i l l a t i o n v e s s e l
a n d m i x w i t h 15 m l . o f a s u i t a b l e s c i n t i l l a t i o n s o l u t i o n (e. g. A q u a s o l , N e w E n g l a n d N u c l e a r
C o r p . ) f o r c o u n t i n g in a l i q u i d s c i n t i l l a t i o n s p e c t r o m e t e r .
F o r r o u t i n e m e a s u r e m e n t s , t h e p r o c e d u r e after t h e e l e c t r o p h o r e t i c s e p a r a t i o n is a s f o l l o w s :
Acetylcholine a n d Choline 1823
Pipette into plastic t u b e s : C o n c e n t r a t i o n in assay mixture
S a m p l e , d r i e d in p l a s t i c t u b e u p to 0.2 m M choline
(choline or acetylcholine
in the tissue)
Water 5 pi
M i x with a drawn-out glass rod
Reagent mixture 5 /d. 64 m M glycylglycine; 2.2 m M

Mg 2 +
; 2 . 3 m M A T P ; 1.82 m g .
choline kinase/ml. = 182 m U
choline kinase/ml.
I n c u b a t e for 6 0 m i n . a t 3 7 ° C , c o o l in a n ice b a t h .
BaCl 2 (IX) 10 pi
Phosphorylcholine solution (X) 5 pi
Centrifuge; introduce 20 /d. o f the supernatant into a
D o w e x - 1 ( f o r m a t e ) c o l u m n , e l u t e w i t h 2 m l . buffer
( X I ) , m i x w i t h 15 m l . s c i n t i l l a t i o n s o l u t i o n , and
measure cpm.
Calculations
Subtract the average of the b l a n k s from the results of all m e a s u r e m e n t s . C o n s t r u c t a calibration curve from
the results of m e a s u r e m e n t s o n t h e s t a n d a r d s (abscissa scale from 5 n m o l e t o 100 n m o l e ) . F i n d the c p m
values corresponding to 1 n m o l e of acetylcholine from the calibration curves.
Since only 20 p\. of the 25 pi. test mixture passes t h r o u g h the D o w e x c o l u m n , it is necessary to multiply by
a factor of 1.25. T h e result from the calibration curve gives the n u m b e r of n m o l e in the v o l u m e of s a m p l e
examined.
The recovery of k n o w n quantities of acetylcholine o r choline ( 0 . 0 1 - 5 n m o l e ) in the e l e c t r o p h e r o g r a m is

9 1 - 1 0 0 % . In a typical r u n , 1 n m o l e samples of a n acetylcholine s t a n d a r d gave an average net value of
29100 c p m with a coefficient of variation of 7 . 5 % . T h e limit sensitivity of the m e t h o d is 10 p m o l e = 0.010
1
nmole of acetylcholine or choline.
Normal Values
All investigations using this m e t h o d were carried out with tissue from the lobster Homarus americanus.
In all the nerve a n d muscle tissue samples investigated, choline was roughly uniformly distributed with a
content of a p p r o x . 1.8 pmole/jUg. protein. Acetylcholine, o n the other h a n d , was not found everywhere.
Sensory nerves contained acetylcholine, whereas n o acetylcholine was detected in muscles a n d m o t o r
nerves. The acetylcholine c o n t e n t was always related to the sensory elements in the nerve tissue. Values
of 1.9 ± 1.1 p m o l e / ^ g . were found for individual a b d o m i n a l muscle a x o n s , 1.51 ± 0 . 8 9 pmole//*g. of protein
for a b d o m i n a l ganglia, a n d 5 . 3 1 + 2 . 4 2 pmole//ig. of protein for a n t e n n a l hair cells (with s u r r o u n d i n g
connective tissue).
Sources of Error
Interference in the assay technique: E r r o r s arise only at very low choline or acetylcholine concentrat
ions. Samples containing less t h a n 50 p m o l e m u s t be p r e p a r e d extremely carefully for correct determination
of (acetyl)choline. Substances t h a t affect the b l a n k s are eluted from some electrophoresis papers. This
can be prevented by preliminary washing of the p a p e r with e l e c t r o p h e r o g r a m elution solution (IV). T h e
blank is excessively high if the A T P - y - P used is old.
3 2
Choline kinase from brewer's yeast is specific for choline. T h e d e t e r m i n a t i o n is n o t significantly influenced
by e t h a n o l a m i n e , m o n o m e t h y l e t h a n o l a m i n e , dimethylethanolamine, carnitine, or betaine. The specificity
is increased by initial electrophoretic isolation of the samples a n d by c o l u m n - c h r o m a t o g r a p h i c isolation
of the labelled p h o s p h o r y l c h o l i n e .
References
1 A. M. Goldberg & R. E. McCaman, J. N e u r o c h e m . 20, 1 [1973].

2 W. D. Reid, D. R. Haubrich & G. Krischna, Analyt. Biochem. 42, 390 [1971].
3 R. E. McCaman, S. A. Dewhurst & A. M. Goldberg, Analyt. Biochem. 42, 171 [1971].
4 M. Toru & M. H. Aprison, J. N e u r o c h e m . 13, 1533 [1966].
5 M. E. Feigenson & J. K. Saelens, Biochem. P h a r m a c o l . 18, 1479 [1969].
Triglycerides and Glycerol
Determination after Alkaline Hydrolysis
Manfred Eggstein and Elisabeth K u h l m a n n
Triglycerides with the p h o s p h a t i d e s , a n d the free a n d esterified cholesterol, m a k e u p the quantitatively m o s t

i m p o r t a n t lipid fractions of b l o o d . T h e y represent 85 t o 9 0 % of the weight of the 200 t o 500 mu size chylo
microns and 5 0 % a n d 11 % respectively of the very low a n d low density l i p o p r o t e i n s of plasma. Daily a b o u t 1
100 to 150 g. of exogenous fat are t r a n s p o r t e d in the p l a s m a as chylomicrons a n d e n d o g e n o u s triglycerides

(synthesized in the liver as very low or low density lipoproteins) and, d e p e n d i n g on the nutritional state, are
taken u p by fat oxidizing or storing tissues . 2
Adipose tissue has the highest triglyceride content, namely between 60 a n d 85 %. It constitutes at least 2 to
3 % of the total b o d y weight, in n o r m a l cases n o t m o r e t h a n 20 to 30% a n d in obese subjects over 50%.
T h e d e t e r m i n a t i o n of triglycerides as t h e difference between gravimetrically o r p h o t o m e t r i c a l l y d e t e r m i n e d
total fat a n d cholesterol, cholesterol esters a n d p h o s p h a t i d e s 3 - 5
or between titratable total fats or p h o t o
metrically m e a s u r e d esterified fatty acids a n d the cholesterol a n d p h o s p h a t i d e - b o u n d fatty a c i d s 6 - 8
requires extraction, purification a n d hydrolysis a n d a precise d e t e r m i n a t i o n of the cholesterol fractions a n d

p h o s p h a t i d e s . T h e m e a s u r e m e n t s of neutral fat by the d e t e r m i n a t i o n of glycerol by chemical or fluori
metric m e t h o d s 9 , 1 0
also requires an extraction a n d the careful isolation of triglycerides followed by h y d r o
lysis 1 1 - 1 3
.
In the enzymatic d e t e r m i n a t i o n of triglycerides in b l o o d serum or p l a s m a 1 4 - 1 8
the isolation or purification
of the triglycerides or the extraction of fat can be omitted. T h e glycerol present in serum or p l a s m a or
Folch extracts of t i s s u e s 2 0 , 2 1
before a n d after ethanolic-alkaline hydrolysis is converted t o g l y c e r o
p h o s p h a t e a n d A D P with A T P a n d glycerokinase, G K ( A T P : glycerol 3-phosphotransferase, E C 2.7.1.30).
T h e A D P reacts with p y r u v a t e kinase, P K ( A T P : pyruvate 2-O-phosphotransferase, E C 2.7.1.40) a n d a d d e d
p h o s p h o e n o l p y r u v a t e t o give p y r u v a t e which is reduced t o lactate with N A D H a n d lactate d e h y d r o g e n a s e ,
L D H (L-lactate: N A D oxidoreductase, E C 1.1.1.27).
Application of Method: In biochemistry, clinical chemistry a n d foodstuff chemistry.
Principle
H C—O—COR
2 H C—OH
2
alkali
(1) H C—O—COR + 3H 0 ^ H(t—OH + 3 R—COOH
2
70 °C*
H i—O—COR
2
H i—OH
2
Triglyceride Glycerol
(2) Glycerol + A T P Glycerol-3-P + ADP
I
(3) ADP + PEP A T P + Pyruvate
7 ^ Lactate + N A D +
T h e d e t e r m i n a t i o n of triglyceride d e p e n d s o n a d o u b l e enzymatic d e t e r m i n a t i o n of g l y c e r o l . T h e free 19
glycerol (determined directly) is subtracted from the total glycerol m e a s u r e d after alkaline hydrolysis of
serum or plasma. T h e difference c o r r e s p o n d s t o the glyceride-glycerol or the n e u t r a l fat c o n t e n t (mole /
mole). Only traces of m o n o - a n d diglycerides are present in plasma. T h e decrease of extinction due to the
oxidation of N A D H is measured.
T h e phosphorylation of glycerol with A T P according to e q u a t i o n (2) is a n exergonic reaction which

proceeds to c o m p l e t i o n ; the auxiliary a n d indicator reactions are q u a n t i t a t i v e ' . T h e equilibrium of
22 23 24
the auxiliary reaction (3) ( K is 2 x 1 0 at 30 °C) is on the side of A T P , the L D H - c a t a l y s e d indicator

3 2 5
reaction (4) is far to the side of lactate with a K value of 1 x 1 0 l . / m o l e ( p H 7.6). T h e assay conditions
4
are 25 ° C ; 71.4 or 81.4 m M t r i e t h a n o l a m i n e buffer ( p H 7.6), 8.5 m M M g 2 +

, 2 m M ATP, 1 m M PEP,
0.4 m M N A D H , 3 m U pyruvate kinase/ml., 3.6 m U lactate dehydrogenase/ml., 0.425 m U glycerokinase/ml.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e f o r p r e c i s e m e a s u r e m e n t s at 3 3 4 ,
3 4 0 o r 365 n m , p r e f e r a b l y w i t h r e c o r d e r a t t a c h m e n t ; b e n c h c e n t r i f u g e ; 7 0 ° C w a t e r b a t h ;
m i c r o test t u b e s , 1.5 m l .
Reagents*
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 7. L a c t a t e d e h y d r o g e n a s e , L D H
2. M a g n e s i u m chloride, M g C l - 6 H 0 , 2 2 from rabbit skeletal muscle, crystalline suspen
A.R. sion in 3.2 M a m m o n i u m sulphate solution;
3. P h o s p h o e n o l p y r u v a t e , P E P = 360 mg. U / m g . (25 °C); commercial p r e p
tricyclohexylammonium salt; commercial p r e p aration, see p . 4 8 1 .
aration, see p . 548. 8. G l y c e r o k i n a s e , G K
4. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o from Candida mycoderma, suspension in 3.2 M
tide, N A D H ammonium sulphate solution: = 85 U/mg.
disodium salt, /?-N A D H - N a ; commercial p r e p
2
(25 °C); commercial p r e p a r a t i o n , see p. 468.
aration, see p . 545. 9. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) ,
5. A d e n o s i n e t r i p h o s p h a t e , A T P s p . g r . 1.67
disodium salt A T P - N a H - 3 H 0 ; commercial
2 2 2 10. P o t a s s i u m h y d r o x i d e , A . R .
preparation, see p . 527. 11. Ethanol, absolute, A . R.
6. P y r u v a t e k i n a s e , P K 12. S o d i u m h y d r o g e n c a r b o n a t e , A . R . ,
from rabbit muscle or crystalline suspension NaHC0 3
in 3.2 M a m m o n i u m sulphate solution; = 150 13. Glycerol

U / m g . (25 ° C ) ; commercial p r e p a r a t i o n , see 14. Triolein
p. 509. doubly distilled, A. R.
Purity of Reagents
Potassium hydroxide, free from glycerol m u s t be used. ( K O H is, o r has been, supplied by several firms
with traces of glycerol a d d e d for technical reasons connected with the p a c k a g i n g without this fact being
disclosed on the label). A T P must be completely free from A D P , a n d P E P free from pyruvate. C o n t a m i n a
tion of the glycerokinase with hexokinase should n o t exceed 0.02% of the G K activity.

Triglycerides a n d Glycerol 1827
U s e o n l y fresh, d o u b l y d i s t i l l e d w a t e r .
I. T r i e t h a n o l a m i n e buffer (0.1 M ; p H 7 . 6 ) :
D i s s o l v e 1 8 . 5 7 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in ca. 8 0 0 m l . d i s t i l l e d w a t e r , a d j u s t t o
p H 7.6 w i t h 1 N N a O H u n d d i l u t e t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r .
II. M a g n e s i u m c h l o r i d e ( 0 . 8 5 ) :
D i s s o l v e 1 7 . 2 5 g. M g C l * 6 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
2 2
III. P h o s p h o e n o l p y r u v a t e ( 5 0 m M ) :
Dissolve 23 mg. P E P - ( C H A ) 3 in 1.0 m l . d i s t i l l e d w a t e r .
IV. R e d u c e d nicotinamide-adenine dinucleotide (20 m M / ? - N A D H ) :
D i s s o l v e 14 m g . N A D H - N a 2 i n 1.0 m l . d i s t i l l e d w a t e r .
V . A d e n o s i n e t r i p h o s p h a t e (0.1 M ) :
D i s s o l v e 6 0 m g . A T P - N a H - 3 H 0 i n 1.0 m l . distilled w a t e r .
2 2 2
VI. Pyruvate kinase (2 m g . p r o t e i n / m l . ) :

If n e c e s s a r y , d i l u t e s t o c k s u s p e n s i o n w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V I I . L a c t a t e d e h y d r o g e n a s e (5 m g . p r o t e i n / m l . ) :
If n e c e s s a r y , d i l u t e t h e s t o c k s u s p e n s i o n w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V I I I . G l y c e r o k i n a s e (1 m g . p r o t e i n / m l . ) :
E a c h d a y j u s t b e f o r e u s e m i x 1 p a r t G K s u s p e n s i o n (5 m g . / m l . ) w i t h 4 p a r t s t r i e t h a n o l
a m i n e buffer ( I ) .
IX. A l c o h o l i c K O H (0.5 N ) :
D i s s o l v e 2 5 g. K O H in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l . D i l u t e 1 1 . 2 m l . o f t h i s
s o l u t i o n t o 1 0 0 m l . w i t h e t h a n o l a n d a l l o w t o s t a n d for c a . 12 hr. ( u n t i l c l e a r ) .
X . Perchloric acid (2.5 N ) :
D i l u t e 21 m l . 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h d i s t i l l e d w a t e r .
X I . Glycerol standard solution (2 m M ) :
Dilute 92.09 m g . glycerol t o 500 ml. with distilled water.
X I I . Triglyceride s t a n d a r d s o l u t i o n ( 2 m M ) :
D i s s o l v e 8 8 . 5 m g . t r i o l e i n in 5 0 m l . e t h a n o l .
Store all solutions, stoppered, in a refrigerator at 0 - 4 ° C . P r e p a r e solution I freshly every t w o m o n t h s

and solutions III, IV, V, I X a n d X I I freshly every week. T h e enzyme suspensions VI a n d VII are stable
for ca. 1 year at 4 ° C ; the glycerokinase solution (VIII) loses ca. 1 0 - 2 0 % of its activity d u r i n g the course
of a year. Solution II is stable indefinitely.
Procedure
T h e c o n c e n t r a t i o n o f t r i g l y c e r i d e in t h e b l o o d is n o t c o n s t a n t t h r o u g h o u t t h e d a y . T h e r e f o r e
c o l l e c t b l o o d 8 t o 10 hr. after a fat-free m e a l a n d u n d e r r e s t i n g c o n d i t i o n s ' . T h e c o n c e n t r a t i o n
6 2 6
o f free g l y c e r o l is a l s o d e p e n d e n t t o a large e x t e n t o n ' t h e t i m e o f c o l l e c t i o n . A d d i t i o n o f o x a -

1 8
late (1 m g . / m l . ) , citrate (1 m g . / m l . ) , fluoride ( 2 m g . / m l . ) , E D T A (1 m g . / m l . ) , h e p a r i n ( 0 . 2 m g . /

m l . ) o r a z i d e (1 m g . / m l . ) d o n o t interfere. O b t a i n p l a s m a o r s e r u m b y c e n t r i f u g a t i o n for 10 m i n .
at 3 0 0 0 g. U s e fresh s a m p l e s , free f r o m h a e m o l y s i s o r freeze u n t i l r e a d y for a s s a y . D i l u t e
lipaemic serum with physiological saline ( 1 + 9 ) .
Hydrolysis :
P i p e t t e 1.0 m l . a l c o h o l i c K O H ( I X ) i n t o s t o p p e r e d m i c r o - t e s t t u b e s , t h e n a d d 0.2 m l . s a m p l e
d r o p w i s e w i t h a r o t a t i n g m o v e m e n t ( s e r u m , p l a s m a , s t a n d a r d s o l u t i o n X I I o r t i s s u e extract)
o r 0.2 m l . distilled w a t e r (or e x t r a c t i n g a g e n t ) for t h e b l a n k . S t o p p e r t u b e s a n d p l a c e in a w a t e r
b a t h at 7 0 ° C for 3 0 m i n . T h e n c o o l in a n ice b a t h a n d n e u t r a l i z e t o p H 7 w i t h c a . 0 . 2 t o 0 . 2 2 m l .
perchloric acid (solution X ) (check p H by spotting o n L y p h a n or Universal indicator paper).
C e n t r i f u g e off t h e p r o t e i n - p e r c h l o r a t e p r e c i p i t a t e at 1 0 0 0 0 t o 1 6 0 0 0 r p m for 1 m i n . U s e t h e
s u p e r n a t a n t fluid ( h y d r o l y s a t e ) for t h e d e t e r m i n a t i o n o f t o t a l g l y c e r o l .
Assay System
Reagent mixtures
M i x daily j u s t b e f o r e u s e :
Buffer (I) 41.8 parts

MgCl 2 solution (ID 1 part
PEP solution (III) 2 parts
N A D H solution (IV) 2 parts
A T P solution (V) 2 parts
P K suspension (VI) 1 part
L D H suspension (VII) 0.2 p a r t
T h e m i x t u r e is s t a b l e in a n ice b a t h o r at 4 ° C for at least 2 4 hr.

W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 1.005 m l . ; r o o m t e m p e r
a t u r e ; read a g a i n s t air. P r e p a r e a b l a n k for e a c h series o f m e a s u r e m e n t s : for the d e t e r m i n a t i o n o f
free g l y c e r o l a b l a n k c o n t a i n i n g d i s t i l l e d w a t e r i n s t e a d o f s a m p l e ; for t h e d e t e r m i n a t i o n o f
total glycerol use the solution from the blank o f the "hydrolysis" (see a b o v e ) instead o f the
hydrolysate.
With extinction changes o f over 0.800 (334, 340 n m ) or 0.400 (365 n m ) dilute the serum or
h y d r o l y s a t e w i t h buffer (I). C h e c k t h e c o r r e c t f u n c t i o n i n g o f t h e a s s a y w i t h s t a n d a r d s o l u t i o n s
X I and X I I (instead o f serum).
Pipette into Free Total C o n c e n t r a t i o n in

micro-test tubes: glycerol glycerol assay mixture
Reagent mixture 0.5 m l . 0.5 m l . 71.4 m M T R A ;

8.5 m M M g 2 +
1.0 m M P E P
0.4 m M NADH;
2.0 m M A T P ;
2 0 /ig. P K / m l .
^3 U/ml.;
10 fig. L D H / m l .
^ 3 . 6 U/ml.
T R A buffer (I) 0.3 ml. 0.4 ml.
Sample (serum) 0.2 ml.
Hydrolysate 0.1 m l .
M i x a n d transfer t o c u v e t t e . F o l l o w o r r e c o r d t h e e x t i n c t i o n
c h a n g e s for 10 m i n . E 0 — E 1 0 = A E.t
G K solution (VIII) 0.005 ml. 0.005 ml. 5 fig. G K / m l .

^ 0 . 4 2 U/ml.
M i x ; follow or record extinction c h a n g e until c o m p l e t i o n of

reaction (10 min.). E 1 0 — E 2 0 = AE . 2 E c o r r = AE 2 — AE . 1
S u b t r a c t t h e a p p r o p r i a t e b l a n k s f o r free a n d t o t a l g l y c e r o l . If
t h e d e c r e a s e in e x t i n c t i o n is r e c o r d e d d e t e r m i n e A E g r a p h i c a l l y
(see p . 3 0 8 ) A E c o r r is u s e d f o r t h e c a l c u l a t i o n s .
Calculations
In this case the calculation formula (2) on p. 312 applies. T h e 1 + 6 dilution of the serum for the hydrolysis
must be corrected for with the dilution factor of 7. Using 0.2 ml. serum for the determination of free
glycerol or 0.1 ml. hydrolysate for the determination of total glycerol a n d a final volume in the cuvette
of 1.005 the concentration of the sample is calculated as follows:
Wavelength : 334 nm 340 nm 365 nm
Free glycerol:
c = 0.824 x AE CI 0.808 x z l E C( 1.478 x z l E c<
[^mole/ml.]
c = 7.59 x AE CI 7.44 x AE cl 13.61 x AE ct
[mg./lOO ml.]
Total glycerol:
c =• 11.53 x Z I E corr . 11.31 x J E c o r r . 20.69 x AECi
[/miole/ml.]
c = 106.2 x AE cnrr 104.2 x J E c o r r . 190.5 x AE rt
[mg./lOO ml.]
Glyceride-glycerol:
Cglyceride- glycerol " ^total glycerol —
^free glycerol
F o r the calculation of triglyceride a molecular weight of 885 is a s s u m e d . 6
Triglyceride:
c = 88.5 x c g l y c e r i d e _ g l y c e r o l [jumole/ml.]
c = 9.62 x c g l y c e r i d e _ g l y c e r o l [mg./100ml.]
Reproducibility of a series18
Coefficient
Mean s of variation
Free glycerol 0.1304 0.0033 ^ m o l e / m l . 2.5%
Total glycerol 5.0916 0.1066 /miole/ml. 2.09%
Glyceride-glycerol 4.9612 0.1065/xmole/ml. 2.15%
Reproducibility from day to day (duplicate assays)
Free glycerol 0.142 0.0037 /miole/ml. 2.59%

Total glycerol 1.808 0.0566/imole/ml. 3.14%
Glyceride-glycerol 1.666 0.0567 ^ m o l e / m l . 3.40%
N o r m a l Values
N o r m a l values in h u m a n s e r u m 18
( M + s):
^mole/ml. mg./lOO ml.

Free glycerol 0.1202 ± 0.065 1.105 ± 0.6 (N 57)
Total glycerol 1.572 ± 0.645 14.5 ± 5.98 (N 59)
Glyceride glycerol 1.388 ± 0.550 12.8 ± 5.06 (N 56)
Triglyceride 123 + 49 ( N = 56)
Further v a l u e s ' ' '
6 1 3 2 8 2 9
Effects of drugs and other therapeutic measures: I n t r a v e n o u s a d m i n i s t r a t i o n of heparin results in a transient

rise in free glycerol in serum, while insulin results in transient fall. High values m a y be found after in
jection of drugs containing glycerol.
Interference in the assay technique: Reagents which are n o t of the requisite purity (see p . 1826) can cause
high values; prolonged storage of solutions IV a n d V at temperatures above 4 °C can give values which
are t o o low. This type of interference is detected by the blanks and s t a n d a r d solutions. (The reagent blank
for the total glycerol d e t e r m i n a t i o n is not m o r e t h a n the equivalent of 10% of the n o r m a l triglyceride
content according to Eggstein.)
Addition of 0.2 ml. hydrolysate a n d / o r of insufficiently neutralized hydrolysate can cause turbidity in 3 0
the assay system. Haemolysed or lipaemic serum which has n o t been diluted gives a value which is t o o
high. A noticeable creep in the d e t e r m i n a t i o n of free glycerol indicates that the glycerokinase is c o n t a m i n a t
ed with hexokinase. This source of error does n o t occur with the determination of total glycerol.
Pure glycerokinase and A T P react also with dihydroxyacetone a n d L-( - )-glyceraldehyde to give dihydroxy
acetone p h o s p h a t e or glyceraldehyde-3-phosphate respectively a n d A D P . As the auxiliary a n d indicator
reactions of the glycerol assay detect A D P , dihydroxyacetone a n d L-( - )glyceraldehyde are determined.
However, these two c o m p o u n d s d o n o t occur in blood. A p a r t from P E P , n o o t h e r substrate for the P K -
catalysed reaction is k n o w n whose p r o d u c t can react with L D H . 3 1
References
1 D. G. Cornwell & G. Schettler: Lipids a n d Lipidoses, Springer-Verlag Berlin, Heidelberg, N e w Y o r k

1967.
2 M. Eggstein: Klinik der G e g e n w a r t , Vol. IX, p . 5 9 3 - 6 3 5 , U r b a n & Schwarzenberg, M u n c h e n u n d
Berlin, 1960.
3 E. Kirk, J. H. Page & D. van Slyke, J. biol. C h e m . 106, 203 [1934].
4 W. R. Bloor, J. biol. C h e m . 170, 671 [1947].
5 / . H. Bragdon, J. biol. C h e m . 190, 513 [1951].
6 M. Eggstein, Klin. Wschr. 43, 1031 [1965].
7 S. J. Thannhauser & H. Reinstcin, Arch. P a t h . 23, 646 [1942].
8 M. J. Albrink, J. Lipid Res. 1, 53 [1959].
9 M. Lambert & A. C. Neish, C a n a d . J. Res. B 28, 83 [1950].
10 D. Mendelson & A. Antonis, J. Lipid Res. 2, 95 [1961].
11 E. van Handel & D. Zilversmit, J. clin. M e d . (China) 50, 152 [1957].
12 W. M. Butler jr., H. M. Mating, M. G. Horning & B. B. Brodie, J. Lipid R e s . 2, 95 [1961].
13 D.H. Blankenhorn, G. Rouser & T. I. Weimer, J. Lipid Res. 2, 281 [1961].
14 O. Wieland, Biochem. Z. 329, 313 [1957].
15 J. H. Hagen & P. J. Hagen, C a n a d . J. Biochem. 40, 1129 [1962].
16 M. Eggstein & F. H. Kreutz, Ergebn. L a b o r a t o r i u m s m e d i z i n , 2, 99 [1965].
17 M. Eggstein & F. H. Kreutz, Klin. Wschr. 44, 262 [1966].
19 F. H. Kreutz, Klin. Wschr. 40, 362 [1962].
20 J. M. Folch & G. / / . Sloane-Stanley, F e d e r a t i o n P r o c . 13, 209 [1954].
21 J. M. Folch 8L G. H. Sloane-Stanley, J. biol. C h e m . 226, 409 [1957].
22 C. E. Bublitz & P. Kennedy, J. biol. C h e m . 277, 951 [1954].
23 H. J. Hohorst, F. H. Kreutz & Th. Bucher, Biochem. Z. 332, 18 [1959].
24 J. T. McQuate & M. F. Utter, J. biol. C h e m . 234, 2151 [1959].
25 H. Adam, Diss. M a r b u r g 1955, cited by H . A d a m in H. U. Bergmeyer: M e t h o d e n der enzymatischen
Analyse, Verlag Chemie, Weinheim, 1st. edn. 1962, p . 573.
26 M. Eggstein, Deutsches medizin. J o u r n a l 19, 12 [1968].
27 K. Doerffel: Beurteilung von Analysenverfahren u n d Ergebnissen. Springer-Verlag Berlin-Heidelberg-
N e w York u. J. F . B e r g m a n n , M u n c h e n [1965], 2nd. edn., p . 48 et. seq.
28 M. Eggstein, Med. u. E r n a h r u n g 2, 1 [1961].
29 / . P. Peters & E. B. Man, J. clin. Invest. 22, 707 [1943].
30 F. H. Schmidt & K. v. Dahl, Z. klin. C h e m . u. klin. Biochem. 6, 156 [1968].
31 R. Czok & H. Eckert in H. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag Chemie
Weinheim, 1st. edn. 1962, p . 224.
32 J. Holzl, Fette, Seifen, Anstrichmittel 69, 328 [1967].
33 G. Berner, D K 637.0 Milchwirtschaftliches Ordnungssystem, Bibliotheca Lactis 24, 284 [1969].
Triglycerides
Determination after Enzymatic Hydrolysis
A u g u s t Wilhelm Wahlefeld
Serum triglycerides can be specifically a n d quantitatively hydrolysed by e n z y m e s 1 - 3

. Lipases hydrolyse
triglycerides only if the substrate is present as an emulsion. Desnuelle* was able to show that the lipase
activity is p r o p o r t i o n a l to the surface areas of the emulsion particles.
Serum triglycerides are present as a quasi-solution in which they are b o u n d to a l b u m i n ; consequently,
n o measurable cleavage is observed with pancreatic lipases or a large n u m b e r of other lipases. Schmidt
and Storck 1
were able to show that the lipase from Rhizopus arrhizus hydrolyses serum triglycerides
quantitatively to glycerol a n d fatty acids. Bucolo et a l . found that the lipase from
2
Chromobacterium
References
1 D. G. Cornwell & G. Schettler: Lipids a n d Lipidoses, Springer-Verlag Berlin, Heidelberg, N e w Y o r k

1967.
2 M. Eggstein: Klinik der G e g e n w a r t , Vol. IX, p . 5 9 3 - 6 3 5 , U r b a n & Schwarzenberg, M u n c h e n u n d
Berlin, 1960.
3 E. Kirk, J. H. Page & D. van Slyke, J. biol. C h e m . 106, 203 [1934].
4 W. R. Bloor, J. biol. C h e m . 170, 671 [1947].
5 / . H. Bragdon, J. biol. C h e m . 190, 513 [1951].
7 S. J. Thannhauser & H. Reinstcin, Arch. P a t h . 23, 646 [1942].
8 M. J. Albrink, J. Lipid Res. 1, 53 [1959].
9 M. Lambert & A. C. Neish, C a n a d . J. Res. B 28, 83 [1950].
10 D. Mendelson & A. Antonis, J. Lipid Res. 2, 95 [1961].
11 E. van Handel & D. Zilversmit, J. clin. M e d . (China) 50, 152 [1957].
12 W. M. Butler jr., H. M. Mating, M. G. Horning & B. B. Brodie, J. Lipid R e s . 2, 95 [1961].
13 D.H. Blankenhorn, G. Rouser & T. I. Weimer, J. Lipid Res. 2, 281 [1961].
14 O. Wieland, Biochem. Z. 329, 313 [1957].
15 J. H. Hagen & P. J. Hagen, C a n a d . J. Biochem. 40, 1129 [1962].
16 M. Eggstein & F. H. Kreutz, Ergebn. L a b o r a t o r i u m s m e d i z i n , 2, 99 [1965].
17 M. Eggstein & F. H. Kreutz, Klin. Wschr. 44, 262 [1966].
20 J. M. Folch & G. / / . Sloane-Stanley, F e d e r a t i o n P r o c . 13, 209 [1954].
21 J. M. Folch 8L G. H. Sloane-Stanley, J. biol. C h e m . 226, 409 [1957].
22 C. E. Bublitz & P. Kennedy, J. biol. C h e m . 277, 951 [1954].
24 J. T. McQuate & M. F. Utter, J. biol. C h e m . 234, 2151 [1959].
25 H. Adam, Diss. M a r b u r g 1955, cited by H . A d a m in H. U. Bergmeyer: M e t h o d e n der enzymatischen
Analyse, Verlag Chemie, Weinheim, 1st. edn. 1962, p . 573.
26 M. Eggstein, Deutsches medizin. J o u r n a l 19, 12 [1968].
27 K. Doerffel: Beurteilung von Analysenverfahren u n d Ergebnissen. Springer-Verlag Berlin-Heidelberg-
N e w York u. J. F . B e r g m a n n , M u n c h e n [1965], 2nd. edn., p . 48 et. seq.
28 M. Eggstein, Med. u. E r n a h r u n g 2, 1 [1961].
29 / . P. Peters & E. B. Man, J. clin. Invest. 22, 707 [1943].
30 F. H. Schmidt & K. v. Dahl, Z. klin. C h e m . u. klin. Biochem. 6, 156 [1968].
31 R. Czok & H. Eckert in H. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag Chemie
Weinheim, 1st. edn. 1962, p . 224.
32 J. Holzl, Fette, Seifen, Anstrichmittel 69, 328 [1967].
33 G. Berner, D K 637.0 Milchwirtschaftliches Ordnungssystem, Bibliotheca Lactis 24, 284 [1969].
Triglycerides
Determination after Enzymatic Hydrolysis
A u g u s t Wilhelm Wahlefeld
Serum triglycerides can be specifically a n d quantitatively hydrolysed by e n z y m e s 1 - 3

. Lipases hydrolyse
triglycerides only if the substrate is present as an emulsion. Desnuelle* was able to show that the lipase
activity is p r o p o r t i o n a l to the surface areas of the emulsion particles.
Serum triglycerides are present as a quasi-solution in which they are b o u n d to a l b u m i n ; consequently,
n o measurable cleavage is observed with pancreatic lipases or a large n u m b e r of other lipases. Schmidt
and Storck 1
were able to show that the lipase from Rhizopus arrhizus hydrolyses serum triglycerides
quantitatively to glycerol a n d fatty acids. Bucolo et a l . found that the lipase from
2
Chromobacterium
viscosum is also active; moreover, the addition of proteases as non-specific esterases h a d a strongly activating
effect o n the complete hydrolysis of the triglycerides. Wahlefeld a n d Roschlau 3
discovered a c o m b i n a t i o n
with a cumulative effect for the lipolysis of serum triglycerides: in addition to the relatively rapid cleavage
to the diglyceride by the lipase from Rhizopus arrhizus in the presence of s o d i u m dodecyl sulphate, a liver
esterase accelerates the total hydrolysis by specific cleavage of the soluble glyceride ester. The indicator
system for the glycerol d e t e r m i n a t i o n in all the cases m e n t i o n e d is similar to that used by Kreutz .
5
Application of Method: In clinical chemistry.
Principle
(1) Triglyceride {d J^Z* , ph te) > 1,2-Diglyceride + F a t t y Acid
(2) 1,2-Diglyceride l i p a s e
*' e s t e r a s e
**) 2 - M o n o g l y c e r i d e + F a t t y Acid
(3) 2-Monoglyceride e s t e r a s e
** • Glycerol + F a t t y Acid
Glycerol is determined as described on p. 1825 with G K * * * , P K \ a n d L D H f t
as auxiliary and indicator
enzymes. The decrease in the extinction at 340, 334 or 365 n m due to the oxidation of N A D H is measured.
T h e free glycerol in the serum is also detected in this m e t h o d . It must* be determined beforehand by the
m e t h o d on p . 1825 a n d subtracted from the result. In routine d e t e r m i n a t i o n s in the clinical laboratory,
10 mg. of neutral fat/100 ml. or 0.11 mmole/1. m a y be subtracted as the correction for this.
T h e p H o p t i m u m of the coupled d e t e r m i n a t i o n is between p H 7.0 a n d 8.0. N o clear dependence on buffer

ions is evident, but anionic buffers are preferable, since they have n o salting-out effect on the liberated
fatty acids. If the time for complete hydrolysis (concentration of the triglycerides u p to 6.6 m M ) at 25 °C
is fixed as 15 min., then 300 U of lipase a n d 30 U of esterase should be used per d e t e r m i n a t i o n , provided
that the buffer contains 0.1 mg. of sodium dodecyl sulphate/ml.
The other conditions are the s a m e as for the d e t e r m i n a t i o n of glycerol on p . 1826.
Equipment
S p e c t r o p h o t o m e t e r or spectrum-line p h o t o m e t e r c a p a b l e of a c c u r a t e m e a s u r e m e n t s at 340,
3 3 4 o r 365 n m .
Reagents****
1. T r i e t h a n o l a m i n e , A . R. 3. P h o s p h o e n o l p y r u v a t e , P E P
2. M a g n e s i u m c h l o r i d e , M g C l * 6 H 0 , A . R.
2 2 tricyclohexylammonium salt; commercial prep
a r a t i o n s see p. 548.
* Triacylglycerol acyl-hydrolase, E C 3.1.1.3

** Carboxylic-ester hydrolase, E C 3.1.1.1.
*** A T P :glycerol 3-phosphotransferase, E C 2.7.1.30
**** Complete reagent kits are commercially available (see p. 558).
f
A T P : p y r u v a t e 2-O-phosphotransferase, E C 2.7.1.40
n
L-Lactate : N A D oxidoreductase, E C 1.1.1.27
4. R e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o 8, P y r u v a t e k i n a s e , P K
tide, N A D H from rabbit skeletal muscle, crystalline sus
disodium salt, / ? - N A D H - N a ; commercial prep
2
pension in 3.2 M a m m o n i u m sulphate solution;
arations, see p . 545. ^ 150 U / m g . (25 °C); commercial p r e p a r a t i o n s ,
5, A d e n o s i n e t r i p h o s p h a t e , A T P see p . 509.
disodium salt A T P - N a H * 3 H 0 ; commercial
2 2 2
9. L a c t a t e d e h y d r o g e n a s e , L D H
p r e p a r a t i o n s , see p . 527. from pig heart, crystalline suspension in 3.2 M
6. E s t e r a s e ammonium sulphate solution; ^400 U/mg.
from pig liver; suspension in 3.2 M a m m o n i u m (25 °C); e.g. from Boehringer M a n n h e i m .
sulphate s o l u t i o n ; ^ 1 0 0 U / m g . (25 °C); e.g. 10. G l y c e r o k i n a s e , G K
from Boehringer M a n n h e i m . from Candida mycoderma, suspension in 3.2 M
7. L i p a s e ammonium sulphate solution; ^85 U/mg.
from Rhizopus arrhizus; suspension in 3.2 M (25 °C); commercial p r e p a r a t i o n s , see p . 468.
a m m o n i u m sulphate solution; ^ 6 0 0 0 U / m g . 11. B o v i n e serum a l b u m i n
(25 °C); e.g. from Boehringer M a n n h e i m . 12. S o d i u m d o d e c y l s u l p h a t e
13. H y d r o c h l o r i c acid, HC1, 2 N , A . R.
14. S o d i u m h y d r o g e n c a r b o n a t e , N a H C Q 3
Purity of Reagents
The enzymes must be substantially free from hexokinase ( < 0 . 0 2 % ) , as well as o t h e r interfering kinases
and p h o s p h a t a s e s ; A T P m u s t be substantially free from A D P , a n d P E P from pyruvate.
M a k e u p all s o l u t i o n s w i t h f r e s h l y p r e p a r e d d i s t i l l e d w a t e r .
I. T r i e t h a n o l a m i n e buffer (0.1 M t r i e t h a n o l a m i n e , p H 7 . 6 ; 4 m M M g C l ; 2 . 0 m g . b o v i n e
2
s e r u m a l b u m i n / m l . ; 0.1 m g . s o d i u m d o d e c y l s u l p h a t e / m l . ) :
D i s s o l v e 3 . 7 3 g. t r i e t h a n o l a m i n e , 2 0 3 . 3 m g . M g C l - 6 H 0 , 5 0 0 m g . b o v i n e s e r u m a l b u m i n ,
2 2
a n d 2 5 m g . s o d i u m d o d e c y l s u l p h a t e , in t h a t o r d e r , in a p p r o x . 2 0 0 m l . w a t e r . A d j u s t t o
p H 7.6 w i t h d i l u t e h y d r o c h l o r i c a c i d ; m a k e u p t o 2 5 0 m l . w i t h w a t e r .
II. C o f a c t o r s o l u t i o n ( 6 m M N A D H ; 3 3 m M A T P ; 11 m M P E P ) :
Dissolve 20.5 mg. N A D H - N a , 80 mg. A T P - N a H , and 20.5 mg. P E P - ( C H A )
2 2 2 3 in 4 m l .
saturated N a H C 0 3 solution.
III. E n z y m e m i x t u r e ( ^ 3 2 0 U L D H / m l . ; ^ 8 0 U P K / m l . ; ^ 3 0 0 0 U l i p a s e / m l . ; ^ 4 0 0 U
esterase/ml.):
M i x appropriate v o l u m e s o f the stock suspensions o f the e n z y m e s and dilute to the
r e q u i r e d v o l u m e a c t i v i t i e s w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
IV. G l y c e r o k i n a s e (170 U / m l . ) :
D i l u t e 5 m g . / m l . s t o c k s u s p e n s i o n t o 2 m g . / m l . w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
Keep all solutions in stoppered containers in a refrigerator (4 °C). M a k e u p fresh solution I every 2 m o n t h s
and fresh solution II every 2 weeks. Suspensions III a n d IV are stable for 1 year at 4 °C.
Procedure
Collection of sample: S e e p . 1 8 2 7 .
Stability of sample: A t least 10 d a y s at 4 ° C , a n d at least 3 d a y s at 2 0 - 2 5 ° C .
Assay System
F o r a series o f d e t e r m i n a t i o n s , it is a d v i s a b l e t o p r e p a r e a d a y ' s s u p p l y o f r e a g e n t b y m i x i n g
s o l u t i o n s I, II, a n d III i n p r o p o r t i o n s o f 5 0 : 2 : 1 . T h e r e a c t i o n m i x t u r e is s t a b l e f o r o n e d a y at
2 0 - 2 5 °C a n d for three days at a b o u t 4 ° C .
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 o r H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 5 . 4 m l . , o r 2 . 5 2 m l . ;
25 ° C ; r e a d a g a i n s t air.
F o r e a c h series o f m e a s u r e m e n t s , carry o u t o n e r e a g e n t b l a n k w i t h w a t e r i n s t e a d o f s e r u m .
A n y difference in e x t i n c t i o n o n a d d i t i o n o f s o l u t i o n I V is s u b t r a c t e d f r o m t h e e x t i n c t i o n
difference o b t a i n e d w i t h t h e s a m p l e .
P i p e t t e i n t o test t u b e s : C o n c e n t r a t i o n in assay mixture
T r i e t h a n o l a m i n e buffer (I) 92.6 m M triethanolamine

3.7 m M M g 2 +
0.1 m g . d o d e c y l s u l p h a t e / m l .
1.85 m g . b o v i n e s e r u m a l b u m i n /
ml.
Cofactor solution (H) 0.22 m M N A D H
1.22 m M A T P
0.41 m M P E P
Enzyme mixture (HI) ca. 6 U L D H / m l .
c a . 1.5 U P K / m l .
ca. 56 U lipase/ml.
c a . 7.5 U e s t e r a s e / m l .
Sample (serum)
M i x , a n d a l l o w t o s t a n d f o r a t l e a s t 15 m i n . a t 2 5 ° C .
Determination of Total Glycerol
Concentration
P i p e t t e i n t o test t u b e s : Blank Sample
in a s s a y m i x t u r e
Hydrolysed sample 2.50 ml. 2.50 ml.

Water 0.02 ml. —
Glycerol kinase (IV) — 0.02 ml. ca. 1.4UGK/ml.
M i x ; after 1 0 - 2 0 m i n . , m e a s u r e e x t i n c t i o n o f t h e b l a n k a n d o f
t h e s a m p l e in t h e s a m e c u v e t t e a g a i n s t air. E - E = J E .
B S
A f t e r e a c h m e a s u r e m e n t o n t h e s a m p l e , rinse t h e c u v e t t e w i t h
d o u b l y distilled w a t e r .
Calculations
F o r m u l a (2) on p . 312 is valid.

The concentration of neutral fat in the serum is calculated as follows:
c = Êx9.07 A E x 8.78 A E x 16.5 [mmole/1.]

c = AEx803 AEx777 AEx 1460 [mg./lOO ml.]
A molecular weight of 885 was used as a basis in the calculations for the triglycerides.
The coefficient of variation (CV) of the m e t h o d , m e a s u r e d in the series, is e.g. 4 . 2 % with a wavelength
of H g 365 n m a n d a c o n c e n t r a t i o n of 85.8 mg. neutral fat per 100 m l . ; the value for a concentration of
263.5 mg. neutral fat per 100 ml. is 1 %.
A coefficient of variation of 5 % was found for the day-to-day accuracy at 152 mg. of neutral fat per 100 ml.
N o r m a l Values, see p . 1830.
Effects of drugs and other therapeutic measures: see p . 1830.
Interference in the assay technique: In addition t o the interference by reagent impurities (e.g. hexokinase
in the glycerokinase) discussed o n p . 1826, haemolysis giving m o r e t h a n 200 mg. haemoglobin/100 ml.
interferes in the present d e t e r m i n a t i o n . Interference in the lipolysis is also caused by detergents of all
kinds and by traces of organic solvents (take care in the case of Fblch-Sperry extracts). Excessively high
protein concentrations in the assay also interfere, a n d the sample dilution m u s t therefore be adhered t o .
N o other glycerides are hydrolysed by the enzyme mixture used for the hydrolysis of the triglycerides;
a-lecithin a n d glycerol-3-phosphate (up to 200 m g . / l 0 0 ml. serum) d o n o t affect the measurement. C o n
cerning the specificity of indicator a n d auxiliary enzymes, see p . 1830.
References
1 F. H. Schmidt & H. Stork, Boehringer M a n n h e i m G m b H , D P D O S 2 0 0 0 1 2 7 [1970].

2 G. Bucolo & H. David, Scand. J. Clin. L a b . Invest., 29, Suppl. 126 [1972], Abstr. 3,19.
3 A. W. Wahlefeld, H. Mollering, W. Gruber, E. Bernt & B. Roschlau, Boehringer M a n n h e i m G m b H ,
Dtsch. P a t e n t m e l d u n g P 2229849.3 [1972].
4 S. Sarda & P. Desnuelle, Biochim. Biophys. A c t a 30, 513 [1958].
D-(-)-3-Hydroxybutyrate
D e r m o t H. Williamson and Jane Mellanby
D-( — )-3-Hydroxybutyrate is present in b l o o d a n d m o s t tissues of the b o d y . It is excreted in the urine

in large quantities by the u n t r e a t e d diabetic in keto-acidosis. Chemical m e t h o d s for the determination
of 3-hydroxybutyrate depend on its oxidation to acetone a n d the colorimetric estimation of the latter;
these m e t h o d s are not specific a n d d o n o t differentiate between the stereoisomers of 3-hydroxybutyrate.
T h e present m e t h o d d e p e n d s on the oxidation of 3-hydroxybutyrate by 3-hydroxybutyrate dehydrogenase,
3 - H B D H ( D - 3 - H y d r o x y b u t y r a t e : N A D oxidoreductase, E C 1.1.1.30) a n d N A D ; a n u m b e r of modifica 1
tions to the original m e t h o d , including fluorimetric m e t h o d s , have been p u b l i s h e d 2 - 4

.
Principle
(1) D-(-)-3-Hydroxybutyrate 4 - N A D +
, 3 - H
^ L Acetoacetate + N A D H + H +
T h e increase in extinction at 340 (334 o r 365) n m d u e to the formation of N A D H is a measure of the

reaction.
T h e equilibrium constant of the reaction (1) is 1.45 x 1 0 - 9

at 25 ° C . A t p H 8.5 approximately 4 0 % of
5
the 3-hydroxybutyrate is oxidized to acetoacetate. In the presence of hydrazine to t r a p the acetoacetate

formed as the h y d r a z o n e , the reaction p r o c e e d s quantitatively from left to right.
D u e to the low activity of the 3-hydroxybutyrate dehydrogenase p r e p a r a t i o n s a n d to avoid the use of
excessive a m o u n t s of enzyme, it is preferable to carry o u t the m e a s u r e m e n t s between 25 °C a n d 30 °C.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at 3 4 0 ,
3 3 4 or 365 n m , b e n c h c e n t r i f u g e .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 8. N i c o t i n a m i d e - a d e n i n e dinucleotide,
2. H y d r o c h l o r i c a c i d , 1 N NAD
3. P e r c h l o r i c a c i d , A . R., s p . gr. 1 . 5 4 ; c a . free acid; commercial p r e p a r a t i o n , see p . 545.
6 0 % (w/v) 9. D L - 3 - H y d r o x y b u t y r i c a c i d
4. P o t a s s i u m h y d r o x i d e sodium salt, c o m m e r c i a l preparation, see p . 543.
5. U n i v e r s a l i n d i c a t o r * 10. 3 - H y d r o x y b u t y r a t e dehydrogenase,
6. H y d r a z i n e h y d r a t e ( 9 9 - 1 0 0 % ) HBDH
7. E t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d , E D T A from Rhodopseudomonas spheroides. Suspension
disodium salt, E D T A - N a H - 2 H 0 2 2 2 in 3.2 M a m m o n i u m sulphate solution; ^3
U / m g . (25 °C); commercial p r e p a r a t i o n , see
p . 475.
* Commercial p r e p a r a t i o n from British D r u g H o u s e s Ltd., Poole, England.

D - ( - )-3-Hydroxybutyrate 1837
Purity of Reagents
3 - H B D H should n o t contain m o r e t h a n 0.1 % L D H a n d 1 - 2 % M D H (relative to the 3 - H B D H activity).
P r e p a r e all s o l u t i o n s w i t h d i s t i l l e d o r d e i o n i z e d w a t e r .
I. Tris buffer (0.1 M ; p H 8 . 5 ) :

D i s s o l v e 2 . 4 2 g. tris in 100 m l . d i s t i l l e d w a t e r , a d d 6 m l . N H C 1 a n d m a k e u p t o 2 0 0 m l .
w i t h distilled w a t e r . C h e c k p H w i t h g l a s s e l e c t r o d e .
II. H y d r a z i n e - t r i s ( p H 8 . 5 ) :
M i x 1 ml. hydrazine hydrate, 20 mg. E D T A - N a H - 2 H 0 a n d 5 ml. N HC1, and dilute
2 2 2
t o 2 0 m l . w i t h tris buffer ( s o l u t i o n I). P r e p a r e freshly e a c h d a y .

III. P e r c h l o r i c a c i d (ca. 1 0 % w / v ) :
D i l u t e 11 m l . 6 0 % H C 1 0 4 to 100 ml. with distilled water.
I V . P o t a s s i u m h y d r o x i d e (ca. 2 0 % w / v ) :
D i s s o l v e 2 0 g. K O H in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
V . N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( c a . 14 m M / ? - N A D ) :
D i s s o l v e 2 0 m g . N A D in 2 m l . d i s t i l l e d w a t e r .
V I . 3 - H y d r o x y b u t y r a t e d e h y d r o g e n a s e , H B D H (5 m g . p r o t e i n / m l . ) :
U s e t h e s t o c k s u s p e n s i o n in 3.2 M a m m o n i u m s u l p h a t e d i r e c t l y .
Prepare the hydrazine-tris buffer (solution II) freshly each day. Store the N A D solution (V) at — 15 °C
and the tris buffer solution (I) at 0 - 4 °C. T h e enzyme suspension is stable for several m o n t h s at 0 - 4 °C.
Procedure
F o r d e t a i l s refer t o " A c e t o a c e t a t e " , p . 1 8 4 0 .
3 - H y d r o x y b u t y r a t e is s t a b l e in b l o o d a n d d e p r o t e i n i z e d s o l u t i o n s .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 3.1 m l . M e a s u r e a g a i n s t
a c u v e t t e c o n t a i n i n g 1.0 m l . h y d r a z i n e - t r i s buffer ( s o l u t i o n II) a n d 2 . 0 m l . distilled w a t e r .
Sample (deproteinized, neutralized) 2.00 ml. 1 0 - 1 0 0 fiM D-3-hydroxybutyrate

H y d r a z i n e - t r i s buffer (II) 1.00 m l .
N A D solution (V) 0.10 ml. ca. 0.4 m M NAD
M i x well and read extinction E . x
H B D H suspension (VI) 0.010 ml. c a . 15 pg. p r o t e i n / m l . =

ca. 50 m U / m l .
M i x a n d r e a d t h e e x t i n c t i o n at 4 0 , 5 0 a n d 6 0 m i n . ;
by extrapolation determine extinction E . 2
E 2 — E x = AE is u s e d for t h e c a l c u l a t i o n s .
T h e e x t i n c t i o n c h a n g e s o c c u r i n g o n a d d i t i o n o f 3 - H B D H s u s p e n s i o n ( V I ) are m e a s u r e d b y
addition o f 0.010 ml. e n z y m e t o a cuvette c o n t a i n i n g distilled water instead o f sample.
Calculations
U n d e r the conditions described here the oxidation of 3-hydroxybutyrate proceeds quantitatively. T h e

calculation formula (2) on p . 312 is used. T h e results are o b t a i n e d in /rniole 3-hydroxybutyrate/ml.
sample. This value m u s t be multiplied by t h e a p p r o p r i a t e factor if t h e sample h a s been deproteinized,
neutralized or diluted in any way. W i t h a dilution factor, F , the following relationships h o l d :

c = AE x 0.254 x F AE x 0.249 x F AE x 0.456 x F [/miole/ml.]
The percentage recovery of 3-hydroxybutyrate a d d e d to blood was 104 + 5.75 (S. D . ) . T h e coefficient of 1
variation is 5 % .
N o r m a l Values
T h e 3-hydroxybutyrate c o n t e n t of b l o o d of n o r m a l fasting subjects is 0 . 0 5 8 - 0 . 1 7 m M . F o r fed rats, 2
the m e a n values range from 0 . 0 8 0 - 0 . 0 8 2 m M ' ; for starved (48 hr.) rats, from 1.82-2.10 m M ' . F o r
3 6 3 6
the 3-hydroxybutyrate content of rat liver refer t o a n d p . 2292. 7
Interference in the assay: C o n t a m i n a t i o n of the 3 - H B D H suspension with excessive a m o u n t s of L D H o r

M D H leads to high values. A p r o c e d u r e for overcoming this source of error has been d e s c r i b e d . 2
D - ( - )-3-Hydroxybutyrate 1839
Specificity
3-Hydroxybutyrate d e h y d r o g e n a s e is n o t completely specific for 3-hydroxybutyrate; the higher h o m o -

logues, 3-hydroxypentanoic a n d 3-hydroxyhexanoic acids also react b u t at m u c h slower r a t e s . 8
Other Methods
A n a u t o m a t e d enzymatic colorimetric m e t h o d for the d e t e r m i n a t i o n of 3-hydroxybutyrate using 3-

h y d r o x y b u t y r a t e dehydrogenase a n d 3-p-nitrophenyl-2-p-iodophenyl-5-phenyltetrazolium chloride ( I N T )
has been described . 9
References
1 D. H. Williamson, J. Mellanby & H. A. Krebs, Biochem. J. 82, 90 [1962].

2 H. U. Bergmeyer & E. Bernt, E n z y m . biol. clin. 5, 65 [1965].
3 D. A. B. Young & A. E. Renold, Clin. C h i m . A c t a 13, 791 [1966].
4 G. Stein & K. H. Bdssler, Z . Klin. C h e m . u n d Klin. Biochem. 6, 27 [1968].
5 H. A. Krebs, J. Mellanby & D. H. Williamson, Biochem. J. 82, 96 [1962].
6 M. N. Berry, D. H. Williamson & M. B. Wilson, Biochem. J. 94, 17 C [1965].
7 Z). H. Williamson, P. Lund & H. A. Krebs, Biochem. J. 103, 514 [1967].
8 H. U. Bergmeyer, K Gawehn, H. Klotzsch, H. A. Krebs & D. H. Williamson, Biochem. J. 102,423 [1967].
9 J. A. Zivin & /. F. Snarr, Analyt. Biochem. 52, 456 [1973].
Acetoacetate
Jane M e l l a n b y and D e r m o t H. Williamson
In 1937, Green et a l . described an insoluble enzyme from pig heart muscle which catalysed the reversible
1
oxidation of D-( —)-3-hydroxybutyrate. T h e discovery that cell-free extracts of certain bacteria which
accumulate poly-3-hydroxybutyrate contain a soluble a n d stable D-( — )-3-hydroxybutyrate dehydrogenase,
3 - H B D H ( D - 3 - H y d r o x y b u t y r a t e : N A D oxidoreductase, E C 1 . 1 . 1 . 3 0 ) " has led to the purification and
2 5
crystallization of this e n z y m e a n d its use as an analytical r e a g e n t '

6 4 7 - 9
.
Principle
(1) D-(-)-3-Hydroxybutyrate + NAD " 4

(
3
~ H B P H
» Acetoacetate + N A D H + H +
T h e decrease in extinction at 340 (334, 365) n m d u e t o the oxidation of N A D H is p r o p o r t i o n a l to the

a m o u n t of acetoacetate present.
T h e equilibrium constant of e q u a t i o n (1),K is 1.45 x 1 0 - 9

at 25 ° C 1 0
a n d 4.93 x 1 0 - 9
at 37 ° C . A t
n
p H 7.0 a n d with a suitable excess of N A D H , at least 9 8 % of the acetoacetate is reduced to 3-hydroxy

butyrate. D u e to the low activity of the 3-hydroxybutyrate dehydrogenase p r e p a r a t i o n s a n d to avoid
the use of excessive a m o u n t s of enzyme, it is preferable to carry o u t m e a s u r e m e n t s between 25 °C a n d
30 °C.
Equipment
3 3 4 or 365 n m ; b e n c h c e n t r i f u g e .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , lithium s a l t ; determine purity enzymatically

1 2
K H P 0 , A . R.
2 4 and manometrically . 12
2. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , 7. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo
K H P 0 , A . R.
2 4 tide, N A D H
3. P e r c h l o r i c a c i d , A . R., s p . gr. 1 . 5 4 ; 6 0 % disodium salt, N A D H - N a ; commercial prep 2
(w/w) a r a t i o n , see p . 545.

4. P o t a s s i u m h y d r o x i d e 8. 3 - H y d r o x y b u t y r a t e dehydrogenase,
5. U n i v e r s a l i n d i c a t o r * HBDH
6. A c e t o a c e t i c a c i d , l i t h i u m salt from Rhodopseudomonas spheroides. Suspension
hydrolyse freshly distilled ethyl acetoacetate in 3.2 M a m m o n i u m sulphate solution ^3
with lithium hydroxide and isolate the crystalline U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , see
p. 475.
* Commercial p r e p a r a t i o n from British D r u g H o u s e s Ltd., Poole, England.

Acetoacetate 1841
Purity of Reagents
H B D H should not be contaminated with more than 0.1 % L D H or 1 - 2 % M D H (relative to the H B D H

activity); less pure preparations may be used if the modifications to the method described under "Sources
of Error" are carried out.
P r e p a r e all s o l u t i o n s w i t h d o u b l y d i s t i l l e d o r d e i o n i z e d w a t e r .
a) D i s s o l v e 1 3 . 6 g. K H P 0 2 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r .
b) D i s s o l v e 1 7 . 4 g. K H P 02 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r .
M i x e q u a l v o l u m e s o f s o l u t i o n s a) a n d b ) . C h e c k t h e p H o f t h e m i x t u r e w i t h a g l a s s
electrode.
II. P e r c h l o r i c a c i d (ca. 1 0 % w / v ) :
D i l u t e 11 m l . 6 0 % p e r c h l o r i c a c i d t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r .
III. P o t a s s i u m h y d r o x i d e (ca. 2 0 % w / v ) :
D i s s o l v e 2 0 g. K O H i n d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
IV. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 6 m M / ? - N A D H ) :
V . 3 - H y d r o x y b u t y r a t e d e h y d r o g e n a s e , 3 - H B D H (5 m g . p r o t e i n / m l . ) :
U s e t h e s t o c k s u s p e n s i o n in 2 . 4 M a m m o n i u m s u l p h a t e s o l u t i o n .
The N A D H solution is stored at —15 °C. The enzyme suspension is stable for months at 0 °C to 4 °C.
Procedure
Collection :
Collect v e n o u s b l o o d without venestasis and deproteinize immediately to minimize the n o n -

e n z y m i c d e c a r b o x y l a t i o n o f a c e t o a c e t a t e t o a c e t o n e ' . If b l o o d m u s t b e t r a n s p o r t e d , c o l l e c t
4 1 4
in a h e p a r i n i z e d t u b e a n d c o o l in ice. T i s s u e s f r o m e x p e r i m e n t a l a n i m a l s s h o u l d b e o b t a i n e d
w i t h freeze c l a m p s (refer t o p . 4 0 0 ) .
Deproteinization :
W h o l e b l o o d : P i p e t t e i n t o a c e n t r i f u g e t u b e 5 m l . i c e - c o l d p e r c h l o r i c a c i d s o l u t i o n (II) a n d
5 m l . w h o l e b l o o d . M i x t h o r o u g h l y ( p r e f e r a b l y w i t h a V o r t e x m i x e r ) a n d t h e n c e n t r i f u g e at
3 0 0 0 g for 15 m i n . P o u r off s u p e r n a t a n t fluid i n t o a g r a d u a t e d , c o n i c a l c e n t r i f u g e t u b e a n d
m e a s u r e t h e v o l u m e . M i x i n t o t h e s u p e r n a t a n t fluid 0 . 0 0 5 m l . U n i v e r s a l i n d i c a t o r a n d s l o w l y
a d d K O H s o l u t i o n (III) u n t i l t h e c o l o u r c h a n g e s f r o m red t o g r e e n o r b l u e - g r e e n ( p H 7 - 8 ) .
A l l o w t o s t a n d for a p p r o x i m a t e l y 3 0 m i n . in a n ice b a t h a n d t h e n c e n t r i f u g e at 3 0 0 0 g for
10 m i n . N o t e v o l u m e , d e c a n t off s u p e r n a t a n t fluid a n d u s e for t h e d e t e r m i n a t i o n o f a c e t o a c e t a t e .

S e r u m : Treat a s for w h o l e b l o o d .
T i s s u e : F o r a full d e s c r i p t i o n o f t r e a t m e n t refer t o 1 1
.
A c e t o a c e t a t e is s l o w l y d e c a r b o x y l a t e d b y p r o t e i n - c o n t a i n i n g s o l u t i o n s . I n c u b a t i o n (at 37 ° C
1 4
for 1 hr.) o f w h o l e b l o o d c o n t a i n i n g 1 m M a c e t o a c e t a t e r e s u l t e d in a 4 0 % l o s s o f t h e o x o
a c i d . A c e t o a c e t a t e is r e l a t i v e l y s t a b l e in d e p r o t e i n i z e d , n e u t r a l s o l u t i o n s at 0 t o 4 ° C .
Assay System

a t u r e ; r e a d a g a i n s t a c u v e t t e c o n t a i n i n g 1.0 m l . p h o s p h a t e buffer s o l u t i o n ( I ) + 2.1 m l . d i s t i l l e d
water.
Sample 2.0 ml. 1 0 - 1 0 0 pM acetoacetate

P h o s p h a t e buffer (I) 1.0 m l . 0.033 M
N A D H solution (IV) 0.1 m l . 0.2 m M
M i x well and read the extinction E . t
H B D H suspension (V) 0.010 ml. ca. 15 pg. p r o t e i n / m l . =

ca. 5 0 m U / m l .
R e a d t h e e x t i n c t i o n at 10, 15 a n d 2 0 m i n . D e t e r m i n e
extinction E 2 by extrapolation from these values.
E l — E 2 = A E is u s e d for t h e c a l c u l a t i o n s .
A c u v e t t e c o n t a i n i n g d i s t i l l e d w a t e r i n s t e a d o f s a m p l e is u s e d t o m e a s u r e the c h a n g e in e x
t i n c t i o n o n a d d i t i o n o f 0.01 m l . H B D H s u s p e n s i o n ( V ) .
Calculations
U n d e r the assay conditions the reaction proceeds stoichiometrically. T h e calculation formula (2) on p . 312
applies. The results are obtained in pmole acetoacetate/ml. sample.
U n d e r the above conditions the following relationships hold.
c - AE x 0.254 AE x 0.249 AE x 0.456 [//mole/ml.]
The percentage recovery of acetoacetate ( 0 . 1 7 - 1 . 0 m M ) added to blood was 99 ± 6.25% ( S . D . ) . T h e 4
coefficient of variation is 6 % .
Acetoacetate 1843
N o r m a l Values
T h e acetoacetate content of whole b l o o d of n o r m a l fasting subjects is 0 . 0 1 8 - 0 . 0 7 8 m M . Values for fed

7
rats range from 0.076 to 0.2 m M ; for starved (48 hr.) rats from 0.31 to 0.74 m M 8 , 9 , 1 5
. F o r the acetoacetate
content of rat liver refer t o 1 1
a n d p . 2291.
Interference in the assay: C o n t a m i n a t i o n of the 3 - H B D H suspension (V) by L D H o r M D H can lead to

falsely high values for acetoacetate if pyruvate or oxaloacetate are present in the sample. Interference
from this source can be overcome by the addition of L D H a n d M D H to the cuvettes before reading
extinction E . x
7
3-Hydroxybutyrate dehydrogenase is n o t absolutely specific for a c e t o a c e t a t e ; the higher h o m o l o g u e s

3-oxopentanoic a n d 3-oxohexanoic acids are reduced, b u t at considerably slower r a t e s . 6
Other Methods
A new enzymatic m e t h o d for the d e t e r m i n a t i o n of acetoacetate has recently been d e s c r i b e d . It involves

16
the use of pigeon liver extract containing acetoacetyl-CoA synthetase, 3-oxoacyl-CoA thiolase a n d
arylamine acetylase. T h e decrease in extinction at 405 n m d u e to the acet>lation of p-nitroaniline is
measured.
References
1 D. E. Green, J. G. Dewan & L. F. LeLoir, Biochem. J. 31, 934 [1937].

2 R. Gavard, D. Combre & A. Tujfet, C. R. hebd. Seances A c a d Sci. 257, 1931 [I960].
3 M. Doudoroff, J. M. Merrick & R. Contopoulou, Feder. P r o c . 20, 272 [1961].
4 D. H. Williamson, J. Mellanby & H. A. Krebs, Biochem. J. 82, 90 [1962].
5 J. Schindler & H. G. Schlegel, Biochem. Z. 339, 154 [1963].
6 H. U. Bergmeyer, K. Gawehn, H. Klotzsch, H. A. Krebs & D. H. Williamson, Biochem. J. 102, 423
[1967].
7 H. U. Bergmeyer & E. Bernt, Enzym. biol. clin. 5, 65 [1965].
9 G. Stein & K. H. Bdssler, Z. Klin. C h e m . u n d Klin. Biochem. 6, 27 [1968].
10 H. A. Krebs, J. Mellanby & D. H. Williamson, Biochem. J. 82, 96 [1962].
11 D.H. Williamson, P. Lund & H. A. Krebs, Biochem. J. 103, 514 [1967].
12 L. M. Hall, Analyt. Biochem. 3, 75 [1962].
13 N. L. Edson, Biochem. J. 29, 2082 [1935].
14 Di A. Rossi, Arch. Sci. biol. (Bologna) 24, 73 [1938].
15 M.N. Berry, D. H. Williamson & M. B. Wilson, Biochem. J. 94, 17C [1965].
16 I. Alkonyi, J. Kerner &. D. Szabo, A c t a Biochim. Biophys. Acad. Sci. H u n g . 7, 143 [1972].
Triacetate and Fumarylacetoacetate
D a v i d J. H . B r o c k a n d D e r m o t H . W i l l i a m s o n
Triacetate (3,5-dioxohexanoate, C H 3 • CO • C H 2 • CO • C H 2 • C O O H ) has been identified as the p r o d u c t

formed by a purified pigeon liver fatty acid synthetase from acetyl-CoA a n d m a l o n y l - C o A in the absence
of N A D P H . Triacetic acid lactone
1
OH
HC'^CH
H C-C^C=0
3
was shown to be the major p r o d u c t formed with a fatty acid synthetase from E. coli . T h e occurrence of 2
these c o m p o u n d s in biological s y s t e m s " 3 5

suggested the need for a rapid a n d simple m e t h o d for their
determination. T h e enzymic m e t h o d described here is based on the cleavage of triacetate to acetoacetate
and acetate by a diketo acid hydrolase (4-fumarylacetoacetate fumarylhydrolase, E C 3.7.1.2) from rat liver 6
The main disadvantage is that the enzyme also reacts rapidly with fumarylacetoacetate, C O O H • C H =
CH • CO • C H 2 • CO • C H 2 • C O O H (an intermediate in tyrosine catabolism) to give acetoacetate a n d
fumarate
Principle
(1) Triacetate <

! ;
ik to
1
acid
) Acetoacetate + Acetate
hydrolase
(2) F u m a r y l a c e t o a c e t a t e ^ ^ 1 ^ Acetoacetate + Fumarate
(3) Acetoacetate + N A D H + H +
, ~ 3 H B D H
* i D-3-Hydroxybutyrate + N A D +
The decrease in extinction at 340 (334 or 365) n m due to the formation of N A D is a measure of the a m o u n t
of triacetate a n d fumarylacetoacetate present in the sample.
The o p t i m u m p H of the diketo acid hydrolase is between p H 6 a n d 7. T h e reduction of acetoacetate with

excess N A D H is quantitative at p H 6 . 8 - 7 . 0 . If a mixture of triacetate a n d acetoacetate is to be analysed
(see p . 1846), it is preferable to t a k e less t h a n 0.10 jumole of triacetate for the d e t e r m i n a t i o n , because
H B D H catalyses the reduction of triacetate at a slow rate ( K M 6.7 m M ) .
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at 3 4 0
(334 or 365 ) n m .
* 3-Hydroxybutyrate d e h y d r o g e n a s e ( D - 3 - H y d r o x y b u t y r a t e : N A D oxidoreductase, E C 1.1.1.30).

Triacetate a n d F u m a r y l a c e t o a c e t a t e 1845
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 5. 3 - H y d r o x y b u t y r a t e d e h y d r o g e n a s e ,
K H P 0 , A . R.
2 4 HBDH
2. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , from Rhodopseudomonas spheroides; suspension
K H P 0 , A . R.
2 4 in 3.2 M a m m o n i u m sulphate solution, ca. 3
3. R e d u c e d nicotinamide-adenine dinucle U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , see
otide, N A D H p . 475.
disodium salt, N A D H - N a ; commercial p r e p
2 6. D i k e t o a c i d h y d r o l a s e
aration, see p. 545. preparation from rat liver , ca. 0.5
6
U/mg.
4 . Triacetic a c i d (25 ° C ; assay with triacetic acid as substrate).
s o d i u m salt, p r e p a r e d according t o and concen 7
F o r isolation, see A p p e n d i x p . 1847.
tration determined enzymatically.
Purity of Reagents
T h e diketo acid hydrolase should be free of triacetic acid lactonase.
P r e p a r e all s o l u t i o n s w i t h d o u b l e d i s t i l l e d w a t e r o r d e i o n i z e d w a t e r .
a) D i s s o l v e 13.6 g. K H P 0 2 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r .
b) D i s s o l v e 1 7 . 4 g. K H P 0 2 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r .
M i x e q u a l v o l u m e s o f s o l u t i o n s a) a n d b ) .
Check the p H o f the mixture with a glass electrode.
II. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( c a . 6 m M j S - N A D H ) :
III. 3 - H y d r o x y b u t y r a t e d e h y d r o g e n a s e , H B D H (5 m g . p r o t e i n / m l . ) :
U s e t h e s t o c k s u s p e n s i o n in 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
I V . D i k e t o a c i d h y d r o l a s e (5 m g . p r o t e i n / m l . ) :
U s e t h e purified p r e p a r a t i o n ( p . 1 8 4 7 ) u n d i l u t e d .
Store the diketo acid hydrolase p r e p a r a t i o n a n d N A D H solution at —15 ° C ; they are stable for several
weeks. T h e H B D H suspension is stable for m o n t h s at 0 - 4 °C.
Procedure
T h e m e t h o d has only been tested o n pure solutions o f triacetate or mixtures o f k n o w n c o m p o s i

t i o n c o n t a i n i n g t r i a c e t a t e . Triacetate is r e l a t i v e l y s t a b l e at 0 - 4 ° C .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 0 7 m l . M e a s u r e
against a cuvette containing water.
Sample 1.0 m l . 5 - 5 0 ^ M triacetate

P h o s p h a t e buffer s o l u t i o n (I) 1.0 m l . 0.05 M
N A D H solution (II) 0.05 ml. 0.15 m M
M i x well a n d read extinction E. t M i x in
H B D H suspension* (III) 0.010 ml. c a . 2 5 fig. p r o t e i n / m l .

ca. 75 m U / m l .
D i k e t o acid hydrolase solution (IV) 0.010 ml. c a . 2 5 fig. p r o t e i n / m l .
c a . 10 m U / m l .
E J - E 2 = A E is u s e d for t h e c a l c u l a t i o n s .
A c u v e t t e c o n t a i n i n g distilled w a t e r i n s t e a d o f s a m p l e is u s e d t o m e a s u r e t h e c h a n g e in e x t i n c t i o n
o n a d d i t i o n o f t h e e n z y m e . T h e p r o c e d u r e for t h e a s s a y o f f u m a r y l a c e t o a c e t a t e is carried o u t in
t h e s a m e w a y e x c e p t t h a t t h e m e a s u r e m e n t s are m a d e at 365 n m .
Calculations
U n d e r the assay conditions the hydrolysis of triacetate to acetoacetate, a n d the reduction of the latter, is
is quantitative with the stoichiometric formation of an equivalent a m o u n t of N A D . T h e calculation formula
(2) on p . 312 applies in this case.

c - AE x 0.339 AE x 0.333 zlE x 0.609 [^mole/ml.]
Fumarylacetoacetate absorbs strongly in the ultraviolet, a n d to minimize its c o n t r i b u t i o n to the extinction

changes the m e a s u r e m e n t s are m a d e at 365 n m . and a m o l a r extinction coefficient of 5.28 c m . / ^ m o l e for 2
N A D H plus fumarylacetoacetate is used for the c a l c u l a t i o n . 6
c - AE x 0.392 [/miole/ml.].
The accuracy of the m e t h o d is 5 % .
Normal Values
None known.
* If the sample contains acetoacetate, wait for the end of the reaction after addition of H B D H and then
a d d diketo acid hydrolase.
Triacetate a n d F u m a r y l a c e t o a c e t a t e 1847
Acetoacetate will react quantitatively in the assay. To avoid this source of e r r o r t h e H B D H can be a d d e d
first, followed by diketo acid hydrolase when the acetoacetate initially present has reacted. C o n t a m i n a t i o n
of the diketo acid hydrolase p r e p a r a t i o n with other N A D - l i n k e d d e h y d r o g e n a s e s m a y lead to errors with
samples of u n k n o w n c o m p o s i t i o n .
As far as is k n o w n the hydrolase only reacts with triacetate and fumarylacetoacetate t o yield acetoacetate.
Appendix
Isolation of Diketo Acid Hydrolase 6
T h e p r o c e d u r e includes the following steps:

Extraction from rat liver with 0.25 M sucrose. - F r a c t i o n a t i o n of extract with a m m o n i u m sulphate
between 40 a n d 75 %. - F r a c t i o n a t i o n of a m m o n i u m sulphate precipitate with e t h a n o l between 50 a n d
7 5 % (v/v). - C h r o m a t o g r a p h y o n D E A E - S e p h a d e x . C h r o m a t o g r a p h y o n C M - S e p h a d e x .
References
1 J. D. Brodie, G. Wasson & J. W. Porter, J. biol. C h e m . 239, 1 346 [1964].

2 D. J. H. Brock & K. Block, Biochem. biophys. Res. C o m m u n . 13, 775 [1966].
3 T. M . Harris, C. M. Harris & R. J. Light, Biochim. biophys. Acta, 121, 420 [1966].
4 R. J. Light, T. M. Harris & C. M. Harris, Biochemistry, 5, 4 0 3 7 [1966].
5 7. E. Nixon, G. R. Putz & /. W. Porter, J. biol. C h e m . 243, 5471 [1968].
6 D. J. H. Brock & D. H. Williamson, Biochem. J. 110, 677 [1968].
7 R. F. Witter & E. Stotz, J. biol. C h e m . 176, 501 [1948].
Hydrolysis of Steroid Conjugates
Klaus-Dieter Voigt and H e l m u t h Schmidt
The steroid h o r m o n e s a n d their metabolites a p p e a r in the urine a l m o s t exclusively as water-soluble

conjugates, a n d are also found to some extent in this form in blood. C o m p o u n d s having a 3 /Miydroxyl
g r o u p are excreted mainly as sulphates, while 3 a-hydroxysteroids are excreted as /?-glycosidically linked
g l u c u r o n i d e s ' ; however, there are exceptions. Steroid conjugates with other acids p r o b a b l y also
1 2
exist .3,4
In o r d e r to determine steroid conjugates they m u s t first be converted to the free alcohols. In the course
of the constant refinement of m e t h o d s for steroid analysis, enzymatic hydrolysis, particularly of steroid
glucuronides, has become very i m p o r t a n t because of its mildness a n d its specificity; it is n o w a d a y s essential
for the accurate d e t e r m i n a t i o n of some labile steroids.
Application of Method: In biochemistry, clinical chemistry, a n d food chemistry.
Principle
(1) Conjugate + H 0 ^± Steroid alcohol + Acid

2
Steroid hydrolases (^-glucuronidase, sulphatase) catalyse the hydrolysis of the steroid conjugate to the
steroid alcohol a n d the free acid.
U n d e r the conditions described here the equilibrium of the reaction lies far to the right. In the case of
biological material, the hydrolysis is followed by further steps for the extraction a n d purification of the
steroid, which is then determined. W i t h p u r e solutions a n d extracts, the acid liberated can also be esti
mated.
Hydrolysis of Steroid Glucuronides

Steroid glucuronides can be hydrolysed with /^-glucuronidase (jft-D-Glucuronide glucuronosohydrolase,
E C 3.2.1.31), which was discovered by Cohen a n d Marrian 5
a n d by Patterson . 6
Three well-proven m e t h o d s a r e described below. These a r e used (1) mainly for the hydrolysis of corti
costeroid a n d oestrogen glucuronides in urine, (2) mainly for the hydrolysis of C 1 9 and C 2 1 steroid glucuro
nides in plasma, a n d (3) especially for the hydrolysis of 17-ketosteroid a n d testosterone glucuronides
in urine.
The following points are of vital i m p o r t a n c e :

1. T h e activity* of the enzyme is generally determined with p h e n o l p h t h a l e i n glucuronide as the s u b s t r a t e . 7
This activity is n o t identical with the activity for the esters of the h o r m o n e steroids t h a t are of interest
here. T h e hydrolase activity for these esters d e p e n d s on the source of the enzyme. ^ - G l u c u r o n i d a s e of
bacterial origin hydrolyses the esters of steroid h o r m o n e s m u c h m o r e rapidly t h a n enzyme p r e p a r a t i o n s
from m a m m a l i a n liver or from m o l l u s c s . T h u s practically quantitative hydrolysis of the steroid glucu
8
ronides in urine is achieved in 24 hr. at 37 °C by incubation with 5 - 1 0 m U of a bacterial p r e p a r a t i o n per
* The activity was formerly expressed in Fishman units. O n e Fishman unit is the enzyme activity t h a t
liberates 1 /ig. of phenolphthalein from phenolphthalein glucuronide in 1 hr. at 37 °C. 20150 Fishman
units correspond to 1 U (37 °C) according to I U B ; these " u n i t s " are n o w used to the exclusion of all
others.
Hydrolysis of Steroid Conjugates 1849
ml. of mixture. O n the o t h e r h a n d , 2 0 - 5 0 m U of a p r e p a r a t i o n from m a m m a l i a n liver or ca. 35 m U

of the enzyme from molluscs is required to achieve the same effect, with i n c u b a t i o n times of 2 - 3 days.
2. T h e o p t i m u m p H also d e p e n d s o n the source of the enzyme. It is a b o u t p H 4.6 for p r e p a r a t i o n s from
bovine liver a n d a b o u t p H 5.2 for those from molluscs, while bacterial ^ - g l u c u r o n i d a s e p r e p a r a t i o n s
exhibit o p t i m u m hydrolysis in the neutral region ( p H 6 . 2 - 7 . 0 ) . T h e reaction is usually carried out at
37 °C.
3. T h e rate of hydrolysis a n d hence the enzyme activity required in the mixture d e p e n d on the structure
of the steroid. T h e glucuronides of pregnanediol a n d related c o m p o u n d s (and of pregnanetriol) are
hydrolysed m o s t rapidly, a n d these are followed by the phenolic steroids (oestrogens), corticosteroids,
and 17-ketosteroids . W i t h a ^-glucuronidase from Patella vulgata, the rate of hydrolysis of glucuronides
8
was found to decrease in the order phenolic, 3 /?-, 17 /?- and 3 a-steroids . 9
A n excess of enzyme is normally used in practice in order to o b t a i n suitable hydrolysis times and because
of the frequent presence of enzyme inhibitors.
Pregnanediol glucuronide is a l m o s t completely hydrolysed in only 6 - 8 hr. at 37 °C by 2 . 5 - 5 m U of
bacterial enzyme or 2 5 - 3 0 m U of the enzyme from Helix pomatia (per ml. of m i x t u r e ) . 8
Phenolic steroid glucuronides can be quantitatively hydrolysed with 5 - 1 0 m U of a bacterial enzyme

(24 hr., p H 6.5 to 7 ) ' , with 37 to 50 m U of /^-glucuronidase from molluscs (24 hr., p H 5 . 2 ) , or with
1 0 1 1 12
50 m U of m a m m a l i a n liver enzyme (72 hr., p H 5 . 0 ) 13

(enzyme activities per ml. of mixture at 37 °C).
T h e corticosteroid glucuronides c a n be reliably hydrolysed in 48 hr. with 10 m U of a bacterial ^-glucuroni
dase (per ml. of m i x t u r e ) , the enzyme being present in excess. Ten to fifteen times this c o n c e n t r a t i o n is
14
necessary in the case of enzymes from other sources.

F o r complete hydrolysis of 17-ketosteroid glucuronides in 48 hr., 7.5 m U of bacterial e n z y m e 1 5 1 6
, 50 to
100 m U of ^-glucuronidase from m o l l u s c s 1 6 1 7
, or 100 m U of the bovine liver p r e p a r a t i o n 16
are used
per ml. of mixture, large excesses of enzyme being present in some cases. C o m p a r a b l e enzyme c o n c e n t r a
tions are also required for the hydrolysis of testosterone glucuronide in u r i n e 1 8 - 2 0
.
Equipment
L a b o r a t o r y c e n t r i f u g e , i n c u b a t o r , w a t e r b a t h , a n d in s o m e c a s e s a s t e a m b a t h .
1. H y d r o l y s i s o f G l u c u r o n i d e s o f C o r t i c o s t e r o i d s and P h e n o l i c S t e r o i d s in U r i n e
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 4. ^-Glucuronidase
2. Acetic acid, c o n e . Serva, Heidelberg (Item N o . 22850): dry p o w
3. C h l o r o f o r m der from E. coli, ca. 2 m U / m g . (37 °C). Sigma
Chemicals C o m p a n y , St. L o u i s : dry p o w d e r
from E. coli, 1250 m U bottles; ca. 0.6 m U / m g .
(37 °C). Activities determined with phenol
phthalein g l u c u r o n i d e as the substrate.
F o r a specific determination of steroid glucuronides, the p r e p a r a t i o n should contain not m o r e t h a n

0.01 % of sulphatase a n d p h o s p h a t a s e impurities (based on the specific activity of the /^-glucuronidase).
In determinations of the total steroid conjugates, such impurities are of n o i m p o r t a n c e .
Sterilize c o n t a i n e r s t o p r e v e n t t h e g r o w t h o f m i c r o - o r g a n i s m s .
I. Tris buffer ( 0 . 3 M ; p H 6 . 5 ) :
D i s s o l v e 3 6 . 3 g. tris in 100 m l . d o u b l y d i s t i l l e d w a t e r a n d a d j u s t t o p H 6.5 b y d r o p w i s e
addition of cone, acetic acid. M a k e u p to 1 0 0 0 ml. with d o u b l y distilled water.
II. ^ - G l u c u r o n i d a s e (ca. 1 2 m U / m l . ) :
D i s s o l v e c o m m e r c i a l p r e p a r a t i o n s as r e q u i r e d in tris buffer ( s o l u t i o n I).
Keep containers closed at 0 to + 4 ° C . T h e dilute enzyme solution keeps for 3 - 4 weeks, while the buffer
solution is stable virtually indefinitely.
Procedure
N o d r u g s t o b e g i v e n for 3 d a y s b e f o r e a n d d u r i n g t h e u r i n e c o l l e c t i o n p e r i o d . U s e a fresh
s a m p l e o f a 2 4 hr. u r i n e ( n o a d d i t i v e s ) . B o i l t h e u r i n e for a s h o r t t i m e t o d e s t r o y s o m e o f t h e
e n z y m e i n h i b i t o r s . If s t e r o i d g l u c u r o n i d e s a n d free s t e r o i d s are t o b e d e t e r m i n e d s e p a r a t e l y , t h e
latter m u s t b e e x t r a c t e d t w i c e w i t h 2 5 m l . o f c h l o r o f o r m .
Enzymatic System
I n c u b a t i o n t e m p e r a t u r e : 37 ° C ; v o l u m e : 2 0 m l .
Pipette into 50 ml. centrifuge tubes with stoppers:
S a m p l e (urine) 4 ml.
^ - G l u c u r o n i d a s e s o l u t i o n (II) 16 m l .
I n c u b a t e for 4 8 hr. in a n i n c u b a t o r . E x t r a c t in t h e
centrifuge tubes with t w o 25 ml. portions o f c h l o r o
form to r e m o v e the liberated steroid a l c o h o l s ; suck
the c h l o r o f o r m out with a 50 ml. glass syringe having
a l o n g b l u n t steel n e e d l e . U s e c o m b i n e d c h l o r o f o r m
p h a s e s for further p u r i f i c a t i o n a n d d e t e r m i n a t i o n
o f the steroids.
Interference due to drugs and other therapeutic measures: G l u c u r o n i d e s of certain drugs (e.g. acetyl-
salicylic acid) act as competitively inhibiting s u b s t r a t e s . D r u g s excreted in the urine can also interfere
21
with the steroid determination after hydrolysis t h r o u g h the formation of foreign c h r o m o g e n s .
Interference in assay technique: If free a n d conjugated steroids are to be determined separately, the urine
must be worked u p immediately, since s p o n t a n e o u s hydrolysis by e n d o g e n o u s o r bacterial glucuronidases
may otherwise occur. A d d i t i o n of E D T A to the urine inhibits the bacterial /^-glucuronidase a n d m a y
thus cause incomplete hydrolysis of the s t e r o i d s . T h e bacterial enzyme is also inhibited in alcohol-ether
22
e x t r a c t s . T h e /^-glucuronidase inhibitors present in biological material (thiol-blocking agents?) c a n n o t

16
be completely eliminated w i t h o u t a great deal of t r o u b l e ; an a d e q u a t e excess of enzyme should therefore

be always used.
Steroid glucuronides are specifically determined if the enzyme p r e p a r a t i o n has the specific purity. Free
steroids must naturally be extracted beforehand if it is particularly desired to determine the ^-glucuronides.
T h o u g h the activity of the /^-glucuronidase t o w a r d s various h o r m o n e steroids varies considerably (refer
to " O p t i m u m C o n d i t i o n s for M e a s u r e m e n t s " ) , specific hydrolysis of the glucuronides of definite g r o u p s
of steroids or even of individual steroids c a n n o t be achieved.
2 . H y d r o l y s i s o f G l u c u r o n i d e s o: C 1 9 and C 2 1 S t e r o i d s in P l a s m a
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 5. Shell s p e c i a l p e t r o l o r C e l l o s o l v e B ( b . p .
2. A c e t i c a c i d , c o n e . 6 8 - 7 3 °C)
3. E t h e r , p e r o x i d e free. 6. S o d i u m h y d r o x i d e s o l u t i o n , A . R . , 0.1 N
4. E t h a n o l , 9 6 % , d e n a t u r e d w i t h 2 . 5 % ( v / v ) 7. /^-Glucuronidase
methanol see p. 460.
Purity o f t h e e n z y m e p r e p a r a t i o n a n d stability f s o l u t i o n s a s in m e t h o d 1, p. 1 8 4 9 , 1 8 5 0 .
Procedure
Take 6 0 - 8 0 m l . * b l o o d f r o m a c o m p r e s s e d b r a c h i a l v e i n . Wet t h e w a l l s o f t h e s y r i n g e a n d o f
the c o l l e c t i n g v e s s e l w i t h 0.5 m l . h e p a r i n s o l u t i o n ( L i q u e m i n " R o c h e " , 5 0 0 0 u n i t s / m l . ) . C e n t r i
f u g e off t h e c o r p u s c u l a r c o m p o n e n t s w i t h i n 3 0 m i n . o f c o l l e c t i o n o f s a m p l e .
Extraction and deproteinization:
F o r t h e s e p a r a t e d e t e r m i n a t i o n o f c o n j u g a t e d a n d free s t e r o i d s , t h e latter m u s t b e e x t r a c t e d a n d
the p l a s m a t h e n d e p r o t e i n i z e d . T h e p r o c e d u r e is t h e s a m e for t h e d e t e r m i n a t i o n o f C 1 9 and C 2 1
s t e r o i d s a n d for g l u c u r o n i d e s a n d s u l p h a t e s .
R e m o v e free s t e r o i d s b y e x t r a c t i n g t w i c e w i t h t h r e e v o l u m e s o f c h l o r o f o r m . If c o m p l e t e
e x t r a c t i o n is r e q u i r e d , u s e t w o further e x t r a c t i o n s w i t h three v o l u m e s o f e t h y l a c e t a t e for C 2 1
s t e r o i d s a n d t w o e x t r a c t i o n s w i t h t h r e e v o l u m e s o f b e n z e n e for C 1 9 steroids. The aqueous

p h a s e c o n t a i n s t h e s t e r o i d c o n j u g a t e s . T o d e p r o t e i n i z e , a d d five v o l u m e s o f 9 6 % e t h a n o l a n d
h e a t t o 4 0 ° C o n a w a t e r b a t h w i t h s h a k i n g . A l l o w t o s t a n d for 2 4 hr. at 4 ° C a n d filter t h e
s u p e r n a t a n t fluid i n t o a r o u n d - b o t t o m flask. W a s h t h e p r e c i p i t a t e w i t h t w o 5 0 m l . p o r t i o n s o f
* Considerably smaller volumes of blood a n d , in consequence, of extraction a n d deproteinization reagents

are necessary when m o d e r n steroid d e t e r m i n a t i o n m e t h o d s with high e n d - p o i n t sensitivity are used.
9 6 % e t h a n o l a n d c e n t r i f u g e . C o m b i n e t h e a l c o h o l i c e x t r a c t a n d distill off t h e e t h a n o l u n d e r
v a c u u m . E x t r a c t t h e a q u e o u s r e s i d u e w i t h t w o 100 m l . p o r t i o n s o f e t h e r t o r e m o v e l i p i d s .
D i s c a r d t h e ether a n d h e a t t h e a q u e o u s l a y e r o n a w a t e r b a t h ( n o t a b o v e 50 °C) t o r e m o v e
d i s s o l v e d ether. A d d e t h a n o l t o g i v e a final e t h a n o l c o n c e n t r a t i o n o f 7 0 % a n d w a s h w i t h t w o
100 m l . p o r t i o n s o f s p e c i a l p e t r o l ( s a t u r a t e d w i t h 7 0 % e t h a n o l ) . D i s c a r d t h e p e t r o l p h a s e .
D i s t i l l off t h e a l c o h o l u n d e r v a c u u m . T h e r e s i d u e m u s t b e p r a c t i c a l l y free f r o m a l c o h o l .
C o n c e n t r a t i o n t o 4 - 5 ml. is g e n e r a l l y sufficient.
T h e steroid c o n t e n t o f t h e clear, h a e m o l y s i s - f r e e p l a s m a d o e s n o t c h a n g e in the c o u r s e o f o n e

d a y at 4 ° C . If t h e a n a l y s i s c a n n o t be c a r r i e d o u t for s e v e r a l d a y s , k e e p the p l a s m a at 16 t o
- 2 0 °C.
Enzymatic System
I n c u b a t i o n t e m p e r a t u r e : 37 ° C , i n c u b a t i o n v o l u m e : 8 3 m l .
Pipette into stoppered centrifuge tubes:
S a m p l e (protein-free extract) 4 ml.

Tris buffer (I) 12 m l .
G l u c u r o n i d a s e s o l u t i o n (II) 67 ml.
I n c u b a t e for 4 8 hr. in a n i n c u b a t o r , e x
tract s o l u t i o n w i t h t w o 150 m l . p o r t i o n s
o f ether (separating funnel), and w a s h
c o m b i n e d e t h e r p h a s e s w i t h three 5 m l .
p o r t i o n s o f 0.1 N N a O H a n d t h e n w i t h
t h r e e 10 m l . p o r t i o n s o f w a t e r . E v a p o r a t e
ether phase to dryness o n a steam bath
(explosion hazard). Use residue for
steroid determination.
S o u r c e s o f Error and Specificity
These are substantially as in m e t h o d 1, p . 1850, 1851.
3. H y d r o l y s i s o f G l u c u r o n i d e s of 1 7 - K e t o s t e r o i d s and T e s t o s t e r o n e in U r i n e
Reagents
1. S u l p h u r i c a c i d , 7 N lyophilized, ca. 950 m U / m g . (37 °C). Serva,

2. S t a n d a r d a c e t a t e buffer ( p H 4 . 6 2 ; 0.1 M ) Heidelberg (Item N o . 22859): dry powder, ca.
3. E t h e r 20 m U / m g . (37 °C). Warner-Chilcott, M o r r i s
4. ^ - G l u c u r o n i d a s e Plains, N . J.: Ketodase® from bovine liver,
from bovine liver, ^ 2 5 0 U/ml. (37 °C). C o m - dissolved in acetate buffer ( p H 5.0) 250 m U / m l .
mercial p r e p a r a t i o n s : Schering A G , Berlin: dry (37 °C). Activity determinations with phenol-
powder, 1 0 - 1 5 m U / m g . (37 °C), practically free phthalcin glucuronide as the substrate,
from sulphatase. Schwarz Bioresearch Inc.,
O r a n g e b u r g , New York (Item N o . 0110-42):
Refer to m e t h o d 1.
In s t o p p e r e d c o n t a i n e r s at + 4 ° C , t h e s o l u t i o n s k e e p p r a c t i c a l l y i n d e f i n i t e l y , u n l e s s a t t a c k e d b y
micro-organisms.
Procedure
N o d r u g s t o b e g i v e n for 3 d a y s b e f o r e a n d d u r i n g t h e u r i n e c o l l e c t i o n p e r i o d . U s e a n a l i q u o t o f a
fresh 2 4 hr. u r i n e . M a y b e k e p t f o r a b o u t 2 4 hr. at + 4 ° C , o t h e r w i s e t h e r e is a d a n g e r o f h y d r o l y s i s
b y e n d o g e n o u s ^ - g l u c u r o n i d a s e o r b a c t e r i a l g r o w t h . If s t e r o i d g l u c u r o n i d e s a n d free s t e r o i d s
are t o b e d e t e r m i n e d s e p a r a t e l y , t h e latter m u s t b e e x t r a c t e d w i t h 3 x 2 5 0 m l . e t h e r .
Enzymatic System
I n c u b a t i o n t e m p e r a t u r e : 37 ° C , i n c u b a t i o n v o l u m e : 3 5 0 m l .
Introduce into an Erlenmeyer flask:
S a m p l e (urine) 250 ml.

Adjust to p H 4 . 6 - 4 . 7 (glass electrode)
with 4 - 5 drops of 7 N H S02 4
S t a n d a r d a c e t a t e buffer 50 ml.
Glucuronidase solution 50 ml.
I n c u b a t e for 4 8 hr., e x t r a c t s o l u t i o n w i t h t h r e e
250 ml. portions o f ether (separating funnel),
c o m b i n e extracts.
Refer to m e t h o d 1, p . 1850.
Specificity
Refer to m e t h o d 1, p . 1851. N o steroid sulphates are hydrolysed with the enzyme p r e p a r a t i o n K e t o d a s e *

from bovine liver.
Other Methods
F o r the hydrolysis of 17-ketosteroid glucuronides, an ether-ethanol extract of the urine m a y be p r e p a r e d

after addition of a m m o n i u m sulphate to the latter. T h e dry residue is then t a k e n u p with acetate buffer
a n d hydrolysed with 50 o r 100 m U of ^-glucuronidase (from molluscs) per ml. of mixture in 1 6 - 2 0 h r . - . 2 3 2 4
In this way, enzyme inhibitors should be eliminated before the enzymatic hydrolysis.
Hydrolysis of Steroid Sulphates

T h e enzymatic hydrolysis of steroid sulphates has not yet attained the same i m p o r t a n c e as that of steroid
glucuronides in biochemical analysis. O n the one h a n d , steroid sulphates can be b r o k e n d o w n u n d e r
relatively mild conditions by non-enzymatic m e t h o d s (e. g. solvolysis or c o n t i n u o u s extraction at p H 1
in the c o l d ) ' . O n the other, the activities of the sulphatases obtained from various molluscs, m a m m a l i a n
2 5 2 6
organs, and bacteria against steroid sulphates depend strongly on the structure of the steroid in question,
so that quantitative hydrolysis of all steroid sulphates in biological fluids c a n n o t be achieved with a
single enzyme p r e p a r a t i o n . However, the use of sulphatases can be of value for the mild hydrolysis of
sulphates of certain groups of steroids or of individual steroids.
A n application of which little use has been m a d e as yet, but which is i m p o r t a n t in connection with certain
clinical problems, is the d e t e r m i n a t i o n of J -androsten-3/?-ol-17-one sulphate ( d e h y d r o e p i a n d r o s t e r o n e
5
sulphate), which can be hydrolysed with a steroid sulphatase from liver. A p r o c e d u r e for this hydrolysis
is given below.
The substrate specificities of the various sulphatases are i m p o r t a n t for o p t i m u m use. T h e arylsulphatases*
hydrolyse almost exclusively sulphates of phenolic steroids, while the steroid sulphatases** p r o p e r also
hydrolyse the sulphates of certain neutral steroids. A m o n g the steroid sulphatases, a p r e p a r a t i o n from
the mold Aspergillus oryzae is of interest, since it is free from ^-glucuronidase, a n d thus allows the specific
hydrolysis of steroid sulphates, a possibility of which practically n o use has been m a d e as yet.
There is also a cortisone sulphatase, which hydrolyses C 2 1 s u l p h a t e s . A c o m p a r i s o n of steroid sulphatases
27
from m o l l u s c s ' 8 28
shows that an enzyme p r e p a r a t i o n from Helix pomatia is still the most suitable for
approximately total hydrolysis of various steroid sulphates. Sulphates of phenolic steroids and of 3 p-A - 5
and 3 ft, 5 a-steroids are rapidly hydrolysed, while the hydrolysis of the other steroid sulphates is slow.
This sulphatase, like others, is ineffective against sulphates of steroids having the 3 a, 5 a configuration
(e. g. a n d r o s t e r o n e , 5 a - p r e g n a n o l o n e ) .
The o p t i m u m p H for the sulphatases from molluscs is a r o u n d 5, while that for sulphatases from m a m m a l i a n
liver is a r o u n d 7.0.
Enzymatic Hydrolysis of Dehydroepiandrosterone Sulphate

Equipment
Extraction apparatus, laboratory centrifuge, water bath, incubator.
Reagents
1. S u l p h u r i c a c i d , A . R . , 4 N 6. S t e r o i d s u l p h a t a s e
2. n-Butanol dry p o w d e r from bovine liver. Commercial pre
3. T r i e t h a n o l a m i n e h y d r o c h l o r i d e p a r a t i o n , Schering A G , Berlin. T h e enzyme is
4. A c e t i c a c i d , A . R., c o n e . b o u n d to cell c o m p o n e n t s , and is therefore in
5. E t h a n o l , 9 6 % ( v / v ) , d e n a t u r e d w i t h b e n z e n e soluble. 0 . 0 6 - 0 . 1 2 m U / m g . (37 ° Q * * * . The pre
p a r a t i o n is free from ^-glucuronidase. Activity
determination with dehydroepiandrosterone
sulphate as the substrate.
* Aryl-sulphate sulphohydrolase, E C 3.1.6.1.

** Sterol sulphatase (Sterol-sulphate sulphohydrolase, E C 3.1.6.2).
*** The units were formerly expressed as the enzyme activity that liberates 1 fig. of d e h y d r o e p i a n d r o s t e r o n e
from its sulphate per h o u r at 37 ° C . 17 280 of these units correspond to 1 U (37 °C) according to I U B
2 9
and only these latter " u n i t s " are n o w used.

C o n t a m i n a t i o n with ^-glucuronidase should not exceed 1 % (relative to the specific activity of the sulpha
tase).
D i s s o l v e 9 2 . 5 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in 5 0 0 m l . d o u b l y d i s t i l l e d w a t e r , a d j u s t t o
p H 7.3 w i t h c o n e , a c e t i c a c i d a n d d i l u t e t o 1 0 0 0 ml. w i t h d o u b l y d i s t i l l e d w a t e r .
I I . S t e r o i d s u l p h a t a s e (ca. 12 m U / m l . ) :
W i t h v i g o r o u s stirring, s u s p e n d 2 g. s t e r o i d s u l p h a t a s e in buffer ( s o l u t i o n 1) a n d m a k e u p t o
10 m l . S h a k e s u s p e n s i o n j u s t b e f o r e u s e .
Stability o f S o l u t i o n s
The enzyme suspension keeps for 3 - 4 weeks at 4 °C. T h e buffer is usuable indefinitely, provided it is n o t
c o n t a m i n a t e d with micro-organisms.
Procedure
U r i n e ( s e e p . 1 8 5 0 ) . P l a s m a ( s e e p. 1 8 5 1 ) .
Extraction and deproteinization:
U r i n e . C o n c e n t r a t e 2 4 hr. u r i n e u n d e r v a c u u m t o 4 0 0 - 5 0 0 m l . a n d a d j u s t t o p H 2.5-3.0
( i n d i c a t o r p a p e r ) w i t h 4 N s u l p h u r i c a c i d . Extract in a Kutscher-Steudel a p p a r a t u s for 16 hr.
w i t h 0.5 v o l u m e o f n - b u t a n o l . W a s h the b u t a n o l e x t r a c t f o u r t i m e s w i t h 1/10 o f its v o l u m e o f
distilled w a t e r c o n t a i n i n g a little s o d i u m c h l o r i d e . E v a p o r a t e off t h e b u t a n o l u n d e r v a c u u m
(temperature < 50 °C).
P l a s m a , sec p . 1851.
U s e urine a n d p l a s m a as s o o n as p o s s i b l e after c o l l e c t i o n . E n d o g e n o u s a n d b a c t e r i a l s u l p h a t a s e s
m a y l e a d t o i n c o r r e c t results if a specific d e t e r m i n a t i o n o f t h e s t e r o i d ester is t o b e carried o u t .
F r e e z e s a m p l e s at — 1 6 t o — 2 0 ° C if n e c e s s a r y .
Enzymatic System
D i s s o l v e e x t r a c t f r o m urine or p l a s m a in 1 0 0 - 2 0 0 ml. t r i e t h a n o l a m i n e buffer ( I ) , a d d 2 ml. (ca.

23 m U ) s t e r o i d s u l p h a t a s e s u s p e n s i o n ( I I ) , a n d i n c u b a t e for 17 hr. at 37 C (incubator) with
stirring t o p r e v e n t s e d i m e n t a t i o n o f the e n z y m e .
A d d 4 0 0 ml. e t h a n o l , c e n t r i f u g e at 2 0 0 0 g for 15 m i n . , a n d w a s h s e d i m e n t w i t h 3 x 50 ml.

portions of ethanol. C o m b i n e ethanol fractions and evaporate to dryness under vacuum. U s e
r e s i d u e for t h e s t e r o i d d e t e r m i n a t i o n .
Sulphates a n d p h o s p h a t e s in urine a n d p l a s m a inhibit the sulphatase activity. These interfering factors

are eliminated by the extraction m e t h o d s described here.
Specificity
W h e n a steroid sulphatase having the r e c o m m e n d e d purity is used, the m e t h o d is largely specific with
respect to sulphate, b u t n o t with respect to the steroid. T h e specificity of the steroid determination must
be ensured by the subsequent fractionation.
Other Applications of Steroid Sulphatases
A n enzyme p r e p a r a t i o n from Helix pomatia (commercially available, see p . 460) is very suitable for the
simultaneous enzymatic hydrolysis of sulphates a n d glucuronides of phenolic steroids (oestrogens). This
p r e p a r a t i o n c o n t a i n s b o t h ^-glucuronidase (ca. 5000 m U / m l . ) a n d arylsulphatase (ca. 2500 m U / m l . ) .
25 m U * / m l . of mixture completely hydrolyses the sulphates of the phenolic steroids within 16 hr. at
p H 5.2 and 37 °C.
If 1 % (v/v) of the enzyme p r e p a r a t i o n m e n t i o n e d above is a d d e d to the urine t o be hydrolysed, complete
hydrolysis of all oestrogen conjugates is achieved in 24 hr. at 37 °C a n d p H 5.2.
References
1 J. R. Pasqualini: C o n t r i b u t i o n a l'Etude biochimique des Corticosteroides. R. F o u l o n , Paris 1962.

2 O. Crepy, O. Judas, F. Rulleau-Meslin & M. F. Jayle, Bull. Soc. C h i m . biol. 44, 327 [1962].
3 K. D. Voigt & W. Schroder, A c t a endocr. ( K b h ) 27, 343 [1956].
4 G. W. Oriel & K. Eik-Nes, A c t a endocr. ( K b h ) 30, 93 [1959].
5 S. L. Cohen & G. F. Marrian, Biochem. J. 29, 1577 [1935].
6 / . Patterson, Brit. med. J. 2, 522 [1937].
7 P. Talalay, W. H. Fishman & C Huggins, J. biol. C h e m . 166, 757 [1946].
8 M. F. Jayle: Analyse des steroides h o r m o n a u x , Vol. I a n d II, M a s s o n , Paris 1961.
9 A. E. Kellie, Biochem. J. 100, 631 [1966].
10 O. W. Smith & N. N. Blackham, A c t a endocr. ( K b h ) 25, 133 [1957].
11 P. A. Katzman, R. F. Straw, H. J. Biihler & E. A. Doisy, Rec. Progr. H o r m o n e Res., A c a d e m i c Press,
N e w Y o r k 9, 45 [1954].
12 M. F. Jayle, R. Scholler, M. Heron & S. Metay, Clin. Chim. Acta 4, 276 [1959].
13 W. R. Slaunwhite & A. A. Sandberg, P r o c . Soc. exp. Biol. 101, 544 [1959].
14 R. H Silber, Acta endocr. ( K b h ) Suppl. 50, 207 [I960].
15 H. J. Biihler, P. A. Katzman, P. P. Doisy & E. A. Doisy, P r o c . Soc. exp. Biol. 72, 297 [1949].
16 P. Vestergaard, Acta endocr. ( K b h ) Suppl. 64, 39, 50 [1962].
17 M. F. Jayle & E. E. Baulieu, Bull. Soc. Chim. Biol. 36, 1391 [1954].
18 W. Futterweit, N. L. McNiven, L. Narcus, C Lantos, M. Drosdowsky & R. I. Dorfman, Steroids 1,
628 [1963].
19 A. Vermeulen & / . C. M. Verplancke, Steroids 2, 453 [1963].
20 K. D. Voigt, U. Volkwein & J. Tamm, Klin. Wschr. 13, 642 [1964].
* T h e activity is often given in P P S units. 1 P P S unit is the enzyme activity t h a t liberates 1 pg. of p h e n o l
phthalein from its sulphate in 1 hr. at 37 °C and p H 5.2. 20150 P P S units correspond to 1 U (37 °C)
according to I U B . These latter units are n o w exclusively used.
21 R. S. Stempfel, J. B. Sidbury & C. J. Migeon, J. clin. E n d o c r . 20, 814 [I960].

22 B. Kritz Kirk, C. B. Felger & P. A. Katzman, Arch. Biochem. Biophys. 98, 206 [1962].
23 A. E. Kellie & A. P. Wade, Biochem. J. 66, 196 [1957].
24 V. H. T. James, J. E n d o c r i n o l o g y 22, 195 [1961].
25 S. Lieberman, B. Mond & E. Smyles, Rec. Progr. H o r m o n e Res. 9, 113 [1954].
26 / . C. De Paoli, E. Nishizawa & K. B. Eik-Nes, J. Clin. E n d o c r . 23, 81 [1963].
27 K. S. Dodgson, Biochem. J. 78, 324 [1961].
28 P. Jarrige, J. Yon & M. F. Jayle, Bull. Soc. C h i m . Biol. 45, 783 [1963].
29 H. Langecker, A c t a endocr. ( K b h ) 23, 12 [1956].
20-Ketosteroids
The analysis of adrenocortical h o r m o n e s in b l o o d plasma, urine, a n d tissues is of clinical a n d biological
importance. T h e most c o m m o n l y used p h o t o m e t r i c m e t h o d s are generally not specific . Preliminary1
c h r o m a t o g r a p h i c purification of the p l a s m a extract was therefore r e c o m m e n d e d ; this m a d e the m e t h o d

2
specific, but also m o r e complicated. T h e enzymatic d e t e r m i n a t i o n of adrenocortical h o r m o n e s is b o t h

3
simple and specific.
Photometric Method 4
Wolfgang Staib*
The 20-ketosteroids in plasma, urine, a n d tissue can be quantitatively determined, after extraction a n d
solvent partition, by a s p e c t r o p h o t o m e t r i c assay with the aid of 3a,20/Miydroxysteroid dehydrogenase.
A selective solvent partition allows the differentiation between 17-hydroxycorticosteroids and 17-deoxy-
corticosteroids. After the partition between c a r b o n tetrachloride-cyclohexane and glycerol buffer, 100%
of the corticosterone a n d h y d r o c o r t i s o n e are found in the glycerol buffer phase, while after partition in the
system methylene chloride-cyclohexane a n d glycerol buffer, the glycerol buffer contains only 3 0 % of the
corticosterone a n d 1 0 0 % of the hydrocortisone.
3a,20/?-Hydroxysteroid dehydrogenase, 2 0 - S T D H (17,20/^21-Trihydroxysteroid: N A D oxidoreductase,
EC 1.1.1.53) specifically catalyses the reduction of 20-ketosteroids to 20-/?F-alcohols**.
Principle
I I
(1) C = O + NADH + H +
, ~
2 0 S T D H
- HO-C-H + NAD +
Jl k
20-Ketosteroid 20-jS -Hydroxysteroid
F
The decrease in the N A D H concentration, measured by the change in extinction at 340 (334, 365) nm, is
the unit of measurement.
The equilibrium constant of the reaction ( p H 7 . 3 ; 25 °C) is 3.8 x 1 0 " for Reichstein's 4
substance S. T h e
equilibrium c o n s t a n t for cortisone a n d h y d r o c o r t i s o n e is even smaller, a n d so c a n n o t be accurately determin
ed. After equilibrium has been reached u n d e r the conditions indicated here, therefore, m o r e t h a n 99 % of the
20-ketosteroid initially present has been converted into 20-/?F-alcohol. A mole of N A D H is oxidized per
mole of 20-ketosteroid.
* This chapter was contributed in the 1st. edition by H. J. Hiibenerf.

** F o r nomenclature (/?F), see L. F. Fieser a n d M. Fieser: Steroide, Verlag Chemie, Weinheim/BergstraBe
1961, p . 372.
20-Ketosteroids 1859
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r c a p a b l e o f a c c u r a t e m e a s u r e m e n t at 3 4 0 , 3 3 4 ,
or 365 n m . L a b o r a t o r y c e n t r i f u g e , c e n t r i f u g e t u b e s w i t h g r o u n d g l a s s s t o p p e r s .
Reagents
1. M e t h y l e n e c h l o r i d e , A . R . 8. H y d r o c o r t i s o n e ( A - p r e g n e n e - 1 7 a , l l / ? ,
4
2. C a r b o n t e t r a c h l o r i d e , A . R . 21-triol-3,20-dione)
3. C y c l o h e x a n e , A . R. 9. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E D T A
4. G l y c e r o l , r e d i s t i l l e d d i s o d i u m salt, E D T A - N a H 2 2 -2H 0
2
5. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 10. H y d r o c h l o r i c a c i d , A . R . , 1 0 % ( w / v )
6. S o d i u m s u l p h a t e , a n h y d r o u s 11. Ethanol, absolute, A . R.
7. R e d u c e d n i c o t i n a m i d e - a d e n i n e 12. 3 a , 2 0 / ? - H y d r o x y s t e r o i d d e h y d r o g e n a s e
dinucleotide, N A D H crystalline from Streptomyces hydrogenans ' ,5 6
as the sodium salt, N A D H - N a ; Commercial 2

suspension in 3.2 M a m m o n i u m sulphate solu
p r e p a r a t i o n , see p . 545. t i o n ; ^ 18 U / m g . (25 °C). C o m m e r c i a l p r e p a r a t
ion, see p . 477.
Purity of Reagents
The enzyme should have a turnover n u m b e r of at least 1500 mole of h y d r o c o r t i s o n e / 1 0 g. of protein x min. 5
It must be practically free from other enzymes. In particular, the activities of lactate dehydrogenase,
glucose-6-phosphate dehydrogenase, a n d malate dehydrogenase must n o t exceed 0.01 % of the activity of
3a,20/?-dehydrogenase.
Extract methylene chloride with 0.5 v o l u m e of concentrated sulphuric acid until n o further yellowing is
detected. Wash with water until neutral, dry with s o d i u m sulphate, a n d distil ( b . p . = 41.6 °C).
Keep c a r b o n tetrachloride over calcium chloride or p o t a s s i u m c a r b o n a t e for several days, a n d then distil
( b . p . - 76.7 °C).
Allow cyclohexane to stand over 0.1 v o l u m e of concentrated sulphuric acid for 24 h with occasional shak
ing. Wash with water until neutral, dry with calcium chloride, a n d distil.
I. Tris buffer ( 5 0 m M ; 0.1 % E D T A ; p H 7 . 3 ) :

D i s s o l v e 6.05 g. tris a n d 1 g. E D T A - N a H - 2 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o
2 2 2
a b o u t 9 0 0 m l . A d j u s t w i t h 1 0 % H C 1 t o p H 7.3 ( ± 0 . 0 5 ) u s i n g a g l a s s e l e c t r o d e , a n d m a k e
up to 1 0 0 0 ml. with distilled water.
II. Glycerol/buffer:
M i x 2 p a r t s o f tris buffer (I) w i t h 1 part o f g l y c e r o l .
TIT. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 1 . 4 m M / ? - N A D H ) :
Freshly dissolve 1 mg. N A D H - N a 2 in 1 m l . tris buffer (I).
IV. 3 a , 2 0 / ? - H y d r o x y s t e r o i d d e h y d r o g e n a s e , 2 0 - S T D H (1 m g . o f p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n as r e q u i r e d w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V. S t a n d a r d h y d r o c o r t i s o n e s o l u t i o n ( 0 . 2 0 m g . / m l . ) :
D i s s o l v e 5 m g . h y d r o c o r t i s o n e in a b s o l u t e a l c o h o l at 4 5 ° C a n d m a k e u p t o 25 m l .
T h e buffer solution is stable virtually indefinitely. T h e N A D H solution should be freshly p r e p a r e d every

week o r frozen in small p o r t i o n s . T h e enzyme solution is stable for a n u m b e r of weeks at 0 °C and p H
approximately 8, even in the dilute state.
Procedure
Treat blood w i t h h e p a r i n i m m e d i a t e l y after c o l l e c t i o n , a n d c e n t r i f u g e w i t h i n t h e n e x t h a l f h o u r

t o o b t a i n the p l a s m a .
D i l u t e 10 m l . p l a s m a w i t h 10 m l . w a t e r a n d e x t r a c t w i t h 160 m l . m e t h y l e n e c h l o r i d e in a
s e p a r a t i n g f u n n e l ( c a p a c i t y 2 0 0 m l . ) for 15 m i n . ; s e p a r a t e t h e o r g a n i c p h a s e a n d c o n c e n t r a t e
u n d e r v a c u u m t o a b o u t 2 0 m l . W a s h t h e m e t h y l e n e c l o r i d e e x t r a c t t w i c e w i t h 0.1 v o l u m e o f
0.1 N s o d i u m h y d r o x i d e s o l u t i o n a n d t w i c e w i t h 0.1 v o l u m e o f w a t e r , dry w i t h s o d i u m s u l p h a t e ,
filter q u a n t i t a t i v e l y , a n d d i v i d e a c c u r a t e l y i n t o t w o e q u a l p o r t i o n s ( A a n d B ) . E v a p o r a t e e a c h
h a l f t o d r y n e s s in a 4 0 m l . c e n t r i f u g e t u b e w i t h a g r o u n d g l a s s s t o p p e r in a c u r r e n t o f n i t r o g e n at
a temperature not exceeding 40 °C.
Urine: P i p e t t e 5 m l . o f a c o m p l e t e 2 4 h o u r urine i n t o a 4 0 m l . c e n t r i f u g e t u b e w i t h a g r o u n d g l a s s
s t o p p e r , adjust t o p H 1 w i t h 0 . 1 - 0 . 2 m l . o f 4 0 % ( v / v ) s u l p h u r i c a c i d , a n d a d d 3 g. a m m o n i u m
sulphate.
E x t r a c t free a n d c o n j u g a t e d c o r t i c o s t e r o i d s w i t h 35 m l . n - b u t a n o l / c h l o r o f o r m (1:10 v/v)
m i x t u r e for 15 m i n . , c e n t r i f u g e , a n d r e m o v e t h e a q u e o u s p h a s e a n d d i s c a r d . S h a k e the b u t a n o l /
c h l o r o f o r m e x t r a c t for 5 m i n . w i t h 0.5 g. s o d i u m c a r b o n a t e a n d 2 g. s o d i u m s u l p h a t e , filter,
a n d adjust t o a final v o l u m e o f 4 0 m l .
E v a p o r a t e a l i q u o t p a r t s o f t h e e x t r a c t in 4 0 m l . c e n t r i f u g e t u b e s w i t h g r o u n d g l a s s s t o p p e r s in a
current o f n i t r o g e n at a t e m p e r a t u r e n o t e x c e e d i n g 4 0 ° C .
Partition against Glycerol
D i s s o l v e dry residue A in 0.5 m l . m e t h y l e n e c h l o r i d e a n d a d d 1.5 m l . c y c l o h e x a n e a n d 0.45 m l .

g l y c e r o l / b u f f e r (II). S t o p p e r t h e c e n t r i f u g e t u b e s . E x t r a c t for 5 m i n . w i t h a Vortex m i x e r
a n d c e n t r i f u g e for 10 m i n . at 2 0 0 0 r p m . R e m o v e t h e u p p e r p h a s e c o m p l e t e l y f r o m the l o w e r
g l y c e r o l p h a s e t h r o u g h a fine p o l y v i n y l c h l o r i d e c a p i l l a r y c o n n e c t e d t o a water-jet p u m p .
A t the s a m e t i m e , r e m o v e as m u c h a s p o s s i b l e o f t h e t h i n e m u l s i o n b e t w e e n t h e t w o p h a s e s .
The (lower) glycerol phase contains the 20-ketosteroids to be analysed.
D i s s o l v e dry residue B in 0.5 m l . c a r b o n t e t r a c h l o r i d e , a d d 1.5 m l . c y c l o h e x a n e a n d 0.45 m l .
g l y c e r o l / b u f f e r , a n d t h e n p r o c e e d a s for p o r t i o n A o f t h e e x t r a c t .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; m i c r o c u v e t t e w i t h c a p a c i t y 0.8 m l . a n d light p a t h
10 m m ; 25 ° C ; a s s a y v o l u m e 0 . 4 7 5 m l . ; m e a s u r e m e n t a g a i n s t w a t e r .
F o r e a c h series o f m e a s u r e m e n t s , p r e p a r e a s t a n d a r d ( 0 . 4 m l . g l y c e r o l / b u f f e r II, 0 . 0 2 5 m l .
s t a n d a r d s o l u t i o n V , a n d 0 . 0 2 5 m l . N A D H s o l u t i o n III) a n d a b l a n k ( 0 . 4 m l . g l y c e r o l / b u f f e r
II, 0 . 0 2 5 m l . e t h a n o l , a n d 0 . 0 2 5 m l . N A D H s o l u t i o n I I I ) , a n d m e a s u r e after a d d i t i o n o f
enzyme solution.
Ethanol abs. 0.025 ml.

N A D H solution 0.025 ml. 0.074 m M
Sample (glycerol extract A or B) 0.400 ml.
M i x , read extinction Ej
3a.20/?-Hydroxysteroid dehydrogenase
(IV) 0.025 ml. 52.5 /ig./ml. ^ approx. 1 U / m l .
M i x , read c o n s t a n t final v a l u e ( a p p r o x . 15 m i n . ) E . 2
A E = Ei - E 2 is u s e d in t h e c a l c u l a t i o n s .
Calculations
The reaction proceeds stoichiometrically u n d e r the conditions indicated. F o r m u l a (1) o n p . 312 can
therefore be used for the calculations. However, various corrections are necessary in the calculation of the
quantity of 20-ketosteroids in p l a s m a .
T h e volume for t h e m e a s u r e m e n t of E is 0.450 ml., a n d t h a t for the m e a s u r e m e n t of E after t h e addition of
x 2
enzyme is 0.475 ml. T h e value found for E is t o o high by an a m o u n t d = E J 1 8 (for details, see ).
x
4
Let the change in the extinction of the blank o n addition of enzyme be a.

Only 0.40 ml. of the total glycerol extract (0.45 ml.) is used as the sample for the assay. T h e correction
factor for conversion to the value for the total glycerol extract is C = 1.125.
Thus
^ E ^ p l e = [zlE-(d-a)] x C.
T h e same correction C is also valid for a s t a n d a r d quantity of corticosteroids if the entire p r o c e d u r e

including extraction a n d solvent partition has been carried out. If the s t a n d a r d q u a n t i t y in 0.025 ml. of
ethanol is a d d e d directly to the enzyme mixture, the correction c o r r e s p o n d s t o :
E£"d„d = JE-(d-a).
The quantity of 20-ketosteroids is n o w found from the e q u a t i o n
A E c o r r
ug. 20-ketosteroids = S a m p l e
x fig. s t a n d a r d .
A F c o r r
a
^Standard
E x a m p l e : 10 ml. of p l a s m a were extracted; 0.4 ml. of glycerol extract A was used after partition in the
system methylene chloride/cyclohexane/glycerol buffer. 2 ug. of h y d r o c o r t i s o n e s t a n d a r d .
F o r portion B of the extract, after partition in the system carbon tetrachioride/cyclohexane/glycerol, it is

calculated that 100 ml. plasma contain 25 fig. 20-ketosteroids. After partition in the system methylene
chloride/cyclohexane/glycerol, 3 0 % of the corticosterone a n d 100% of the hydrocortisone are present in
the glycerol.
After partition in the system c a r b o n tetrachloride/cyclohexane/glycerol, 100% of the corticosterone a n d
100% of the hydrocortisone are present in the glycerol.
The difference between the results for p o r t i o n s A and B, i.e. 3.9 fig., corresponds to 7 0 % of the total
3 9 x 100
quantity of corticosterone, a n d 100 ml. plasma contain —'•—— = 5.6 fig. corticosterone.
The hydrocortisone content is then found to be
B) 2 5 . 0 - 5 . 6 = 19.4 jig. a n d
A) 21.1 - 1 . 7 * = 19.4/ig.
Ei AE d = E /18t a d - a
Blank 0.410 0.390 0.020 0.023 0.003 0.020

Standard(2/ig. hydrocortisone) 0.405 0.320 0.085 0.022 0.003 0.019
Sample (5 ml. plasma) 0.510 0.450 0.060 0.028 0.003 0.025
Êsu"d.rd = (0.085 - 0 . 0 1 9 ) x 1.125 - 0.074

ÊSSpie = ( 0 . 0 6 0 - 0 . 0 2 5 ) x 1.125 = 0.039
0 039
ug. 20-Ketosteroids in 5 ml. plasma = — • 2 = 1.054
0.074
or 21.1 fig. 20-ketosteroids in 100 ml. plasma.
U n d e r the conditions described, pure 20-ketosteroids in quantities of 0 . 2 5 - 0 . 5 /ig. can be determined with
an accuracy of 5 % ,
The accuracy of the m e t h o d described has been checked in recovery experiments With 2 fig. hydr o
cortisone and 1 ^ g . corticosterone a d d e d to 10 ml. plasma, recoveries of 98 and 9 1 % respectively were
obtained.
According to multiple determinations, the methodical error with both partition s y s t e m s is 2 . 8 % .
The sensitivity of the m e t h o d depends on the p h o t o m e t r i c equipment and the cuvetre dimensions. U n d e r
the conditions indicated, 1 fig. hydrocortisone can be determined with sufficient a c c u r a c y with a change
in extinction of 0.036.
N o r m a l Values
Plasma: On average, 19 fig. hydrocortisone ( 1 2 - 2 5 /xg.)/100 ml. and 4 u g . corticosterone ( 3 . 6 - 4 . 4 /ig.)/

100 ml. were found.
U r i n e : 8.5-19.2 fig. 17-hydroxycorticosteroids in the 24 h o u r u r i n e . 4
Rat liver: 0 . 3 - 0 . 4 fig. hydrocortisone/g. of liver (fresh weight).
* 1.7 = 3 0 % of t h e q u a n t i t y of c o r t i c o s t e r o n e (— 5.6 fig.).

Emulsions that occasionally occur o n extraction can be eliminated by centrifugation. Progesterone is

completely removed from the p l a s m a extract by the solvent partition. T h e o p t i m u m enzyme reaction is
guaranteed only with absolutely clean glassware.
Specificity
T h e specificity a n d sensitivity of the m e t h o d d e p e n d on the high specificity of the p u r e enzyme a n d the

special concentration of the p l a s m a corticosteroids by solvent partition. In the optical test, 1 //mole of
N A D H is transformed per //mole of steroid.
3a,20/?-Hydroxysteroid d e h y d r o g e n a s e specifically reduces the 20-keto g r o u p s of corticosteroids. N o
dependence on substitution o n C a t o m s 3 , 1 1 , o r 17 has been observed, a p a r t from a decrease in the convers
ion with conjugation on C-3 a n d substitution on C - l l . F o r example, the conversion for 11-ketosteroid
(cortisone) is only half of that for 11-hydroxysteroids (hydrocortisone). T h e only reaction p r o d u c t s t h a t
have been isolated a n d identified , b o t h from cortisone a n d from h y d r o c o r t i s o n e , are the c o r r e s p o n d i n g
4
20/Miydroxy derivatives. A d d i t i o n a l carbonyl g r o u p s in the steroid molecule have n o effect o n the stoichio
metric oxidation of N A D H , which d e p e n d s only o n the quantity of 20-ketosteroids present in the mixture.
C a r b o n y l groups in o t h e r positions are not reduced. T h e oxido-reduction o n C-20 is blocked by esterifica-
tion of the hydroxyl g r o u p on C-21. Corticosteroid 21-sulphates must be hydrolysed before the enzymatic
r e a c t i o n . See also p . 1854.
7
In principle, 20-ketosteroids can also be quantitatively determined in purified urine a n d tissue extracts by
the enzymatic test described after hydrolysis with ^-glucuronidase, solvolysis, solvent partition, a n d
column, thin layer, a n d p a p e r c h r o m a t o g r a p h y . In this case, 2 to 3 g. of tissue frozen in liquid air are
extracted twice for two h o u r s with methylene chloride. T h e further t r e a t m e n t a n d the d e t e r m i n a t i o n are as
described for plasma.
3a,20/?-Hydroxysteroid d e h y d r o g e n a s e can also be successfully used for the microchemical identification of
steroids . T h e 20-ketosteroids are converted into the c o r r e s p o n d i n g m o r e polar, easily acetylated 20 p-
8
hydroxy c o m p o u n d s . T h e hydroxy a n d the acetate derivative differ in their c h r o m a t o g r a p h i c b e h a v i o u r

from the 20-ketosteroids.
The absence of 20-ketosteroids from an extract of biological material can also be d e m o n s t r a t e d if the
substrate remains u n c h a n g e d on t r e a t m e n t with 3a,20/?-hydroxysteroid d e h y d r o g e n a s e a n d N A D H as the
coenzyme . 8
3a,20/?-Dehydrogenase have p r o v e d particularly valuable in the identification of 20-ketosteroids containing

only a few reactive g r o u p s , such as progesterone, pregnane-3,20-dione a n d 17a-hydroxyprogesterone. T h e
microchemical identification of such non-acetylatable steroids is greatly facilitated by enzymatic conversion
into the corresponding hydroxy c o m p o u n d followed by acetate formation. If the acetylation is carried o u t
with [ C]-labelled acetic a n h y d r i d e , with a sufficiently high specific activity, quantitative determinations are
14
possible in a range that was inaccessible with the physical m e t h o d s formerly in c o m m o n u s e . 8
Finally, 3a,20/?-dehydrogenase can also be used for the identification of 20/^-hydroxysteroids, since the
reverse reaction takes place in the presence of N A D or N A D P at p H 8 - 8.5 . F o r this d e t e r m i n a t i o n ,
8
dissolve the steroid dry residue in 1 d r o p of ethanol, a d d 1.5 ml. of 0.15 M p h o s p h a t e buffer ( p H 8.0 - 8.5
with addition of 1 g. of E D T A / 1 . of buffer), 0.03 ml. of N A D solution ( a b o u t 15 equivalents of N A D are
sufficient for 1 equivalent of substrate), a n d 0.03 ml. of 3a,20/?-dehydrogenase (6 ug. of enzyme u p to 5 ug.
of steroid, 12 ug. of enzyme u p to 50 ug. of steroid, a n d 60 ^ g . of enzyme a b o v e 50 ug. of steroid), a n d shake
in a closed vessel for 2 h o u r s at 37 °C. Inclusion of an N A D regenerating system, consisting of 0.1 ml.
sodium pyruvate (1.76 mg./ml.) a n d 0.02 ml. L D H (5 mg./ml.), in the incubation m e d i u m improves the
recovery. Dilute the reaction mixture with 3 - 5 ml. of water, a n d extract three times with the same volume
of ethyl acetate. D r y the extract with s o d i u m sulphate a n d c h r o m a t o g r a p h . T h e enzymatic oxidation is
particularly valuable if the steroid h a s a hydroxyl g r o u p which can be acetylated only on C-20 (e. g. A - 4
pregnene-20/?-ol-3-one, /d -pregnene-ll/?,20/?-diol-3-one, zl pregnene-17a,20/?-diol-3-one), in such cases,

4 4
the oxidation p r o d u c t s can n o longer be acetylated, whereas the original substrates form m o n o a c e t a t e s .
Epimer mixtures of 20a- a n d 20/?-hydroxysteroids that are difficult to separate can be converted into
c o m p o u n d s that can be m o r e easily distinguished chromatographically by enzymatic conversion of the
20/?-hydroxysteroids into 20-ketosteroids.
References
1 R. H. Silber & C. C. Porter, J. biol. C h e m . 210, 923 [1954].

2 A. M. Bongiovanni: S t a n d a r d M e t h o d s of Clinical Chemistry, A c a d e m i c Press, N e w York 1958, Vol. II,
p . 61
3 H. J. Hiibener & F. G. Sahrholz, Naturwissenschaften 46, 112 [1959].
4 H. W. Margraf, Ch. O. Margraj'& Th. E. Weichselbaum, Steroids 2, 143 [1963].
5 F. Lindner, R. Junk, G. Nesemann & J. Schmidt-Thome, Hoppe-Seylers Z. physiol. C h e m . 313,117 [1958].
6 G. Nesemann, H. J. Hiibener, R. Junk & J. Schmidt-Thome, Biochem. Z. 333, 88 [I960].
7 J. R. Pasqualini, M . F. Jayle, Biochem. J. 81, 147 [1961].
8 H. D. Henning & J. Zander, H o p p e Seyler's Z. physiol. Chemie 330, 31 [1962].
Fluorimetric Method
Wirnt Rick
Principle
Reduction of 20-oxosteroids by 3a, 20/?-hydroxysteroid dehydrogenase a n d N A D H gives stoichiometric

a m o u n t s of N A D , see e q u a t i o n (1) p . 1858. T h e N A D can be determined fluorimetrically by its fluorescence
in strongly alkaline s o l u t i o n . If the residual N A D H after complete enzymatic reduction of the 20-oxo
1
steroids is destroyed, the N A D formed in the reaction can be determined (and therefore the 20-oxosteroids)
after alkalinization of the assay mixture.
T h e equilibrium of the reaction is completely in favour of the steroid alcohols at p H 7.3 and 25 °C, so
that in the case of cortisone a n d Cortisol over 9 9 % of the a d d e d steroid is reduced at equilibrium. T h e t u r n
over n u m b e r of the enzyme (1700 mole cortisone per mole per min. at p H 7.3 a n d 25 °C) is low and therefore
relatively large a m o u n t s of enzyme must be a d d e d to the assay.
Equipment
S p e c t r o p h o t o m e t e r or s p e c t r u m - l i n e p h o t o m e t e r w i t h f l u o r e s c e n c e a t t a c h m e n t ; b e n c h c e n t r i
f u g e ; c o n s t a n t t e m p e r a t u r e w a t e r b a t h , 38 ° C .
Reagents
A s for the p h o t o m e t r i c m e t h o d , p . 1 8 5 9 ( g l y c e r o l is n o t r e q u i r e d ) ; In a d d i t i o n ;
13. N i c o t i n a m i d e - a d e n i n e dinucleotide, 14. Q u i n i n e s u l p h a t e .

NAD 15. H y d r o c h l o r i c a c i d , A . R., 2.2 N .
commercial p r e p a r a t i o n , s e e p . 545. 16. S u l p h u r i c a c i d , A . R . , 0.1 N .
17. S o d i u m h y d r o x i d e , A . R . , 10 N .
Purity of Reagents
T h e enzyme p r e p a r a t i o n should n o t c o n t a i n m o r e t h a n 0 . 1 % N A D H oxidase (related to the specific

activity of the 20 /^-hydroxysteroid dehydrogenase).
I. Tris buffer ( 5 0 m M ; p H 7 . 3 ) ; see p. 1 8 5 9 .
II. Tris buffer ( 0 . 2 M ; p H 7 . 3 ) :

D i s s o l v e 2 4 . 2 g. tris a n d 1 g. E D T A - N a H • 2 H 0 in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p
2 2 2
t o a b o u t 9 0 0 m l . , a d j u s t t o p H 7.3 ( g l a s s e l e c t r o d e ) w i t h 1 0 % H C 1 a n d d i l u t e w i t h d i s t i l l e d
water to 1 0 0 0 ml.
III. Tris buffer (ca. 15 m M ; 0 . 2 % E D T A ; p H 8 . 0 ) :
D i s s o l v e 2 g. E D T A - N a H • 2 H 0 in distilled w a t e r a n d m a k e u p t o 1 0 0 0 m l . A d j u s t
2 2 2
this s o l u t i o n t o p H 8 + 0 . 2 w i t h c a . 18 m l . o f a s o l u t i o n o f 10 g. tris in 1 0 0 m l . d i s t i l l e d
water.
I V . R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (2 m M / 2 - N A D H ) :
D i s s o l v e 1.6 m g . N A D H - N a 2 in 1 m l . tris buffer ( I ) ; p r e p a r e freshly.
V. Cortisol standard solution (0.2 m M ) :
D i s s o l v e 7 2 . 4 m g . Cortisol in a b s o l u t e e t h a n o l at 4 5 ° C a n d m a k e u p t o 10 m l . I m m e d i a
tely b e f o r e u s e d i l u t e 0.1 m l . o f this s o l u t i o n in a 10 m l . v o l u m e t r i c flask w i t h tris buffer
(III) t o 1 0 . 0 m l .
V I . 3 a , 2 0 / ? - H y d r o x y s t e r o i d d e h y d r o g e n a s e , 2 0 - S T D H (1 m g . p r o t e i n / m l . ) :
D i l u t e the s t o c k s u s p e n s i o n a c c o r d i n g l y w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V I I . N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 150 pM a n d c a . 15 pM /?-NAD):
a) D i s s o l v e 5.0 m g . N A D in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p t o 5 0 m l . D e t e r m i n e
the e x a c t N A D c o n t e n t a c c o r d i n g t o p. 2 0 4 8 .
b ) D i l u t e o n e v o l u m e s o l u t i o n a) w i t h n i n e v o l u m e s d o u b l y d i s t i l l e d w a t e r .
V I I I . F l u o r e s c e n c e s t a n d a r d (0.1 m g . q u i n i n e s u l p h a t e / 1 . ) :
D i s s o l v e 10.0 m g . q u i n i n e s u l p h a t e in 0.1 N H S 0 2 4 and m a k e up to 1000 ml.; dilute
10.0 m l . o f this s o l u t i o n t o 1 0 0 0 m l . w i t h 0.1 N H S0 .
2 4
Prepare solutions IV a n d VII freshly. Stock solution V is stable for a b o u t 8 weeks at 4 °C, p r e p a r e the
dilution with tris buffer freshly each day. Solutions, I, II, III a n d VI are stable for at least 1 year in a refrige
r a t o r if bacterial growth is prevented. Solution VIII is stable virtually indefinitely.
Procedure
A d d c a . 0.1 ml. h e p a r i n ( L i q u e m i n R o c h e ) t o c a . 2 5 - 3 0 m l . freshly o b t a i n e d b l o o d . C e n t r i f u g e

off t h e e r y t h r o c y t e s w i t h i n 3 0 m i n . P l a s m a c a n b e s t o r e d f o r at least 4 w e e k s at - 2 0 ° C w i t h o u t
loss of corticosteroids.
If the p l a s m a is n o t s e p a r a t e d f r o m t h e e r y t h r o c y t e s w i t h i n 3 0 m i n . after c o l l e c t i o n o f the b l o o d
the c o n t e n t o f t h e c o r t i c o s t e r o i d s d e c r e a s e s d u e t o their u p t a k e b y t h e e r y t h r o c y t e s .
Extraction:
S h a k e 10 m l . p l a s m a in a 5 0 m l . g l a s s c e n t r i f u g e t u b e , s t o p p e r e d w i t h a s u i t a b l e p o l y e t h y l e n e
s t o p p e r , for 3 0 m i n . w i t h 2 0 m l . m e t h y l e n e c h l o r i d e . A d d 10 m l . tris buffer ( I I I ) c o n t a i n i n g
0 . 0 3 m l . 0 . 2 m M Cortisol s o l u t i o n ( V ) in a n o t h e r t u b e a n d e x t r a c t w i t h 2 0 m l . m e t h y l e n e c h l o r i d e .
T h i s m i x t u r e c o n t a i n s 6 n m o l e Cortisol.
C e n t r i f u g e for 10 m i n . at 3 0 0 0 g, s u c k off t h e m e t h y l e n e c h l o r i d e p h a s e w i t h a g l a s s syringe a n d
a l o n g V 2 A n e e d l e , filter i n t o a 5 0 m l . g l a s s c e n t r i f u g e t u b e a n d d r y w i t h a n h y d r o u s N a S 0 . 2 4
Repeat the extraction with methylene chloride. Take the c o m b i n e d methylene chloride ex
tracts t o d r y n e s s at 4 5 ° C u n d e r n i t r o g e n .
Partition against cyclohexane:
Dissolve the dry residue from the methylene chloride extraction (equivalent t o 10 ml. plasma)
in 0 . 3 0 m l . m e t h y l e n e c h l o r i d e , a d d 1.50 m l . c y c l o h e x a n e a n d 0 . 3 0 m l . tris buffer ( s o l u t i o n 1),
s h a k e for 3 0 m i n . a n d c e n t r i f u g e f o r 15 m i n . at 4 0 0 0 g. D i s c a r d t h e u p p e r p h a s e ; t h e l o w e r
aqueous phase contains about 90% of the plasma corticosteroids.
Assay System
I n c u b a t i o n v o l u m e : 0 . 1 2 m l . ; - E x c i t a t i o n w a v e l e n g t h : 365 n m line o f t h e H g v a p o u r l a m p ;
w a v e l e n g t h f o r m e a s u r e m e n t s : f o r fluorescent light 4 3 0 - 3 0 0 0 n m o r m o n o c h r o m a t i c a l l y w i t h
q u a r t z m o n o c h r o m a t o r at 4 6 0 n m ; s l i t - w i d t h : 0 . 2 m m . ; h a l f - b a n d w i d t h : a b o u t 5 n m ; l i g h t
p a t h : 1 c m . ; r o o m t e m p e r a t u r e ; final v o l u m e : 3 . 4 3 m l . R e a d a g a i n s t a c u v e t t e c o n t a i n i n g
fluorescence s t a n d a r d s o l u t i o n ( V I I I ) . R e a d off t h e v a l u e s f r o m t h e t r a n s m i s s i o n s c a l e .
To d e t e r m i n e t h e s m a l l a m o u n t o f N A D in t h e N A D H p r e p a r a t i o n u s e a b l a n k c o n t a i n i n g
w a t e r i n s t e a d o f s a m p l e . T h e Cortisol s e r v e s t o c h e c k e x t r a c t i o n a n d p a r t i t i o n s t e p s o f t h e p r o
cedure.
Pipette into Concentration

Experimental Control Blank
10 m l . test t u b e s in assay mixture
Sample 0.10 ml. 0.10 ml. u p t o 8 0 pM

N A D H solution (IV) 0.01 m l . 0.01 m l . 0.01 m l . 0.17 m M
2.2 N H C 1 0.01 m l .
Enzyme suspension (VI) 0.01 m l . 0.01 m l . 0.01 m l . 80 pg./ml =
1 500 m U / m l .
M i x a n d a l l o w t o s t a n d for 3 0 m i n . at r o o m t e m p e r a t u r e .
T h e e n z y m e r e a c t i o n is c o m p l e t e .
2.2 N H C 1 0.01 m l . 0.01 m l .

10 N N a O H 0.30 ml. 0.30 ml. 0.30 ml. 7N
M i x a n d i n c u b a t e for 3 0 m i n . at 38 ° C .
Distilled water 3.00 ml. to each tube
Mix and measure fluorescence.
S i m u l t a n e o u s l y p r e p a r e N A D s t a n d a r d s c o n t a i n i n g 0 . 1 5 t o 7.5 n m o l e N A D / t u b e : m a k e u p
0.01 - 0 . 0 5 m l . N A D s t a n d a r d s o l u t i o n ( V I I a o r V I I b ) , 0 . 0 2 m l . tris buffer (II) t o 0 . 1 0 m l . w i t h
d o u b l y d i s t i l l e d w a t e r . Treat a s f o r t h e b l a n k .
Standard Curve
P l o t the r e a d i n g s for t h e N A D s t a n d a r d s a g a i n s t t h e n m o l e N A D . T h e i n t e n s i t y o f t h e f l u o r e s
c e n c e is l i n e a r l y p r o p o r t i o n a l t o t h e a m o u n t o f N A D b e t w e e n 0 . 1 5 a n d 7.5 n m o l e N A D p e r t u b e .
Calculations
Subtract the readings for the fluorescence of the b l a n k from those for the experimental a n d Cortisol c o n t r o l
tubes. R e a d off the a m o u n t of N A D corresponding to the corrected values from the s t a n d a r d curve.
Multiplication of this value by 3 gives the 20-oxosteroid concentration in nmole/10 ml. plasma.
P u r e Cortisol in a m o u n t s of a r o u n d 0.5 n m o l e can be determined with a s t a n d a r d deviation of 0.025 nmole.

T h e coefficient o f variation is 4 % .
N o r m a l Values
A value o f 12 + 5 pg. Cortisol has been found in plasma o f healthy subjects. This value is in g o o d agreement
with t h e value o f 12 ± 6 pg. f o r m e n a n d 15 + 6 pg. f o r w o m e n reported by Wu a n d Mason . 2
References
1 O. W. Lowry, N. R. Roberts & J. I. Kapphahn, J. biol. C h e m . 224, 1047 [1957].

2 C. Wu & H. L. Mason, P r o c . M a y o Clin. 33, 627 [1958].
Steroid Alcohols in Urine
Wolfgang Staib
At present the most c o m m o n m e t h o d of determining urinary steroids is the specific estimation of 17-
ketosteroids according to Zimmermann 1
or a modification of this m e t h o d ' . However, as the majority of
2 3
urinary steroids are hydroxysteroids, they can also be determined with steroid d e h y d r o g e n a s e s . 4
Application of Method: In biochemistry a n d in clinical biochemistry.
Principle
3a-Hydroxysteroid d e h y d r o g e n a s e ( 3 a - H y d r o x y s t e r o i d : N A D ( P ) oxidoreductase, E C 1.1.1.50) catalyses

the following reaction :
(1) 3a-Hydroxysteroid + N A D +
, 3-Ketosteroid + N A D H + H +
3/?,17/?-Hydroxysteroid d e h y d r o g e n a s e (3(or 17)/?-Hydroxysteroid : N A D ( P ) oxidoreductase, EC 1.1.1.51)

catalyses several steroid o x i d a t i o n s :
(2) 3^-Hydroxysteroid + N A D . , +
3-Ketosteroid + N A D H + H +
(3) 17^-Hydroxysteroid + N A D +
., 17- Ketosteroid + N A D H + H +
(4) 16£-Hydroxysteroid + N A D +
16-KetosteroidNADH -h H +
The lip- and 16/Mrydroxyl g r o u p s react only when the adjacent c a r b o n a t o m s have n o hydroxyl or keto
g r o u p s ; for example, oestriol (3,16a, 17/?-OH) is n o t oxidized. T h e 16^-hydroxyl g r o u p reacts at an apprecia
bly slower rate t h a n the 3p a n d 17/Mvydroxyl g r o u p s .
The ketosteroids already present in the urine a n d those formed by the action of the enzymes are " t r a p p e d " as
the hydrazones. In this way the equilibrium of the oxidative process is displaced in favour of the quantitative
formation of the ketosteroids. T h e increase in extinction at 340 n m d u e t o the formation of N A D H is a
measure of the r e a c t i o n . If the 3a-hydroxysteroid dehydrogenase is a d d e d to the reaction mixture first a n d
5
then the 3/?,17/Miydroxysteroid d e h y d r o g e n a s e , this allows the successive d e t e r m i n a t i o n of the c o r r e s p o n d

ing hydroxysteroids in the same reaction mixture.
The hydroxysteroids are present only in very low c o n c e n t r a t i o n s in urine a n d they must therefore first be
concentrated by extraction with organic solvents.
C o m p l e t e oxidation of 0.1 fimole of 3a, 3/?, a n d lip hydroxysteroids is achieved at p H 9.5 in the presence of
0.5 U of a- or /^-enzyme a n d 0.5 //mole of N A D in 3 ml. assay mixture by addition of a substance that binds
ketones, such as hydrazine. U n d e r these conditions, 9 9 % of the testosterone used is converted into A - 4
androstene-3,17-dione, a n d 9 5 % of the 3a-hydroxy-5a-androstan-17-one a n d 9 9 % of the 3/?-hydroxy-5a-

androstan-17-one are converted into 5 a - a n d r o s t a n e - 3 , 1 7 - d i o n e . 4
Steroid Alcohols in U r i n e 1869
Reagents
1. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , 9. 3 /?, 1 7 / ? - H y d r o x y s t e r o i d d e h y d r o g e n a s e
NAD 1 5 - 5 0 U / m g . Isolation a n d purity of the p r e p
free acid; c o m m e r c i a l p r e p a r a t i o n s , see p. 545. aration, see p. 1874. C o m m e r c i a l p r e p a r a t i o n ,
2. H y d r a z i n e s u l p h a t e , A . R . see p . 477.
3. S u l p h u r i c a c i d , A . R . , 2 N 10. M e t h y l e n e c h l o r i d e , A . R .
4. S o d i u m h y d r o x i d e , A . R., 1 N 11. S o d i u m hydrogen carbonate, NaHC0 , 3
5. M e t h a n o l , A . R. A . R.
6. Glycine 12. n-Hexane
7. ^-Glucuronidase 13. A m b e r l i t e M B 1*
ca. 0.27 U/ml. (5000 Fishman units /ml.);
6
14. D i s o d i u m h y d r o g e n p h o s p h a t e ,
commercial p r e p a r a t i o n , see p. 460. Na HP0 -2H 0
2 4 2
8. 3 a - H y d r o x y s t e r o i d d e h y d r o g e n a s e 15. P o t a s s i u m d i h y d r o g e n p h o s p h a t e ,
1 5 - 5 0 U / m g . Isolation a n d purity of the p r e p KH P0 2 4
aration, see A p p e n d i x p. 1874. Commercial 16. G l a c i a l a c e t i c a c i d , A . R .

p r e p a r a t i o n , see p. 476. 17. E t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d , E D T A
disodium salt, E D T A - N a H • 2 H 0 . 2 2 2
Purity of Reagents
Allow m e t h a n o l to stand in the d a r k with 2,4-dinitrophenylhydrazine h y d r o c h l o r i d e (0.5 g./l.) and 0.5 ml.
cone. HC1 for 1 5 - 1 8 hr. a n d then distil in a Vigreux c o l u m n .
Extract methylene chloride with 0.5 vol. of cone, sulphuric acid until n o further yellow colour can be
detected. Then wash with water until neutral, dry with sodium sulphate, a n d distil.
Allow n-hexane to stand over 0.1 vol. cone, sulphuric acid for 24 hr. with repeated shaking. Then wash with
water until neutral, dry with calcium chloride, and distil.
Wash Amberlite M B 1 in large c o l u m n s with distilled water, then treat with m e t h a n o l until m e t h a n o l
leaving the c o l u m n exhibits n o measurable extinction at 340 n m .
U s e o n l y fresh d o u b l y d i s t i l l e d w a t e r
I. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 6 m M / ? - N A D ) :
D i s s o l v e 41 m g . N A D in 10 m l . d o u b l y distilled w a t e r . S t o r e t h e s o l u t i o n at 0 ° C a n d
freeze a g a i n after u s e .
II. G l y c i n e buffer (1 M ; p H 9 . 4 ) :
D i s s o l v e 7.5 g. g l y c i n e , 5.2 g. h y d r a z i n e s u l p h a t e a n d 0.2 g. E D T A - N a H - 2 H 0 in 85 m l .
2 2 2
1 N N a O H , a d j u s t t o p H 9 . 4 w i t h 1 N N a O H a n d d i l u t e t o 100 m l . w i t h d o u b l y d i s t i l l e d
water.
III. P h o s p h a t e buffer ( 3 0 m M ; p H 7 . 2 ) :
a) D i s s o l v e 5 . 3 4 g. N a H P 0 - 2 H 0 in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2 4 2
b) D i s s o l v e 4 . 0 8 g. K H P 0 2 4 in d o u b l y distilled w a t e r a n d m a k e u p t o 1 0 0 0 m l .
M i x s o l u t i o n s a) a n d b ) in t h e r a t i o o f 7 2 . 6 t o 2 7 . 4 p a r t s b y v o l u m e .
I V . A c e t a t e buffer (1 N ; p H 4 . 5 ) :
M i x 4 3 m l . 1 N N a O H w i t h 100 m l . 1 N a c e t i c a c i d .
* Mixed bed ion exchange resin (anion-cation) manufactured by R o h m & H a a s C o m p a n y , Philadelphia,

Pa., U S A .
V . 3 a - H y d r o x y s t e r o i d d e h y d r o g e n a s e (ca. 2 5 U / m l . ) :
D i s s o l v e t h e e n z y m e p r e p a r a t i o n i n p h o s p h a t e buffer ( s o l u t i o n III).
V I . 3 / ? , 1 7 / ? - H y d r o x y s t e r o i d d e h y d r o g e n a s e (ca. 2 5 U / m l . ) :
D i s s o l v e t h e e n z y m e p r e p a r a t i o n i n p h o s p h a t e buffer ( s o l u t i o n III).
VII. ^-Glucuronidase (0.27 U / m l . ) :
U s e the commercial preparation undiluted.
The buffer solutions are stable practically indefinitely. If turbidity or a sediment (micro-organisms) is
observed in the buffer solutions, then they should be prepared afresh. P r e p a r e the N A D solution freshly
each week or dispense in small p o r t i o n s a n d store frozen. D u r i n g the analysis keep the dilute enzyme
solutions at 0 °C a n d then store frozen. In this state they are stable for several weeks.
Procedure
Pipette into 60 ml. centrifuge tubes with tapered polyethylene stoppers:
25 ml. urine
A d j u s t t o p H 4.5 w i t h 2 N H S 0 . A d d 2 4
1.3 m l . a c e t a t e buffer ( s o l u t i o n I V )
5.0 m l . ^ - g l u c u r o n i d a s e s o l u t i o n ( V I I )
a n d i n c u b a t e at 30 ° C ( i n c u b a t o r o r w a t e r b a t h ) for 2 4 h o u r s . E x t r a c t t h r e e t i m e s ( s h a k e 30 t i m e s
for e a c h e x t r a c t i o n ) w i t h
15 m l . m e t h y l e n e c h l o r i d e
e a c h t i m e ; if n e c e s s a r y , c e n t r i f u g e for 10 m i n . at 4 0 0 0 g t o s e p a r a t e t h e p h a s e s . S u c k off the

m e t h y l e n e c h l o r i d e ( l o w e r p h a s e ) w i t h a 2 0 m l . R e c o r d s y r i n g e a n d a l o n g n e e d l e ( a s for l u m b a r
p u n c t u r e ) . A d j u s t t h e a q u e o u s r e s i d u e t o p H 1.0 w i t h 2 N H S 0 2 4 and allow to stand 24 hours
at r o o m t e m p e r a t u r e . A g a i n e x t r a c t t h r e e t i m e s w i t h
15 m l . m e t h y l e n e c h l o r i d e
as d e s c r i b e d a b o v e . E v a p o r a t e t h e c o m b i n e d e x t r a c t s t o d r y n e s s in a r o t a t o r y e v a p o r a t o r u n d e r
r e d u c e d p r e s s u r e o r w i t h a s t r e a m o f n i t r o g e n at a b o u t 50 ° C . D i s s o l v e t h e r e s i d u e in
5 ml. methanol
a n d w i t h i n c a . 10 m i n . p a s s o n c e o r t w i c e t h r o u g h a n i o n e x c h a n g e c o l u m n ( 0 . 7 c m . d i a m e t e r ,
10 c m . h i g h , A m b e r l i t e M B 1) t o a b s o r b t h e a c i d i c o e s t r o g e n s . E l u t e t h e c o l u m n w i t h
25 ml. m e t h a n o l .
E v a p o r a t e t h e e l u a t e s (ca. 3 0 m l . ) t o d r y n e s s a s p r e v i o u s l y d e s c r i b e d a n d d i s s o l v e t h e r e s i d u e in
15 m l . a q u e o u s m e t h a n o l ( 7 0 % v / v ) .
Extract the solution three times with
5 ml. n-hexane
each time* (separating funnel or centrifuge tube), evaporate the m e t h a n o l / w a t e r phase to

d r y n e s s (see a b o v e ) a n d d i s s o l v e in
0.5 m l . m e t h a n o l .
U s e p o r t i o n s o f t h i s s o l u t i o n for t h e a s s a y .
Assay System
Wavelength: 340 n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 3 m l . ; t e m p e r a t u r e : 25 ° C . M e a s u r e

against a control cuvette containing m e t h a n o l instead of urine extract.
G l y c i n e buffer (II) 2.00 ml. 0.67 M

N A D solution (I) 0.10 ml. 0.2 m M
Sample (urine extract) + methanol
+ water 0.86 ml.
M i x ; read extinction E ^
3a-Dehydrogenase solution (V) 0.02 ml. ca. 0.16 U / m l .
M i x . A s s o o n as e x t i n c t i o n r e a c h e s a
constant value, read E . 2
3/?,17/?-Dehydrogenase solution (VI) 0.02 ml. ca. 0.16 U / m l .
M i x , r e a d c o n s t a n t final v a l u e E . 3
Calculations
T h e extinction changes E — E = A E a n d E — E = A Ep are used for the calculations. A E c o r r e s p o n d s to

2 x a 3 2 a
the concentration of 3a-hydroxysteroid in the cuvette, a n d AE fi to the s u m of the 3/?-, 17/?- and 3/?,17/?-
hydroxysteroids (abbreviated to 3/?, 17/?-hydroxysteroids in the following). T h e concentration of 3a-
hydroxysteroid or 3/?,17/Mrydroxysteroid** in the assay mixture is
AE x 3 I/I,
c = [/imole/ml.]
r
6 2 2
where
3 = volume of the assay mixture [ml.]
6.22 = extinction coefficient of N A D H at 340 n m [cm. /jumole]
2
* Lipids are extracted; otherwise they would cause turbidity in the a q u e o u s assay mixture a n d so interfere
with the determination.
** Strictly speaking this is ^ m o l e 3/?-hydroxysteroid + ^ m o l e 17/Miydroxysteroid + 0.5 //mole 3/?,17/?-
hydroxy steroid.
The accuracy of the m e t h o d described is given by recovery experiments. F o r p u r e steroids such as androste-
rone and tetrahydrocortisone, as well as a n d r o s t e r o n e and epiandrosterone added to the urine extract, the
recovery is 1 0 0 % + 1%. W h e n a n d r o s t e r o n e a n d tetrahydrocortisone are a d d e d to urine, the recovery
after hydrolysis, extraction, t r e a t m e n t with ion exchange resin, a n d hexane partition is 93 %.
The methodical error (reproducibility) in multiple determinations o n the same urine is less than 5 % (co
efficient of variation).
A strictly linear relation exists between the volume of the urine extract (0.025 - 0.3 ml.) and the observed
change in extinction.
The sensitivity is largely dependent on the p h o t o m e t r i c equipment a n d on the cuvette dimensions. In the
3 ml. mixture described, as little as 2.8-3.7 ug. ( = 0.01 /rniole) of hydroxysteroid can be determined with
sufficient accuracy in a 10 m m . cuvette at 340 n m . T h e extinction change in this case is a b o u t 0.021. W i t h
microcuvettes and a final volume of 0.2 ml., the sensitivity is increased by a factor of 10, so that 0.28-0.37 ug.
of steroid can be determined. T h e sensitivity of the m e t h o d can be still further increased by fluorimetric
measurements of the quantity of N A D H formed.
N o r m a l Values
With this m e t h o d Talalay* found the following n o r m a l values:
N u m b e r of
3a- H y d r o x y steroids 3ft 17£-Hydroxysteroids
Subjects examined determinat
[/miole/24 nr.] Lumole/1.] Lumole/24 hr.] [/miole/1.]
ions
M e n (20 to 47 years) 13 43.7 + 13.0 41.8 + 14.1 7.52 + 2.71 6.90 + 2.76
(29.1 to 70.7) (18.7 to 66.7) (3.16 t o 13.7) (3.29 to 13.2)
Women (17 to 37 years) 11 42.4 + 18.5 44.1 + 16.1 6.07 + 2.65 6.44 + 2.87
(16.4 to 74.2) (26 t o 78) (2.61 to 11.6) (2.90 to 14.6)
The average values a n d the s t a n d a r d deviations are given. T h e figures in brackets show the range of the
analytical values. In the adrenogenital s y n d r o m e the 3/?,17/Miydroxysteroids increase considerably, while
in virilizing adrenocortical hyperplasia the increase is less m a r k e d . 4
The assay mixture m a y become turbid if the extract contains water-insoluble lipids or as a result of precipi
tation of the enzyme protein by the urine extract. T h e water-insoluble lipids are then divided by further
partition of the urine extract between a q u e o u s m e t h a n o l and n-hexane. Steroid extracts are occasionally
not completely soluble in small volumes. In such cases the volume is increased a n d cuvettes of suitable
capacity are used. E n z y m e p r e p a r a t i o n s having low specific activities (less t h a n 15 U/mg.) readily lead to
turbidity, since the quantities of protein a d d e d are t o o large. T h e specific activity of the enzyme can be
increased by treatment with calcium p h o s p h a t e gel ( s e e ) . T h e enzymes must be added in the o r d e r
18
indicated, since the /?-enzyme still contains 1 - 2 % of a-enzyme activity, whereas the a-enzyme contains only
0.1%of ftenzyme.
Specificity
The two hydroxysteroid dehydrogenases possess a high catalytic activity with a high steric and positional
specificity. The a-enzyme is specific for 3a-hydroxysteroids of the C , C , a n d C series and the /?-enzyme
2 4 2l 1 9
for 3/?-hydroxysteroids of the C a n d C series, as well as for 17/?-hydroxysteroids of the C and C

2 1 1 9 1 9 1 8
Steroid Alcohols in Urine 1873
series. F o r a survey of reactive a n d non-reactive steroids, see . T h e oxidation rate is reduced by an oxygen
7
function on C . Of m a n y aliphatic a n d cyclic alcohols tested, only a few bicyclic c o m p o u n d s u n d e r g o slow

n
o x i d a t i o n ' . Pregnane-3a,20a-diol c a n n o t be determined quantitatively, since even in very low concentra

24 25
tions (1 fiM) it precipitates in the cuvette.

The specificity of the m e t h o d is based on the high specificity of the purified enzymes, the special concentra
tion of the urinary steroids, and the c o m b i n a t i o n with the optical d e t e r m i n a t i o n . 1 /miole of N A D is
converted stoichiometrically into N A D H per /miole of steroid. Even crude urine extracts a n d the small
quantities of enzyme protein influence the b a c k g r o u n d only slightly. C 1 9 steroids, which have b o t h a 3/? a n d
a 1 ip hydroxyl g r o u p , convert 2 equivalents of N A D into N A D H in the enzymatic oxidation with /?-enzyme.
T h e reactive 3a, 3p, a n d lip hydroxysteroids of the C , C , C , a n d C 1 8 1 9 2 1 2 4 series can also be determined in
various body fluids with the aid of an easily o b t a i n a b l e c r u d e enzyme extract instead of with highly purified
e n z y m e . This m e t h o d still has the same sensitivity, accuracy, and reproducibility. T h e specificity of the
7
enzymatic determination d e p e n d s o n the purification a n d separation of the reactive steroids.
Procedure: After /^-glucuronidase hydrolysis and solvolysis, extract C 1 9 and C 2 1 urinary steroids exhaustively
with chloroform or methylene chloride. Wash the extract with sodium hydroxide solution a n d water, dry
with sodium sulphate, concentrate u n d e r v a c u u m , a n d purify on a Florisil or silica gel c o l u m n . Then 8 9
separate the 17-ketosteroid a n d corticosteroid fractions by p a p e r or thin layer c h r o m a t o g r a p h y .

T h e Bush 10
a n d Zaffaroni11
systems ( r e v i e w s ~ ) are particularly suitable for separation by paper c h r o m a
12 1 4
tography. After complete separation in at least two different c h r o m a t o g r a p h y systems, elute the individual
steroids with ethyl a c e t a t e / m e t h a n o l (3 : l ) 1 3
a n d determine enzymatically after c o n c e n t r a t i o n of the eluate.
Unstable b l a n k values t h a t occasionally arise when large elution volumes are used can be eliminated by
purification of the eluates o n a silica gel c o l u m n (3 cm. x 1 cm.). F o r this p u r p o s e , elute the 17-ketosteroids
with 6 % m e t h a n o l in c h l o r o f o r m a n d the corticosteroids with 2 0 % m e t h a n o l in c h l o r o f o r m .
Thin layer c h r o m a t o g r a p h y can also be successfully used in c o m b i n a t i o n with p a p e r c h r o m a t o g r a p h y for
the separation of the urinary steroids (reviews, s e e 1 5 1 6
) . After elution of the steroid fractions with ethanol,
filtration t h r o u g h a sintered glass filter, a n d concentration, the enzymatic d e t e r m i n a t i o n can be carried out
directly. Dissolve the dry residues of the steroids obtained by p a p e r and thin layer c h r o m a t o g r a p h y in
0.1 ml. m e t h a n o l (distilled twice over 2,4-dinitrophenylhydrazine)

1.0 ml. N a p y r o p h o s p h a t e buffer (0.1 M ; p H 9.5)
0.1 ml. N A D solution (5 m M )
1.0 ml. hydrazine h y d r a t e (1 M ; p H 9.5)
0.75 ml. water
and m a k e u p to 2.95 ml.
Carry out the determination at r o o m t e m p e r a t u r e , starting the reaction with 0.05 ml. enzyme solution.
After exactly 20 min., m e a s u r e extinction at 340 n m (light p a t h 1 cm.) against control (without steroid). T h e
mixture is designed for 2 - 1 0 0 /ig of hydroxy steroid. T h e n u m b e r of /miole of steroid per 3 ml. mixture is
E/2.07, and the n u m b e r of fig. of steroid is given by /miole of steroid multiplied by the molecular weight.
Separation of the a activity from the P activity of the crude enzyme p r e p a r a t i o n is unnecessary, since the
a-enzyme is completely inhibited by 0.66 m M p - h y d r o x y m e r c u r i b e n z o a t e in the 3 ml. mixture (0.2 ml. of a
10 m M solution in 0.2 M tris buffer, p H 9.5), while the /?-enzyme activity is not affected. In this way 3/?-
hydroxysteroids can be determined in the presence of 3a-hydroxysteroids in 2 mixtures. 4 - 2 0 fig. of
epiandrosterone (5a-androstan-3/?-ol-17-one) have been determined by this m e t h o d with a recovery of
100 ± 3 % in the presence of 10 fig. of tetrahydrocortisol (5/?-pregnane-3a,ll/?,17a,21-tetrol-20-one).
Sensitivity and Accuracy: T h e recovery of p u r e C , C , C , a n d C

2 4 2 1 1 9 1 8 steroids in the enzymatic assay is
8 0 - 1 0 3 % , that of 2 - 4 0 fig. of a n d r o s t e r o n e is 100 ± 1.2%, a n d that of 4 - 4 0 fig. of tetrahydrocortisol is
100.1 + 1.3%. As little as 0.5 pg. of steroid can be determined in the 1 ml. mixture in 1 cm. microcuvettes;
recovery 100 ± 1.8 %. A loss of a b o u t 5 - 1 0 % must be expected in any p a p e r or thin layer c h r o m a t o g r a p h y .
Tetrahydrocortisone (5/?-pregnane-3a,17a,21-triol-ll,20-dione) a n d t e t r a h y d r o c o r t i s o l a d d e d t o urine
samples were found with recoveries of 7 2 - 9 3 % after hydrolysis, extraction, passage t h r o u g h a Florisil
column, and t w o p a p e r - c h r o m a t o g r a p h i c separations.
A quantitative enzymatic determination of bile acid in blood plasma has been described by Iwata and
Yamasak . 11
Enzyme Preparation: Extract 1 g. Pseudomonas testosteroni (Worthington Chemical Corp., Sigma Chemical
Company, Nutritional Chemical Corp.) for 1 hr. with 10 ml. 0.2 N tris buffer ( p H 7.5) at 4 °C, and add 50 ml.
acetone to the extract at - 1 5 °C. Filter off precipitate at 4 °C in a Buchner funnel, wash twice with cold
acetone ( - 1 5 °C), and extract for 20 min. with 10 ml. 0.2 N tris buffer ( p H 9.5); then centrifuge at 10000 g
for 30 min. at — 4°C. D e c a n t s u p e r n a t a n t fluid a n d extract residue again with tris buffer. T h e combined
s u p e r n a t a n t fluids contain the a a n d p enzymes, a n d can be kept for several m o n t h s in t h e frozen state
without loss of activity.
Appendix
Preparation of 3 a-Hydroxysteroid Dehydrogenase
Method: The enzyme is purified from Pseudomonas testosteroni by repeated a m m o n i u m sulphate precipita
tion, p r o t a m i n e precipitation, acetone precipitation and calcium p h o s p h a t e gel a d s o r p t i o n to yield a
preparation with a turnover n u m b e r of a b o u t 2500 to 5000 mole a n d r o s t e r o n e / m i n . / 1 0 5
g. protein
(25 ° C ) " . In particular, it is separated from the 3j5,17jS-dehydrogenase by a m m o n i u m sulphate fraction
1 8 2 0
ation : the 3/?,17/?-dehydrogenase precipitates between 30 a n d 4 0 % saturation, while the 3a-dehydrogenase

is only precipitated between 40 and 55 % saturation. T h e two enzymes can also be separated by gel filtration
(Sephadex G - 1 0 0 ) . 21
Purity: T h e 3a-dehydrogenase contains practically n o 3/?,17/?-dehydrogenase or alcohol dehydrogenase.

However, it is c o n t a m i n a t e d with a J - 3 - k e t o s t e r o i d isomerase.
5
Substrates of the enzyme are 3a-hydroxysteroids containing 19, 21 a n d 24 c a r b o n a t o m s (A/B-cis and A / B -

trans). C steroids d o not react.
2 7
Equilibrium Constant:
= [androstane-3,17-dione] x [ N A D H ] x [H + ] = 1 Q _ 9 1 9
H
[androsterone] x [ N A D ] +
' v F
At p H 9.1 a n d with a ten-fold excess of N A D , a n d r o s t e r o n e is practically quantitatively oxidized to

a n d r o s t a n e d i o n e . It is therefore possible in this way t o estimate 3a-hydroxysteroids w i t h o u t the addition of
hydrazine.
T h e Michaelis constant for a n d r o s t e r o n e is 1.6 x 1 0 ~ M ( p H 9 . 1 a n d 2 5 ° C ) ; K f o r N A D is 1.04 x 1 0 " M
6
M
4
( p H 9 . 1 ; 1 0 " M a n d r o s t e r o n e as substrate).
5
Assay of Activity: see u n d e r the 3/?,17/?-dehydrogenase. A n d r o s t e r o n e is used as the substrate instead of

testosterone.
Stability: Solutions of the enzyme containing at least 50 mg. protein/ml. are stable for years at —20 °C.
Preparation of 3 p, 17 p-Dehydrogenase
Method: The formation of 3/?,l 7/?-dehydrogenase is induced in Pseudomonas testosteroni by the addition of
t e s t o s t e r o n e . T h e cells are disintegrated by exposure to ultrasonic r a d i a t i o n a n d the enzyme is purified by
22
a m m o n i u m sulphate precipitation, p r o t a m i n e precipitation a n d acetone precipitation to yield a p r e p a r a t i o n

with a turnover n u m b e r of a b o u t 2500 to 5000 moles t e s t o s t e r o n e / m i n . / 1 0 g. p r o t e i n " .
5 1 8 2 0
Purity: T h e p r e p a r a t i o n is free from alcohol dehydrogenase, but still contains a steroid isomerase which
catalyses the conversion of J - a n d r o s t e n e - 3 , 1 7 - d i o n e to zl -androstene-3,17-dione. It is also c o n t a m i n a t e d
5 4
with a b o u t 1 to 2 % of the 3a-dehydrogenase.
Equilibrium Constant:
= [zl -androstene-3,17-dione] x [ N A D H ] x [H + ]
4
= 3 J % x 1 Q _ 8
[testosterone] x [ N A D ]
(25 ° C ; p H 6 t o 1 0 ) ' . 1 9 2 4
The enzymatic assay is carried out at p H 9.0 with a ten-fold excess of N A D in relation to the a m o u n t of
testosterone. At equilibrium there is a b o u t 378 times as m u c h a n d r o s t e n e d i o n e as testosterone present. This
m e t h o d is therefore also suitable for the quantitative determination of testotesterone.
Affinity Constant ':

22
Relatively low concentrations of testosterone (6 fiM) inhibit the 3/7,17/Miydroxy-
steroid dehydrogenase. In addition to the Michaelis complex between the substrate and the enzyme (ES), a
further complex is formed with increasing testosterone concentration, which is c o m p o s e d of two molecules
of substrate a n d one molecule of enzyme ( E S ) . The affinity constant K for ES (25 °C a n d testosterone as
2 A
substrate) is 0.93 x 1 0 " M ; the c o n s t a n t K for E S is 39.0 x 1 0 ~ M . K

6
2 2
6 2 3
M for N A D at 25 °C and p H 9.8
is 0.3 to 8 x 1 0 _ 5
M (depending on the steroid used).
Inhibitors: T h e enzymatic reaction is strongly inhibited by natural and synthetic oestrogens (e.g. diethyl-
stilboestrol).
Assay of Activity: Pipette into a 1 cm. cuvette 1 ml. p y r o p h o s p h a t e buffer (0.1 M ; p H 8.9), 0.5 /miole N A D
and 15 /xg. testosterone. Dilute with doubly distilled water to 3 ml. The reaction mixture is p H 9.1. Start the
reaction (at 25 °C) by the addition of 0.02 to 0.1 ml. enzyme solution. One unit is the a m o u n t of enzyme
that causes an extinction change of 0.001 /min. at 340 n m .
Stability .Solutions of the enzyme containing at least 50 mg. protein per ml. are stable for years at —20 °C.
References
1 W. Zimmermann, Hoppe-Seylers Z. physiol. C h e m . 245,47 [1936]; See L. F. Fieser & M. Fieser: Steroide.
Verlag Chemie, Weinheim/Bergstr. 1961, p. 570.
2 H L. Mason & W. W. Engstrom, Physiol. Rev. 30, 321 [1950].
3 P. L. Munson & A. D. Kenny, Recent Prog. H o r m o n e Res. 9, 135 [1954].
4 B. Hurlock & P. Talalay, Endocrinology 62, 201 [1958]; Proc. Soc. Exp. Biol. M e d . 93, 560 [1956];
Enzymic Analysis of S t e r o i d h o r m o n e s in D. Glick: M e t h o d s of Biochemical Analysis, Intersciences
Publisher Inc., N e w York 1960, Vol. 8, p . 119.
5 O. Warburg & W. Christian, Biochem. Z. 287, 291 [1936].
6 P. Talalay, W. H. Fishman & Ch. Huggins, J. biol. C h e m . 166, 757 [1946]; see also p . 463 & 873.
7 R. S. StempJel&J. B. Sidburg, J. clin. E n d o c r . 24, 367 [1964].
8 E. M. Glenn & D. H. Nelson, J. clin. Endocr. 13, 911 [1953].
9 L. P. Romanoff, R. S. Wolf M. Constandse & G. Pincus, J. clin. Endocr. 13, 928 [1953].
10 /. E. Bush, Biochem. J. 50, 370 [1952].
11 A. Zaffaroni, R. B. Burton & E. H. Keutman, Science 111, 6 [1950].
12 R. Neher, J. C h r o m a t o g r . 1, 123 [1958].
13 I.E. Bush: T h e c h r o m a t o g r a p h y of steroids, P e r g a m o n Press, Oxford, L o n d o n , New Y o r k , Paris 1961.
14 H.J. Hiibener & W. Staib, Biochemie der N e b e n n i e r e n r i n d e n - H o r m o n e , in G. Weitzel & N. Zollner:
Biochemie u n d Klinik, G e o r g Thieme-Verlag, Stuttgart 1965.
15 E. Stahl: D u n n s c h i c h t c h r o m a t o g r a p h i e , Springer-Verlag, Berlin, G o t t i n g e n , Heidelberg 1967.
16 K Randerath: D u n n s c h i c h t c h r o m a t o g r a p h i e , Verlag Chemie, Weinheim 1962.
17 T. Iwata & K. Yamasak, J. of Biochemistry 56, 424 [1964].
18 P.I. Marcus & P. Talalay, J. biol. C h e m . 218, 661 [1956].

19 P. Talalay & P. I. Marcus, J. biol. C h e m . 218, 675 [1956].
20 B. Hurlock & P. Talalay, J. biol. C h e m . 227, 37 [1957].
21 J. Deliw & /. Porath, Biochim. Biophys. A c t a 67, 197 [1963].
22 P. Talalay, M. M. Dobson&L D. F. Tapley, N a t u r e [ L o n d o n ] 170, 620 [1952].
23 P. J. Marcus & P. Talalay, Proc. R o y . Soc. [ L o n d o n ] Ser. B. 144, 116 [1955].
24 P. Talalay, R e c o r d C h e m . Progr. K r e s g e - H o o k e r Sci. Lib. 18, 31 [1957].
25 P. Talalay & H. R. Levy in G. E. W. Wolstenhome & C. M. O Conner: T h e Steric C o u r s e of M i c r o
biological Reactions (Ciba F o u n d a t i o n Study G r o u p N o . 2) Churchill, L o n d o n 1959, p . 53.
Prostaglandins
Erik Anggard and Bengt Samuelsson
Prostaglandins are a class of closely related lipids formed from essential fatty acids, which exert their action
on s m o o t h muscle, lipid metabolism a n d m e m b r a n e t r a n s p o r t (for a review, see ). T h e y occur in high 1
c onc e ntra tion (ca. 1 0 " M ) in h u m a n seminal fluid a n d in lower concentration ( 1 0

5 - 8
to 1 0 " 1 0
M ) in various
tissues, such as lung, brain, kidneys, intestine, thyroid glands, the iris, in the e n d o m e t r i u m a n d in m a n y
other tissues . Stimulation of the nerves or injection of certain drugs releases p r o s t a g l a n d i n s into the b l o o d
1
stream at c o n c e n t r a t i o n s of 1 0 " to 1 0 "

8 1 0
M . 1
Prostaglandins are oxidized by a N A D - l i n k e d 15 h y d r o x y - p r o s t a g l a n d i n d e h y d r o g e n a s e , 1 5 - O H - P G D H

( l l a , 1 5 - D i h y d r o x y - 9 - o x o p r o s t - 1 3 - e n o a t e : N A D 15-oxidoreductase, E C 1.1.1.141); the secondary alcohol
g r o u p on C 1 5 is oxidized to a k e t o n e . This enzymatic reaction is specific for p r o s t a g l a n d i n s a n d is the
2
basis of the m e t h o d described here.
Application of Method: A t the present stage of the development of this field the m e t h o d is suitable for the
assay of assorted mixtures of prostaglandins. It should be suitable for the identification of p r o s t a g l a n d i n s
in biological material a n d to differentiate the stereoisomers in mixed synthetic p r o s t a g l a n d i n s .
Principle
(1) Prostaglandin + N A D + 1 5
~ Q H
~ P G D H
> 15-Dehydroprostaglandin + N A D H + H +
Glyceraldehyde-3-P G A P D H
* > Glycerate-3-P
(2) Glutamate < 2-Oxoglutarate + NH 3
(3) Glutamate + Hydrazine + N A D + G 1 P H

* * > 2-Oxoglutarate h y d r a z o n e + N A D H + NH 4
+
T h e N A D H which is formed in stoichiometric a m o u n t s is m e a s u r e d in a fluorimeter. W i t h 1 0 " 1 0

mole P G
only first reaction is used. W i t h smaller a m o u n t s ( 1 0 " 1 0
to 1 0 " 1 2
mole) the " e n z y m a t i c r e c y c l i n g " m e t h o d
3
of Matschinsky et a l . is used to detect the N A D H (reaction 2 a n d 3). In this m e t h o d the N A D H formed in

4
the first reaction is amplified so t h a t it can be measured by fluorimetry. T h e principle is discussed further o n
p . 135.
* Glyceraldehyde-3-phosphate dehydro genase (D-glyceraldehyde-3-phosphate: N A D oxidoreductase,

p h o s p h o r y l a t i n g , E C 1.2.1.12).
** G l u t a m a t e de hydro genase ( L - g l u t a m a t e : N A D oxidoreductase, d e a m i n a t i n g , E C 1.4.1.2).
The rate of the reaction is o p t i m u m at p H 9.0. Higher p H values should not be used because some prosta
glandins are alkali-labile. T h e presence of S H protective reagents, such as m e r c a p t o e t h a n o l o r dithiothreitol
(Cleland's reagent) is necessary for steps (1) and (2). The Michaelis constants of P G D H for prostaglandin 5
E is 7.7 / / M ; for prostaglandin E 12 uM \ for prostaglandin E 13 uM; for prostaglandin F

x 2 3 x 25 uM; for
prostaglandin F ^ 31 / i M ; for prostaglandin A
2 x 14 fiM and for prostaglandin A 25 /iM. 2
Equipment
S p e c t r o f l u o r i m e t e r o r filter f l u o r i m e t e r . F o r t h e latter t h e filter s h o u l d h a v e m a x i m u m t r a n s -

m i t t a n c e a t 3 6 0 n m ( p r i m a r y filter) o r 4 5 6 n m ( s e c o n d a r y filter). F o r i n c r e a s e d s e n s i t i v i t y a n d
l o w e r c o n s u m p t i o n o f r e a g e n t s m i c r o c u v e t t e s ( 1 0 0 /d.) c a n b e u s e d . T h e F a r r a n d fluorimeter,
m o d e l A - 3 is s u i t a b l e for this a s s a y . P r i m a r y filter: C o r n i n g 5 8 6 0 ; s e c o n d a r y filters: C o r n i n g
3387, 4308 and 5562.
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , 16. G l y c e r a l d e h y d e - 3 - p h o s p h a t e , GAP
tris, A . R . crystalline d i c y c l o h e x y l a m m o n i u m salt of the
2. S o d i u m h y d r o x i d e , 0.1 N , A . R . diethylacetal; commercial p r e p a r a t i o n , see p. 539.
3. H y d r o c h l o r i c a c i d , 5 N a n d 1 N , A . R . 17. P r o s t a g l a n d i n s u b s t r a t e s
4. 2-Mercaptoethanol experimental samples in small a m o u n t s can be
5. D i t h i o t h r e i t o l , Cleland's reagent obtained from D r . J. E. Pike, T h e Upjohn Co.,
e. g. from Calbiochem, A grade K a l a m a z o o , Mich., U S A .
6. D i s o d i u m h y d r o g e n a r s e n a t e , 18. G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o
Na HAs0 -7H 0,
2 4 2 A.R. genase, G A P D H
7. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , from yeast, crystalline suspension in 3.2 M a m
K HP0 ,
2 4 A.R. m o n i u m sulphate solution, ^ 8 0 U / m g . (25 °C);
8. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , commercial p r e p a r a t i o n , see p . 466.
K H P 0 , A.R.
2 4 19. G l u t a m a t e d e h y d r o g e n a s e , G I D H
9. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E D T A from ox liver, solution in 5 0 % glycerol, free from
disodium salt, EDTA-Na H 2 2 •2H 0,
2 A.R. a m m o n i u m ions, ^ 4 5 U / m g . (25 °C); c o m m e r c
10. A m m o n i u m a c e t a t e , C H C O O N H , 3 4
ial p r e p a r a t i o n , see p. 4 6 1 .
A.R. 20. 15-Hydroxyprostaglandin dehydrogen
11. H y d r o g e n peroxide, H 0 , 2 2 A.R., 30% ase, 1 5 - O H - P G D H
( w / w ) , s p . gr. 1.11 from pig lung. P r e p a r a t i o n according to Anggard
12. H y d r a z i n e h y d r a t e , N H O H , a b o u t
2 5 a n d Samuelsson , 2
see A p p e n d i x , p . 1883. G o o d
24% (w/w) preparations have only slight e n d o g e n o u s fluor
13. 2 - O x o g l u t a r a t e , O x o G escence. ;> 2 m U / m g . (37 ° C ; p H 7.4) with
crystalline free a c i d ; commercial p r e p a r a t i o n , prostaglandin E as substrate).
x
see p. 548. 21. Sulphuric acid, 2 M

14. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , 22. Ethanol, 9 6 % (v/v)
NAD 23. Albumin
free acid; commercial p r e p a r a t i o n , see p. 545. from bovine serum.
15. A d e n o s i n e d i p h o s p h a t e , A D P 24. Sodium hydrogen carbonate, NaHC0 , 3
disodium salt, A D P - N a ; commercial p r e p

2 A.R.
aration, see p . 525.
Prostaglandins 1879
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y d i s t i l l e d o r d e i o n i z e d w a t e r . T h e e n d o g e n o u s fluor

e s c e n c e o f t h e s o l u t i o n s s h o u l d b e l o w , if n o t treat t h e s o l u t i o n s w i t h a c i d - w a s h e d a c t i v a t e d
charcoal to remove the fluorescent contaminants.
I. Tris buffer ( 5 0 m M , p H 9 . 0 ; 1 m M d i t h i o t h r e i t o l ) :
D i s s o l v e 0 . 6 0 5 g. tris in d i s t i l l e d w a t e r , a d j u s t t o p H 9.0 w i t h c a . 0.5 m l . 1 N H C 1 ( g l a s s
electrode), a d d 15.4 m g . dithiothreitol a n d dilute to 100 ml. with distilled water.
II. P o t a s s i u m p h o s p h a t e buffer (0.1 M ; p H 7 . 5 ) :
D i s s o l v e 1 7 . 4 g. K H P 0 2 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r a n d 1 3 . 6 g. K H P 0
2 4 in 1 0 0 0 m l .
distilled water. M i x 852 ml. o f the K H P 0 2 4 s o l u t i o n w i t h 148 m l . o f t h e KH P0 2 4
solution.
III. H y d r a z i n e buffer ( 0 . 2 M ; p H 9 . 0 ) :
D i l u t e 4 . 1 9 m l . h y d r a z i n e h y d r a t e w i t h c a . 30 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 9.0 w i t h
c a . 12 m l . 1 N H C 1 ( g l a s s e l e c t r o d e ) a n d d i l u t e t o 100 m l . w i t h d i s t i l l e d w a t e r .
IV. Prostaglandin E x or F lflt (1 m M ) :
D i s s o l v e 3.5 m g . c r y s t a l l i n e p r o s t a g l a n d i n in a f e w d r o p s o f e t h a n o l , a d d 2 m l . tris (1 M ;
p H 7.5) a n d d i s t i l l e d w a t e r t o 10 m l .
V. N i c o t i n a m i d e - a d e n i n e dinucleotide (10 m M ) :
D i s s o l v e 7 4 m g . N A D in d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
VI. A d e n o s i n e d i p h o s p h a t e (0.1 M ) :
Dissolve 54 mg. A D P - N a 2 in 0.6 m l . 0.5 N N a O H t o g i v e p H 13 a n d w a r m for 2 0 m i n .
at 6 0 ° C t o d e s t r o y a n y N A D c o n t a m i n a t i n g t h e A D P . A d j u s t t o p H 7.0 w i t h c a . 0 . 1 5 m l .
1 N HC1 a n d dilute with distilled water to 1 ml.
VII. G l y c e r a l d e h y d e - 3 - p h o s p h a t e (50 m M D - G A P ) :
Dissolve 47.3 mg. D L - G A P ( C H A ) 2 in 1 m l . distilled w a t e r , a d j u s t to p H ca. 2 w i t h
2 M H S0 2 4 a n d i n c u b a t e for 2 m i n . in a w a t e r b a t h at a b o u t 8 0 ° C . A d j u s t s o l u t i o n
c a u t i o u s l y t o p H c a . 5 w i t h s o d i u m b i c a r b o n a t e . S t o r e at - 2 0 ° C .
V I I I . S t o c k s o l u t i o n for " e n z y m a t i c r e c y c l i n g " (0.1 M p h o s p h a t e , p H 7 . 5 ; 10 m M N a H A s 0 ; 2 4
5 m M 2 - o x o g l u t a r a t e , 0.3 m M A D P ; 10 m M E D T A ; 5 m M m e r c a p t o e t h a n o l ; 5 m M
C H C O O N H ; 0.02% albumin):
3 4
Dissolve 780 mg. N a H A s 0 - 7 H 0 , 2 4 2 180 m g . 2-oxoglutarate, 9 3 0 m g . EDTA-Na ; 2
96 mg. C H C O O N H 3 4 a n d 5 0 m g . a l b u m i n in ca. 2 0 0 m l . p h o s p h a t e buffer ( I I ) ; a d d

0 . 7 5 m l . A D P s o l u t i o n ( V I ) a n d 0 . 0 8 8 m l . m e r c a p t o e t h a n o l . D i l u t e t o 2 5 0 m l . w i t h buffer
II. D e e p - f r e e z e in 2 t o 4 m l . p o r t i o n s at —20 ° C o r b e l o w .
I X . G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e (ca. 10 m g . p r o t e i n / m l . ) :
P i p e t t e 0.1 m l . o f t h e e n z y m e s u s p e n s i o n i n t o a s m a l l c e n t r i f u g e t u b e , c e n t r i f u g e , p i p e t t e
off t h e s u p e r n a t a n t fluid a n d d i s s o l v e t h e p r e c i p i t a t e in 0 . 0 9 m l . p h o s p h a t e buffer II
(containing 0.1% albumin, 5 m M E D T A and 5 m M mercaptoethanol).
X . G l u t a m a t e d e h y d r o g e n a s e ( c a . 10 m g . p r o t e i n / m l . ) :
If n e c e s s a r y , d i l u t e t h e s o l u t i o n w i t h 5 0 % g l y c e r o l .
XI. P r o s t a g l a n d i n d e h y d r o g e n a s e (ca. 10 m g . / m l . ) :
P i p e t t e 0.1 m l . e n z y m e s u s p e n s i o n i n t o a s m a l l c e n t r i f u g e t u b e , c e n t r i f u g e , d e c a n t t h e
s u p e r n a t a n t fluid a n d d i s s o l v e t h e p r e c i p i t a t e in 0 . 0 9 m l . p h o s p h a t e buffer (II) c o n t a i n i n g
5 m M E D T A and 1 m M dithiothreitol.
X I I . H y d r o g e n peroxide/hydrochloric acid (ca. 0.44 M H 0 ; 5 N HC1): 2 2
A d d 0.45 m l . 3 0 % H 0 2 2 t o 10 m l . c o l d 5 N H C 1 .
Store solutions I - I V , IX and X in a refrigerator at ca. 0 - 4 °C. They are stable for a b o u t 1 m o n t h , except for
solution IX which is stable for a week. Store solutions V-VIII at —15 °C. Use solution X I I within 1 hr.
Procedure
Collection:
D i s s o l v e t h e m a t e r i a l t o b e a n a l y s e d in 9 6 % e t h a n o l o r in a s o l u t i o n buffered at p H 8.0. S m a l l
a m o u n t s o f s a m p l e in e t h a n o l i c s o l u t i o n c a n b e p i p e t t e d d i r e c t l y i n t o a 100 /d. m i c r o c u v e t t e
a n d t h e e t h a n o l r e m o v e d in a s t r e a m o f n i t r o g e n . E t h a n o l c o n c e n t r a t i o n s l o w e r t h a n 2 % d o
n o t i n h i b i t t h e r e a c t i o n . F o r the i s o l a t i o n o f p r o s t a g l a n d i n f r o m b i o l o g i c a l m a t e r i a l , s e e ' . 6 7
Assay System
T h e direct a s s a y is u s e d for ca. 1 0 " 1 0

m o l e p r o s t a g l a n d i n / a s s a y . It a l l o w s t h e r e a c t i o n t o b e
f o l l o w e d in t h e c u v e t t e . P r e f e r a b l y a large a m o u n t o f s a m p l e is a d d e d first t o c h e c k w h e t h e r
it c o n t a i n s a n y s u b s t r a t e for P G D H . T h e n a c c o r d i n g t o t h e result o f this p r e l i m i n a r y a s s a y ,
t h e a m o u n t a d d e d is r e d u c e d t o g i v e a c u v e t t e c o n c e n t r a t i o n o f 0 . 5 - 5 0 pM a n d t h e n t h i s is u s e d
as t h e a c t u a l a s s a y .
P r i m a r y w a v e l e n g t h : 3 6 0 n m ; s e c o n d a r y w a v e l e n g t h : 4 5 6 n m ; v o l u m e : 0.11 m l . ; room
temperature; read against a blank (distilled water instead o f substrate). A l w a y s analyse a
standard.
Pipette into fluorimeter micro-cuvettes: C o n c e n t r a t i o n in a s s a y m i x t u r e
Tris/buffer/dithiothreitol (I) 9 0 iA. 5 0 m M tris,

1 m M dithiothreitol
1 5 - O H - P G D H solution (XI) 5 /il. 5 0 0 fig. P G D H / m l . = 1 m U / m l .
N A D solution (V) 10 /il. 1 mM
Read fluorescence F t
Sample or standard solution (IV) 5 pi. 0 . 5 - 5 0 /iM PG
M i x . Wait for c o m p l e t i o n o f t h e r e a c t i o n (ca. 4 5 m i n . )

a n d read fluorescence F . A F = F — F , is u s e d for
2 2
the calculations.
If o n l y a s e m i - q u a n t i t a t i v e e s t i m a t i o n o f p r o s t a g l a n d i n is r e q u i r e d , s t o p t h e r e a c t i o n after
3 0 m i n . i n c u b a t i o n at 37 ° C b y b o i l i n g a n d after c o o l i n g , m e a s u r e t h e fluorescence.
Prostaglandins 1881
Calculations
Co , = ^ ^Sample x c
^Sample i p ' x
^Standard
^ Standard
r
c = fig. p r o s t a g l a n d i n / m l . c u v e t t e v o l u m e
T h e c a l c u l a t i o n s d e p e n d o n t h e fact t h a t t h e e q u i l i b r i u m p o s i t i o n o f t h e P G D H r e a c t i o n for
t h e p r o s t a g l a n d i n in t h e s a m p l e s o l u t i o n a n d in t h e s t a n d a r d s o l u t i o n is i d e n t i c a l , a s i t u a t i o n
which does not always apply.
Assay System (Determination by "enzymatic cycling")
Stage 1: Enzymatic conversion of prostaglandin
I n c u b a t i o n t e m p e r a t u r e : 37 ° C ; i n c u b a t i o n v o l u m e : 6 fi\.
XIII. Reagent mixture:
Immediately before use mix and dissolve:
Tris buffer (I) 100 ml.
Albumin 30 mg.
Mercaptoethanol 14 fil
NAD 12.4 mg.
M i x 1 m l . o f this s o l u t i o n w i t h 0.1 m l . 1 5 - O H - P G D H s o l u t i o n ( X I )
P r e p a r e s t a n d a r d s a n d s a m p l e s in d u p l i c a t e in t h e o r d e r o f 1 0 " 1 2
to 5 x 10" 1 1
mole. Also
a n a l y s e a b l a n k (distilled w a t e r i n s t e a d o f s a m p l e ) .
Pipette into micro-tubes: C o n c e n t r a t i o n in a s s a y m i x t u r e
Reagent mixture (XIII) 5 fil 5 0 m M tris, 0 . 0 3 % a l b u m i n ,

2 m M mercaptoethanol,
0.2 m M N A D ,
1 mg. 1 5 - O H - P G D H / m l . = 2 m U / m l
Sample or standard solution (IV) 1 ixl 0 . 5 - 1 0 fiM prostaglandin
M i x a n d i n c u b a t e f o r 3 0 m i n . at 37 ° C .
NaOH, 1 N 1 ill ca. 0.2 N
M i x , h e a t for 30 m i n . at 6 0 ° C , a l l o w t o c o o l a n d
u s e t h e s o l u t i o n for S t a g e 2.
Stage 2: "Enzymatic cycling"/Stage 3: Glutamate determination.
Prepare immediately before u s e :
X I V . R e a g e n t mixture for "cycling"

T h e e n z y m e activity gives a cycling factor o f a b o u t 1 6 0 0 .
Stock solution for

"enzymatic cycling" (VIII) 2.000 ml. (80% o f the cone, o f solution
VIII)
G A P solution (VII) 0.222 ml. (5 m M )
G A P D H solution (IX) 0.10 ml. (50/ig./ml.)
G I D H solution (X) 0.020 ml. (100 /ig./ml.)
XV. R e a g e n t mixture for glutamate determination
H y d r a z i n e buffer (III) 47.50 ml. (0.2 M ; p H 9.0)

A D P solution (VI) 0.10 ml. (0.2 m M )
N A D solution (V) 2.50 ml. (0.5 m M )
G I D H solution (X) 0.15 ml. (30 fig./ml)
Pipette into ice-cold 3 ml. fluorimeter tubes: Concentration in assay mixture
R e a g e n t mixture for "cycling" (XIV) 100 m M p h o s p h a t e , 10 m M

Na HAs0 ,
2 4
5 m M O x o G , 0.3 m M A D P ,
10 m M E D T A ,
5 m M mercaptoethanol, 5 m M
C H C O O N H , 0.02% albumin,
3 4
5 mM GAP,
50 pg. G A P D H / m l . , 1 0 0 pg.
GIDH/ml. = 4 U GAPDH/ml.;
4.5 U G I D H / m l .
Portion o f stage 1 0 . 0 5 - 1 pM p r o s t a g l a n d i n
M i x , incubate for 60 m i n . and t h e n c o o l .
H 0 / H C 1 solution
2 2
(XII) 5 pi 0.15% H O ; 0 . 5 N H C l
2 2
M i x , p l a c e t u b e s f o r 10 m i n . in a b o i l i n g w a t e r b a t h
and then allow to cool.
R e a g e n t mixture for glutamate

determination (XIV) 200 m M hydrazine, 0.2 m M A D P ,
0.5 m M N A D , 3 0 pg. G I D H / m l . =
1.4 U / m l .
M i x a n d i n c u b a t e s a m p l e s for 1 hr. at r o o m t e m p e r a
ture o r 3 0 m i n . at 37 ° C . R e a d t h e fluorescence a g a i n s t
the blank.
Prostaglandins 1883
Calculations
f> — x
Sample y c
^Sample p ^ ^Standard
^Standard
c ug. prostaglandin/ml. cuvette volume.
T h e purity of the 1 5 - O H - P G D H is a decisive factor in determining the e n d o g e n o u s fluorescence of the assay

system. I m p u r e enzyme p r e p a r a t i o n s have also the tendency to slowly reduce N A D in the absence of
prostaglandins. F o r information on the technique of enzymatic cycling a n d h o w to detect errors, see p . 2059.
Sensitivity
U n d e r the conditions described here the lower limit of the d e t e r m i n a t i o n s is 1 0 ~ 1 0

mole p r o s t a g l a n d i n El
in the direct assay a n d 1 0 " 1 2

mole p r o s t a g l a n d i n E in the cycling m e t h o d .
l
Appendix
Isolation of Prostaglandin Dehydrogenase from Pig Lung 2
Tissues, Reagents and Apparatus
Place lungs from healthy pigs (male or female, even castrated), immediately after slaughter in dry ice and
store at — 20 °C until w o r k e d u p .
Sephadex G-100 a n d D E A E - S e p h a d e x A-25 from P h a r m a c i a , U p p s a l a (Sweden).
Preparative disc electrophoresis a p p a r a t u s from Buchler Instruments, N e w Jersey ( U S A ) . Ultra-Turrax
homogenizer model 45/6 and 18/2 from J a n k e & K u n k e l , Stauffen i. Br. ( G e r m a n y ) . Ultracentrifuge Spinco
L-2-65 with r o t o r 19. Ultrafiltration a p p a r a t u s , Diaflow with 600 ml. cells, filter m e m b r a n e s U M - 1 0 ,
A m i c o n C o r p . , C a m b r i d g e , Mass. ( U S A ) .
Buffer A : 0.1 M potassium p h o s p h a t e buffer; 1 m M E D T A ; 0 . 0 5 % m e r c a p t o e t h a n o l ; p H 7.4.
Buffer B : as for buffer A, but with 10 m M p h o s p h a t e .
Glass distilled a n d deionized water for all stages of the m e t h o d .
Determination of Activity
Oxidized prostaglandin E develops a strong but unstable colour after t r e a t m e n t with alkali.
x
Incubate 0.01 - 0 . 1 0 ml. enzyme sample with 0.25 /rniole N A D and 30 n m o l e p r o s t a g l a n d i n E for 45 min. at t
p H 7.4 and 37 °C. (final volume 0.10 ml.). A d d 0.5 ml. 0.5 N N a O H and read extinction at 500 n m when the
highest value is reached (after ca. 1 min.). A n arbitary unit is defined as A E = 1, which c o r r e s p o n d s to the
oxidation of ca. 30 n m o l e p r o s t a g l a n d i n Ei in 45 min., o r 6.7 x 1 0 ~ U ( I n t e r n a t i o n a l ) .
4
Isolation
Stage 1. Cut u p 2 kg. lung in the frozen state, add 6000 ml. cold buffer A a n d pass t h r o u g h a mincer.
H o m o g e n i z e the suspension in 2000 ml. p o r t i o n s in an Ultra-Turrax 45/6 a n d then h o m o g e n i z e again in
250 ml. portions in an U l t r a Turrax 18/2. Centrifuge at 2500 g for 30 min. a n d pass the s u p e r n a t a n t fluid
t h r o u g h a wire sieve. Centrifuge the filtrate at ca. 45 000 for 1 hr.
Stage 2. A d d 180 g. ( N H ) S 0 / l i t r e to the clear red s u p e r n a t a n t fluid and stir overnight. Centrifuge off the
4 2 4
precipitate and add a further 160 g. ( N H ) S 0 / l i t r e to the s u p e r n a t a n t fluid. After 4 - 6 hr. collect the
4 2 4
precipitate by centrifugation at 10000 g and dissolve in the smallest possible volume of buffer B. First dialyse
against buffer B and then against buffer A.
Stage 3. P r e p a r e a c h r o m a t o g r a p h y c o l u m n (10 cm. x 100 cm.), with a bed v o l u m e of ca. 81., a n d e q u i p m e n t
for ascending c h r o m a t o g r a p h y . Equilibrate Sephadex G-100 for several days with buffer A, deaerate u n d e r
v a c u u m a n d a d d to the c o l u m n . Bring the dialysed solution ( 2 0 0 - 4 0 0 ml.) from stage 2 to the b o t t o m of the
c o l u m n by m e a n s of a p u m p ( 1 - 2 ml./min.). Collect fractions of 8 0 - 1 0 0 ml. In every second fraction
determine protein (measurement at 280 n m ) a n d enzyme activity. T h e enzyme a p p e a r s rather near to the
position of h a e m o g l o b i n , which can be used to locate the enzyme by its a b s o r p t i o n at 465 n m . C o m b i n e the
active fractions a n d dialyse t h o r o u g h l y against buffer B.
Stage 4. Fill a c h r o m a t o g r a p h y c o l u m n (2 cm. x 25 cm.) with D E A E - S e p h a d e x a n d equilibrate with buffer
B. Allow the dialysed solution from stage 3 to run quickly o n t o the c o l u m n . T h e enzyme is adsorbed, while
the h a e m o g l o b i n runs t h r o u g h . Start a gradient elution with 700 ml. buffer B in the mixing cylinder and
700 ml. 0.15 M N a C l + 30 m M p h o s p h a t e buffer, p H 7.4 in the reservoir. K e e p the flow rate at 30 ml./hr.
with a p u m p . Collect 10 ml. fractions. C o m b i n e the fractions with the highest specific activity. Reduce the
v o l u m e to 3 0 - 5 0 ml. by ultrafiltration.
Stage 5. S t a n d a r d c o n d i t i o n s are used for the preparative disc-gel electrophoresis, except that all buffer
8
solutions contain 0 . 0 5 % m e r c a p t o e t h a n o l a n d 1 m M E D T A . Migration to a n o d e at p H 10.3. Dialyse the

enzyme against the buffer of the u p p e r reservoir, a d d sucrose to a final c o n c e n t r a t i o n of 3.5% a n d layer the
concentrating gel with this solution. T h e acrylamide c o n c e n t r a t i o n in the gel is 7 . 5 % . A p p l y 3 0 0 - 6 0 0 volts
at 5 0 - 1 0 0 Watts. Elute the protein b a n d s with a p u m p and collect in a cooled fraction collector. Keep the
t e m p e r a t u r e in all the cooling systems at 4 °C. T h e enzyme is generally eluted after a m a i n protein band
24 hr. after the start. T h e purity can be increased by further electrophoresis with less protein a n d higher
voltage; this results in less spreading of the b a n d s by diffusion. C o m b i n e the active fractions a n d concentrate
by ultrafiltration to a b o u t 10 ml. T h e enzyme can be stored at —20 °C deep-frozen or as a suspension in
2 . 5 - 3 . 0 M a m m o n i u m sulphate solution at 4 °C. It is stable for u p to 1 m o n t h .
T h e following Table gives the result of a representative purification.
Fraction Units Protein Specific Yield

mU(37°C) g. activity %
m U / m g . (37 °C)
Soluble fraction 342 95 0.004 100

(stage 1)
( N H ) S 0 precipitate
4
(stage 2)
2 4
* 16 — —
After Sephadex G-100 c h r o m a t o g r a p h y 315 3.2 0.10 92

(stage 3)
After D E A E - S e p h a d e x c h r o m a t o g r a p h y 309 1.1 0.28 90
(stage 4)
After disc-electrophoresis 215 0.12 1.80 63
(stage 5)
* N o sufficiently accurate m e a s u r e m e n t s were m a d e because of inhibitors.
References
1 S. Bergstrom, L. A. Carlsson & /. E. Weeks, P h a r m a c o l . Revs. 20, 1 [1968].

2 Anggard&B. Samuelsson, Arkiv for K e m i 25, N r . 27, 293 [1966].
Prostaglandins 1885
3 O. H. Lowry, J. V. Passonneau, D. W. Schultz & M . K. Rock, J. biol. C h e m . 236, 2746 [1961].

4 F. M. Matschinsky, D. L. Rutherford & L. Guerra, Proc. 3rd Internat. C o n g r . H i s t o c h e m . C y t o c h e m . ,
N e w York 1968.
5 W. W. Cleland, Biochemistry 3, 480 [1964].
6 B. Samuelsson, J. biol. C h e m . 238, 3329 [1963].
7 S. W. Holmes, E. W. Horton & M. J. Stewart, Life Sciences 7, 349 [1968].
8 L. Ornstein & B. Davis, A n n . N e w York A c a d . Sciences 121, 321 & 404 [1964].
Bile Acids
Friedrich Wilhelm K o s s , Dieter Mayer and H a n s Haindl
T h e bile acids which occur mainly in h u m a n s are the glycine a n d taurine conjugates of chenodeoxycholic
acid, deoxycholic acid a n d cholic acid. These mixtures of substances can be determined q u a n t i t a t i v e l y ' by 1 2
oxidation of the 3a-hydroxyl g r o u p c o m m o n to all bile acids with 3a-hydroxysteroid dehydrogenase

from Pseudomonas testosteroni ( 3 a - H y d r o x y s t e r o i d - N A D ( P ) oxidoreductase, EC 1.1.1.50).
Application of Method: In biochemistry a n d in clinical chemistry for the d e t e r m i n a t i o n of bile acids in

bile, d u o d e n a l juice and serum.
Principle
-NH-CH -COOH 2
-NH-CH -CH -S0 H

2 2 3
3 a-Hydroxycholanic acids are oxidized to 3-keto acids. If the 3-keto acids are t r a p p e d as the hydrazones
the reaction is quantitative a n d o n e mole N A D H c o r r e s p o n d s to 1 mole of bile acid. F o r the determination
of bile acids in serum it is first necessary t o extract the bile acids before the enzymatic assay.
Quantitative oxidation is obtained at p H 9.5 with addition of hydrazine to t r a p the oxo-acids formed.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r for m e a s u r e m e n t s at 3 4 0 , 3 3 4 o r 3 6 5 n m ;
b e n c h c e n t r i f u g e for 100 m l . a n d 1 m l . t u b e s .
Reagents
For the enzymatic assay:
1. G l y c i n e , A . R . 4. 3 a-Hydroxysteroid dehydrogenase
2. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , from Pseudomonas testosteroni, purified, ca.
NAD 0.5 U / m g . (25 °C). C o m m e r c i a l preparation, see
free acid; c o m m e r c i a l p r e p a r a t i o n , see p . 545. p. 476.
3. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E D T A 5. S o d i u m h y d r o x i d e , 2.5 N
d i s o d i u m salt, E D T A - N a H - 2 H 02 2 2 6. H y d r a z i n e s u l p h a t e , A . R.
Bile Acids 1887
For the extraction of bile acids from serum:
7. A n i o n e x c h a n g e r e s i n 10. E t h a n o l , A . R .
D o w e x 1 x 8 (chloride form), 2 0 - 5 0 mesh 11. Diethyl ether, A . R .
8. C a t i o n e x c h a n g e r e s i n 12. M e t h a n o l , A . R .
D o w e x 50 W x 2 ( H - f o r m ) , 5 0 - 1 0 0 mesh
+
13. Ethyl acetate, A . R.
9. T e t r a - n - h e p t y l a m m o n i u m i o d i d e 14. S o d i u m h y d r o x i d e , A . R.
e. g. Eastman Organic Chemicals
U s e o n l y fresh, d o u b l y d i s t i l l e d w a t e r .
I. G l y c i n e buffer, (1 M ; p H 9 . 5 ) :
D i s s o l v e 7.5 g. g l y c i n e , 0 . 8 7 g. h y d r a z i n e s u l p h a t e , 0 . 2 g. E D T A i n c a . 2 0 m l . d i s t i l l e d
w a t e r . A d j u s t t o p H 9.5 w i t h 2.5 N N a O H a n d d i l u t e t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r .
II. 3 a - H y d r o x y s t e r o i d d e h y d r o g e n a s e ( 2 m g . p r o t e i n / m l . ) :
D i s s o l v e 2 m g . o f t h e e n z y m e p r e p a r a t i o n in 1 m l . buffer (I).
III. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 5 0 m M / ? - N A D ) :
D i s s o l v e 4 0 m g . N A D in 1 ml. distilled water.
IV. Tetra-n-heptylammonium chloride, T H A C 3
(5% w/v):
D i s s o l v e 5 g. t e t r a - n - h e p t y l a m m o n i u m i o d i d e in c a . 1 5 0 m l . e t h a n o l / w a t e r 85 : 15 ( V I I )
and convert to the chloride form by passing through D o w e x 1 x 8 (CI"form; column
c a . 2 0 c m . h i g h , i n t e r n a l d i a m e t e r c a . 1.2 c m . ) . A f t e r e v a p o r a t i o n o f t h e s o l v e n t t h e T H A C
r e m a i n s a s a n o i l y r e s i d u e . P r e p a r e a 5% s o l u t i o n o f T H A C i n e t h y l a c e t a t e .
V . E t h a n o l / d i e t h y l e t h e r ( 3 0 : 10)
VI. Methanol/water (70 : 30)
V I I . E t h a n o l / w a t e r ( 8 5 : 15)
Stability of Solutions and Enzyme
3<x-Hydroxysteroid dehydrogenase is stable for ca. 6 m o n t h in the dry state at - 2 0 °C. P r e p a r e the
enzyme solution freshly a n d , if necessary, store for u p t o 1 week at 0 - 4 °C. P r e p a r e the buffer a n d N A D
solution freshly each week a n d store at 0 - 4 °C.
Procedure
A s s a y bile a n d d u o d e n a l j u i c e w i t h o u t further t r e a t m e n t .
Extraction and purification of bile acids from serum :
D e p r o t e i n i z e 1 - 5 m l . s e r u m in a 1 0 0 m l . t u b e , c e n t r i f u g e w i t h 2 x 5 0 m l . e t h a n o l / d i e t h y l
e t h e r 3 0 : 10 ( V ) (bile a c i d s are i n t h e s u p e r n a t a n t fluid after c e n t r i f u g a t i o n ) . E v a p o r a t e t h e
s u p e r n a t a n t fluid t o d r y n e s s , t a k e u p t h e r e s i d u e in 5 m l . d i s t i l l e d w a t e r a n d s h a k e t w i c e w i t h
5 m l . e t h y l a c e t a t e t o r e m o v e n o n - p o l a r l i p i d s (bile a c i d s are in t h e a q u e o u s p h a s e ) .
E x t r a c t t h e a q u e o u s p h a s e t w i c e w i t h 5 m l . 5% T H A C s o l u t i o n ( I V ) (bile a c i d s are in the o r g a n i c

phase).
E v a p o r a t e t h e o r g a n i c p h a s e t o d r y n e s s , t a k e u p t h e o i l y r e s i d u e in 5 m l . m e t h a n o l / w a t e r 7 0 : 3 0
( V I ) . R e c o v e r t h e bile a c i d s o n D o w e x 5 0 x 2 ( h e i g h t o f c o l u m n 5 t o 7 c m . , i n t e r n a l d i a m e t e r
c a . 1 c m . ) a n d w a s h t h e resin w i t h 2 0 - 3 0 m l . m e t h a n o l / w a t e r 7 0 : 3 0 ( V I ) .
E v a p o r a t e off t h e s o l v e n t a n d t a k e u p t h e r e s i d u e in 0.7 m l . buffer (I). A c c o r d i n g t o t h e bile
a c i d c o n t e n t , d i l u t e 0 . 1 - 0 . 6 m l . o f t h e s o l u t i o n t o 0 . 6 m l . w i t h buffer (I). P r o c e e d w i t h t h e
s p e c t r o p h o t o m e t r i c a s s a y w i t h o u t further d i l u t i o n w i t h buffer.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 o r H g 3 6 5 ) n m ; s e m i - m i c r o c u v e t t e s , light p a t h : 1 c m . ; final v o l u m e :
6 4 0 pi.; 30 ° C . P r e f e r a b l y f o l l o w t h e c o u r s e o f t h e r e a c t i o n w i t h a r e c o r d e r .
Pipette into cuvettes : C o n c e n t r a t i o n in a s s a y m i x t u r e
Sample 2 0 ptl.
Buffer (I) 6 0 0 pi. ca. 0.95 M
N A D solution (III) 10 ca. 1 m M
M i x a n d a s s o o n a s t h e e x t i n c t i o n is c o n s t a n t r e a d
extinction E . t
Enzyme solution (II) 10/il. c a . 32 / / g . / m l .
T h e r e a c t i o n is c o m p l e t e after 3 - 1 5 m i n . R e a d e x
tinction E . 2
Determine t h e i n c r e a s e in e x t i n c t i o n due to the

e n z y m e b y a further a d d i t i o n o f 10 fil. e n z y m e s o l u t i o n
(II) o n c o m p l e t i o n o f t h e r e a c t i o n . R e a d e x t i n c t i o n E . 3
( E - E j ) ( E - E ) = A E is u s e d for t h e c a l c u l a t i o n s .
2 3 2
Calculations
U n d e r the conditions given above the reaction proceeds stoichiometrically a n d therefore the general
formula (2) on p. 312 applies for the calculation of the concentration of the bile acids in the solution used
for the assay. In the case of serum the factor F resulting from the extraction of the bile acids must be
included.
c = AEx 5.25 x F AE x 5.14 x F AE x 9.41 x F [^mole/ml.]
F o r the determination of bile acids in bile the s t a n d a r d deviation of the individual values is 2 . 5 % (n = 10).
In the extraction of bile acids from serum 8 0 % recovery is obtained. T h e s t a n d a r d deviation for the
extraction and enzymatic assay is 4 . 3 % (n = 10).
Bile Acids 1889
N o r m a l Values
N o r m a l values in serum < 0.005 /zmole/ml.

Pathological values in serum u p t o 0.3 ^ m o l e / m l .
N o r m a l value in bile (T-drain) 2 0 - 4 0 ^mole/ml.
S o u r c e s o f Error and Specificity o f M e t h o d
All 3a-hydroxysteroids of the C , 19 C2l and C 2 4 series are oxidized in this m e t h o d .
Special Details
F o r special studies the bile acids can be separated by thin-layer c h r o m a t o g r a p h y a n d then determined
enzymatically (extraction from the kiesel gel with m e t h a n o l ) .
2
References
1 T. Iwata & K. Yamasaki, J. Biochem. 56, 424 [1964].

2 D. Mayer, H. Haindl, F. W. Koss & W. Lamprecht, Z. analyt. C h e m . 243, 242 [1968].
3 A. F. Hofmann, J. Lipid Res. 8, 55 [1967].
Cholesterol and Esterified Cholesterol
Peter R 6 s c h l a u , Erich Bernt and Wolfgang Gruber
+
Cholesterol, an essential c o m p o n e n t of the h u m a n organism, is a building material for cell m e m b r a n e s

of all tissues a n d the parent substance of n u m e r o u s h o r m o n e s of the adrenals and sexual organs. Cholesterol
occurs in a relatively high concentration in the blood, where it is p r e d o m i n a n t l y esterified with fatty acids.
It is eliminated by the liver, mainly in the form of bile acids.
The determination of cholesterol is of great i m p o r t a n c e for clinical diagnosis, since a high serum cholesterol
level is one of the i m p o r t a n t risk factors for arteriosclerosis a n d myocardial infarction.
In the procedures used until now, cholesterol is determined almost exclusively by Watson's modification 1
of the Liebermann-Bur chard m e t h o d or by the m e t h o d of Zlatkis et a l . . However, neither of these m e t h o d s

2
is specific, a n d they b o t h have a wide range of error a n d are seriously affected even by small quantities of
moisture. T h e m a i n disadvantage, however, is p r o b a b l y the use of strongly caustic reagents. T h e enzymatic
m e t h o d s using cholesterol o x i d a s e ' d o not have these d i s a d v a n t a g e s ; there is n o need to use a cholesterol
3 4
standard, since the cholesterol concentration in the sample can be readily calculated via the m o l a r extinction
coefficient of zl -cholestenone.
4
Application of Method: In biochemistry, clinical chemistry, a n d foodstuff chemistry.
Principle
Esterified Cholesterol eth

- K Q H
-> Cholesterol + F a t t y acid
or cholesterol esterase*
(2) Cholesterol + 0 2
c h o l e s t e r o 1 o x i d a s e
**) J -Cholestenone + H 0
4
2 2
T h e formation of cholestenone, which is m e a s u r e d by the increase in extinction at 240 n m , is p r o p o r t i o n a l

to the quantity of cholesterol present.
Esterified cholesterol is b r o k e n d o w n into free cholesterol and fatty acid with ethanolic K O H 5
or by
enzymatic cleavage with cholesterol esterase. It is therefore possible to determine the free cholesterol, the
esterified cholesterol, a n d the t o t a l cholesterol (in a single assay system when cholesterol esterase is used).
T h e procedure in which the esterified cholesterol is hydrolysed with ethanolic K O H is described below.
The p H o p t i m u m of the enzyme is p H 7 - 8 . T h e enzymatic reaction proceeds fastest in 0.5 M p h o s p h a t e

buffer in the presence of a solubilizing agent (e. g. Polyethylenglycol-monododecyl-ether***). T h e reaction
equilibrium lies on the side of cholestenone.
Equipment
S p e c t r o p h o t o m e t e r c a p a b l e o f a c c u r a t e m e a s u r e m e n t s at 2 4 0 n m ; w a t e r b a t h 37 ° C .
+
Technical assistance by R e i n h a r d Herz
* Sterol-ester hydrolase, E C 3.1.1.13
** Cholesterol:oxygen oxidoreductase, E C 1.1.3.6
*** e.g. Thesit®
Cholesterol a n d Esterified Cholesterol 1891
Reagents
1. S o d i u m d i h y d r o g e n p h o s p h a t e , 5. A b s o l u t e e t h a n o l , A . R .
NaH P0 H 02 4 2
6. C h o l e s t e r o l o x i d a s e f r o m Nocardia
2. D i s o d i u m h y d r o g e n p h o s p h a t e , N a H P 0 2 4
erythropolis , 6
3. Polyethylenglycol-monododecyl-ether, suspension in 1 M a m m o n i u m sulphate s o l u t i o n ;

e.g. Thesit® of Messrs. Klinke, Hamburg, ^ 25 U / m g . (25 °C). C o m m e r c i a l preparations,
Germany see p . 440.
4. P o t a s s i u m h y d r o x i d e , K O H , A . R.
Purity of Reagents
T h e sodium p h o s p h a t e s , p o t a s s i u m hydroxide a n d e t h a n o l must be of A. R. quality.
P r e p a r e all s o l u t i o n s w i t h freshly d i s t i l l e d w a t e r .
D i s s o l v e 6 0 . 5 g. N a H P 0 H 0 , 1 0 . 2 g. N a H P 0 , a n d 4 g. T h e s i t ® in w a t e r a n d m a k e
2 4 2 2 4
up to 1 0 0 0 ml.
II. P o t a s s i u m h y d r o x i d e s o l u t i o n ( 3 3 % w / v ) :
D i s s o l v e 33 g. K O H in w a t e r a n d m a k e u p t o 100 m l .
III. C h o l e s t e r o l o x i d a s e ( 0 . 2 5 m g . / m l . ) :
Dilute stock suspension as required with 1 M a m m o n i u m sulphate solution.
Solutions I a n d III are stable for at least 6 m o n t h s in stoppered vessels at a b o u t 4 °C. Solution II is stable
for at least 6 m o n t h s at r o o m t e m p e r a t u r e .
Procedure
Collection of sample: F o r c h e c k i n g variation, the b l o o d sample s h o u l d always be taken by

t h e s a m e m e t h o d , s i n c e t h e p o s i t i o n o f t h e b o d y a n d a n y v e n e s t a s i s m a y falsify t h e c h o l e s t e r o l
values . 7
Stability of sample: C h a n g e s b e t w e e n free a n d esterified c h o l e s t e r o l h a v e b e e n observed

b e c a u s e o f t h e c h o l i n e s t e r a s e p r e s e n t in t h e s e r u m . H o w e v e r , t h e s a m p l e s are s t a b l e w i t h o u t
8
c h a n g e s for t w o h o u r s at 2 5 ° C , 1 - 2 d a y s at 5 ° C , a n d 6 m o n t h s at - 1 5 ° C .
1892 M e t a b o l i t e s : F a t t y Acids Metabolism, etc.
Hydrolysis of esterified cholesterol: I n t r o d u c e 0.5 m l . s a m p l e , 4.5 m l . e t h a n o l , a n d 0.3 m l .

p o t a s s i u m h y d r o x i d e s o l u t i o n (II) i n t o s t o p p e r e d test t u b e s a n d swirl a r o u n d carefully. P l a c e
the s t o p p e r e d test t u b e s in a w a t e r b a t h at 37 ° C for 6 0 m i n . C e n t r i f u g e the s u s p e n s i o n a n d u s e
the clear s u p e r n a t a n t for t h e d e t e r m i n a t i o n .
Assay System
W a v e l e n g t h : 2 4 0 n m ; q u a r t z c u v e t t e s : light p a t h : 1 c m . ; final v o l u m e : 3 . 5 2 m l . ; read a g a i n s t

blank value of the sample.
F o r e a c h series o f m e a s u r e m e n t s , carry o u t o n e d e t e r m i n a t i o n o f t h e r e a g e n t b l a n k v a l u e w i t h
buffer (I) i n s t e a d o f s a m p l e ( s e e b e l o w ) .
D i l u t e 0.2 m l . h y d r o l y s a t e ( t o d e t e r m i n e t h e t o t a l c h o l e s t e r o l ) o r 0 . 0 5 m l . s a m p l e ( t o d e t e r m i n e
the free c h o l e s t e r o l ) w i t h 10 m l . p h o s p h a t e buffer ( I ) ; u s e 3 . 5 0 m l . in t h e d e t e r m i n a t i o n for
sample and sample blank.
Sample and Sample Blank
Pipette successively o n t o the A B Concentration

b o t t o m o f test t u b e s : Sample Sample blank in a s s a y m i x t u r e
Cholesterol oxidase solution (III) 0.02 ml. — a p p r o x . 1.4 pgjml. = 36

U/l.
Water — 0.02 ml.
Diluted sample 3.50 ml. 3.50 ml. c a . 0.5 M p h o s p h a t e
buffer, 5 0 pM cholesterol
M i x , a l l o w t o s t a n d for a b o u t 15 m i n . at 2 0 - 2 5 ° C . L a b e l t w o
c u v e t t e s A a n d B. F o r t h e m e a s u r e m e n t s , p o u r t h e s a m p l e i n t o
c u v e t t e A a n d t h e s a m p l e b l a n k i n t o c u v e t t e B. Set e x t i n c t i o n o f
cuvette B to zero, and measure extinction of cuvette A. T h e
extinction E s a m p l e is o b t a i n e d .
Reagent Blank
Pipette o n t o the b o t t o m
Cuvette A Cuvette B
o f the cuvette:
Cholesterol oxidase solution (III) 0.02 ml.

Water — 0.02 ml.
P h o s p h a t e buffer (I) 3.50 ml. 3.50 ml.
M i x , set e x t i n c t i o n o f c u v e t t e B t o z e r o a n d m e a s u r e e x t i n c t i o n o f
cuvette A ( E ) . R B
Esampie E —
R B = zl E is u s e d in t h e c a l c u l a t i o n s .
Calculations
The reaction proceeds quantitatively u n d e r the above conditions. T h e extinction coefficient of chole
s t e n o n e at 240 n m is e = 15.5 c m . / / i m o l e .
9 2
Cholesterol a n d Esterified Cholesterol 1893
The calculation formula (2) from page 312 is applicable. Taking into a c c o u n t the dilution of "the sample*
the following relationships are valid for this p r o c e d u r e :
Total cholesterol:
c=^1Ex35.1 [/zmole/ml.]
c=zlExl3.6 [mg./ml.]
Free cholesterol:
c= JExl3.0 [/miole/ml.]
c = zJEx 5.04 [mg./ml.]
W i t h an average value of 200 mg. cholesterol/100 ml. serum, a s t a n d a r d deviation s = 4.7 mg. cholesterol/
100 ml. was found. T h e coefficient of variation is 2 . 4 % .
Normal Values
N o n o r m a l values have yet been established for this m e t h o d . T h e Liebermann-Bur chard m e t h o d g i v e s 10
1 8 0 - 2 5 0 mg. total cholesterol/100 ml. in s e r u m ; 6 0 - 8 0 % of this is esterified.
Interference in the assay technique: Since the m e a s u r e m e n t at 240 n m is disturbed by even the slightest
turbidity, only perfectly clear solutions m a y be used. To avoid c o n t a m i n a t i o n of the b l a n k cuvette B with
cholesterol oxidase, always use cuvette A for the sample.
Differences in the t r a n s p a r e n c y of the cuvettes are also eliminated in the m e a s u r e m e n t of the reagent blank.
If this difference is greater t h a n the reagent blank, the extinction of cuvette A m a y be smaller than zero. In
this case set cuvette B to extinction E = 0.100 a n d m e a s u r e the extinction of cuvette A . T h e measured
difference in extinction m u s t then be a d d e d to E s a m p l e .
References
1 D. Watson, Clin. C h i m . Acta. 5, 637 [I960].

2 A. Zlatkis, B. Zak & A. J. Boyle, J. L a b . Clin. M e d . 41, 486 [1953].
3 W. Richmond, Scand. J. Clin. L a b . Invest., Vol. 29, Suppl. 126 [1972].
4 K. Beaucamp, H. U. Bergmeyer, E. Bernt, W. Gruber & P. Roschlau, unpublished c o m m u n i c a t i o n .
5 E. H. Martensson, Scand. J. Clin. L a b . Invest. Vol. 75, Suppl. 69 [1963].
6 G. Holz, unpublished c o m m u n i c a t i o n .
7 R. Richterich, Klinische Chemie, S. Karger, 3rd edn., M u n c h e n / B a s e l , 1971, p . 89.
8 D. B. Tonks, Clin. Biochem. 1, 12 [1967].
9 P. Roschlau & R. Herz, unpublished c o m m u n i c a t i o n .
10 N. Zoellner, Dtsch. m e d . Wschr. 84, 389 [1959].
Transfer Ribonucleic Acids:
Determination of the Acceptor Activity for Amino Acids
Hans G. Zachau
Transfer ribonucleic acids ( t R N A ) transfer the activated a m i n o acids to the growing peptide chains in the
biological synthesis of proteins. They are specific for the various a m i n o acids ( t R N A P h e
, tRNA S e r
, etc.), a n d
differ in their structure from one species to a n o t h e r (tRNASJu, t R N A ^ ! ^ , etc.). F o r reviews,see ~ . T h e 1 3
mixture of the t R N A ' s of a species ( t R N A C o l i , tRNA Y e a s t , etc.) was formerly k n o w n as "soluble ribonucleic
acid". The t R N A ' s can be esterified with a m i n o acids in the absence of the o t h e r c o m p o n e n t s of the cell-free
protein synthesis system with the aid of the a m i n o - a c y l - t R N A synthetases (EC 6.1.1.-) ( " t R N A l o a d i n g " ,
" a m i n o acid i n c o r p o r a t i o n " ) . This reaction forms the basis of the following m e t h o d , which is identical
with, or differs only slightly from, the m e t h o d s used in several l a b o r a t o r i e s 4 - 7
.
Application of Method: F o r the d e t e r m i n a t i o n of the content of a given t R N A in a t R N A separation, for

investigation of the biological activity of physically, chemically, or enzymatically modified t R N A , etc.
Principle
(1) A m i n o acid + t R N A + A T P amino- a c y i-^ Amino-acyl-tRNA + A M P + Pyrophosphate

v
' tRNA synthetase J J
T h e incorporation of radioactive a m i n o acids into the acid-precipitable a m i n o - a c y l - t R N A is measured.
T h e conditions indicated below are o p t i m u m for the determination of the acceptor activity of t R N A from
brewer's yeast for Phe, Ser, Val, a n d Lys with the aid of an unfractionated a m i n o - a c y l - t R N A synthetase
preparation.
T h e specific radioactivity of the a m i n o acid must be chosen o n the basis of the expected acceptor activity
and the quantity of t R N A used to give a radioactivity in the assay sample t h a t lies within a favourable
measuring range for the counter.
Equipment
1. U V s p e c t r o p h o t o m e t e r
2. L a b o r a t o r y c e n t r i f u g e
3 . R a d i o a c t i v i t y c o u n t e r , e . g . Tricarb s c i n t i l l a t i o n c o u n t e r m a d e b y t h e Packard Corp.
4 . F i l t e r h o l d e r , e . g . "Pyrex Micro-filtration Unit" N o . X X 1 0 0 2 5 0 0 (Millipore Filter Corp.,
Bedford, M a s s . 01730, U S A ) or a p p a r a t u s for the s i m u l t a n e o u s filtration of n u m e r o u s
s a m p l e s , e. g. a f t e r Leder a n d Byrne* o r greatly simplified h o m e - m a d e versions m a d e f r o m
m e t a l o r p l a s t i c f o r s e v e r a l filter h o l d e r s .
5. C e n t r i f u g e t u b e s , e . g . 1.0 x 7.5 c m . , c l e a n e d w i t h b i c h r o m a t e / s u l p h u r i c a c i d mixture.
6. M i c r o p i p e t t e s , e . g . f r o m Netheler a n d Hinz GmbH., H a m b u r g , o r H. E. Pedersen, Somer-
s t e d g a d e 7, C o p e n h a g e n V
7. I c e b a t h ; w a t e r b a t h , 3 7 ° C ; d r y i n g o v e n , 8 0 ° C .
t R N A : A c c e p t o r Activity for A m i n o Acids 1895
8. G l a s s fibre filter,diameter 2 . 5 c m . , e . g . Whatman t y p e G F / C ; m e m b r a n e filter ( t y p e H A ,

0 . 4 5 / m i . , Millipore, o r t y p e M F 5 0 ) Membranfiltergesellschaft GmbH, 34 Gottingen)
m a y also be used, but occasionally give higher blank values, particularly with aromatic
a m i n o acids.
9. S p e c i a l f o r c e p s f o r filter, e. g. Millipore N o . 6200006.
10. G l a s s s a m p l e b o t t l e s f o r s c i n t i l l a t i o n c o u n t e r , w h i c h c a n b e r e u s e d after w a s h i n g w i t h
d e t e r g e n t s a n d d e i o n i z e d w a t e r , d r y i n g , a n d h e a t i n g f o r 6 hr. a t 6 0 0 ° C ; t h e s c r e w c a p s
are u s e d r e p e a t e d l y .
Reagents
1. Tris-hydroxymethyl-aminomethane, 7. [ C ] - o r [ H ] - a m i n o a c i d
1 4 3
A . R . , o r o t h e r buffer s u b s t a n c e s A m i n o acids with activities of 2 0 - 6 0 curie/mole

2. A d e n o s i n e t r i p h o s p h a t e , A T P can be used in m a n y cases, see " O p t i m u m
2 2 2 C o n d i t i o n s for M e a s u r e m e n t s "
preparations, see p. 527. 8. A m i n o - a c y l - t R N A s y n t h e t a s e
3. M a g n e s i u m c h l o r i d e , e.g. a p r e p a r a t i o n p r e p a r e d from yeast in 50%
7
M g C l - 6 H 0 , A . R.
2 2
glycerol. A p p r o x . 60 O D 2 8 0 units/ml.
4. S o d i u m d i h y d r o g e n p h o s p h a t e , 9. T r i c h l o r o a c e t i c a c i d , A . R .
N a H P 0 H 0 , A.R.
2 4 2
10. E t h a n o l ( m a y b e d e n a t u r e d )
5. A m m o n i u m c h l o r i d e , N H C 1 , A . R . 4
11. Scintillator
6. S o d i u m h y d r o x i d e s o l u t i o n , A . R . , 1 N e.g. Omnifluor, N e w E n g l a n d N u c l e a r C o r p . ,
Boston, M a s s .
12. T o l u e n e , t e c h n i c a l g r a d e , d i s t i l l e d o v e r
sodium.
Purity of Reagents
T h e content of nucleases, proteases, a n d c o n t a m i n a t i n g a m i n o acids in t h e reagents should be a s low a s

possible. See also t h e sections " S o u r c e s of E r r o r " a n d " R e m a r k s " .
Prepare solutions I - I I I with water distilled f r o m quartz apparatus. A v o i d the accidental

introduction o f nucleases with reagents or glass apparatus.
I. B u f f e r / A T P s o l u t i o n ( 0 . 1 M A T P ; 0 . 1 5 M N H C 1 ; 0 . 1 5 M M g C l
4 2 ; 0.25 M tris; p H 7 . 5 ) :
D i s s o l v e 3 1 0 m g . A T P i n 1.0 m l . d i s t i l l e d w a t e r + 0 . 7 5 m l . 1 M N H C 1 ( 5 . 3 5 g / 1 0 0 m l . )
4
+ 0.75 ml. 1 M M g C l 2 ( 2 0 . 3 g./lOO m l . ) a n d a d j u s t t o p H w i t h a p p r o x . 1 m l . 1 N N a O H

( p H p a p e r ) ; a d d 1.25 m l . o f tris buffer ( 1 2 . 1 g. tris a d j u s t e d t o p H 7 . 5 w i t h 1 N H C 1 a n d
m a d e u p t o 100 ml. with H 0 ) , a n d m a k e u p t o 5.0 ml. with distilled water.
2
II. [ C ] - o r [ H ] - a m i n o a c i d s o l u t i o n (1 m M D L - a m i n o a c i d o r 0 . 5 m M L - a m i n o a c i d ) :
1 4 3
D i s s o l v e t h e s o l i d s u b s t a n c e i n w a t e r . If it is i n h y d r o c h l o r i c a c i d s o l u t i o n , t h i s m u s t b e n e u
tralized.
III. P h o s p h a t e - m a g n e s i u m buffer ( 0 . 1 M p h o s p h a t e , 1 m M M g C l 2 ; p H 7.0):
D i s s o l v e 1 3 . 8 g. N a H P 0 - 2 H 0 i n d i s t i l l e d w a t e r , a d d 1 m l . 1.0 M M g C l , a d j u s t t o
2 4 2 2
p H 7.0 with 1 N N a O H , a n d m a k e u p t o 1000 ml. with distilled water.

1896 M e t a b o l i t e s : Nucleic Acids, Purines, Pyrimidines, Nucleosides, Coenzymes
IV. T r i c h l o r o a c e t i c a c i d (5 % w / v ) :
D i s s o l v e 5 g. C C l C O O H in w a t e r a n d m a k e u p t o 100 m l .
3
V. Scintillator s o l u t i o n :
D i s s o l v e 4 g. O m n i f l u o r in 1 0 0 0 m l . t o l u e n e .
VI. A m i n o - a c y l - t R N A synthetase (60 mg. of protein/ml.):
U s e stock solution prepared according t o 7
undiluted.
Store solutions I and II at —18 °C a n d t h a w only for removal of p o r t i o n s . If the g r o w t h of bacteria is

avoided, A T P buffer solution I can be kept for several m o n t h s , and a m i n o acid solution II for several years.
In the case of H - a m i n o acids, the radioactive decay must be t a k e n into a c c o u n t . K e e p solutions III a n d IV
3
at a b o u t + 4 °C. Store solution V at r o o m t e m p e r a t u r e ; after use, collect the contents of several sample
bottles, filter, and reuse. Solution V can be used as long as the radioactivity m e a s u r e d in a 5 ml. p o r t i o n is
low in relation to the radioactivity to be measured on the filter. Fitness for reuse is frequently limited by the
moisture that accumulates with repeated use.
Procedure
D i s s o l v e t R N A in w a t e r ( 2 - 1 0 m g . / m l . o r c o r r e s p o n d i n g l y l e s s o f a n e n r i c h e d a m i n o a c i d -
specific t R N A ) . D e t e r m i n e t h e e x a c t R N A c o n t e n t o f t h e s o l u t i o n b y U V s p e c t r o p h o t o m e t r y
o n a p o r t i o n o f the s o l u t i o n t h a t h a s b e e n g r e a t l y d i l u t e d w i t h t h e phosphate-magnesium
buffer, a n d e x p r e s s t h e result in e x t i n c t i o n u n i t s ( 2 6 0 n m ) / m l . ( A g o o d t R N A in this buffer
should have E 2 5 0 /E 2 6 0 = a p p r o x . 0.9 a n d E 2 8 0 /E 2 6 0 = approx. 0.5; the E 2 6 0 value of t R N A a s t
Y e
in w a t e r is a p p r o x . 1.15 t i m e s t h a t in t h e a b o v e buffer.) S t o r e t h e t R N A s o l u t i o n at — 18 ° C , a n d
t h a w o n l y for r e m o v a l o f p o r t i o n s . If t h e s o l u t i o n is free f r o m n u c l e a s e , t h e a c c e p t o r activity c a n
r e m a i n u n c h a n g e d for several y e a r s .
t R N A : Acceptor Activity for A m i n o Acids 1897
Assay System
R e a c t i o n v o l u m e : 0.1 m l . ; i n c u b a t i o n t e m p e r a t u r e : 37 ° C . B l a n k w i t h w a t e r i n s t e a d o f t R N A
sample.
C o o l all r e a g e n t s o l u t i o n s for u s e . A c c o r d i n g t o t h e p u r p o s e o f t h e e x p e r i m e n t , several s o l u t i o n s
m a y b e c o m b i n e d t o f o r m o n e r e a g e n t m i x t u r e ( a d d s y n t h e t a s e s o l u t i o n last). T h e c e n t r i f u g e
t u b e s r e m a i n in t h e s a m e test t u b e rack for p i p e t t i n g , i n c u b a t i o n , a n d p r e c i p i t a t i o n .
P i p e t t e i n t o 3 m l . c e n t r i f u g e t u b e s s t a n d i n g i n a n ice
bath:
Buffer/ATP solution (I) 10 10 m M A T P ;

15 m M N H C 1 ; 4
15 m M M g 2 +
2 5 m M tris
A m i n o acid solution (II) 10 fil 0.05 m M L-amino acid
t R N A s a m p l e + w a t e r dist. f r o m 7 8 - 7 9 |il. 0.1-0.8 O D 2 6 0 units
quartz apparatus
Synthetase solution (VI) 1-2 fil
M i x (e. g. w i t h v i b r a t o r ) . C e n t r i f u g e briefly, p l a c e in
37 ° C w a t e r b a t h for 1 5 - 2 0 m i n . , c o o l in ice b a t h .
Trichloroacetic acid, cold (IV) approx.

25 ml.
A d d i t i o n f r o m p l a s t i c s q u e e z e b o t t l e until a p p r o x .
1 c m . b e l o w t h e r i m o f t h e t u b e . M o i s t e n filter w i t h
trichloroacetic acid. Suck contents o f tube t h r o u g h
filter ( o n e filter p e r t u b e ) w i t h w e a k v a c u u m ( w a t e r
p u m p ) . Wash tube 2 to 3 times with 3 ml. portions o f
t r i c h l o r o a c e t i c a c i d (last w a s h i n g after r e m o v a l o f
u p p e r part o f filter h o l d e r ) . T h e n w a s h glass-fibre
filter o n c e w i t h a p p r o x . 3 m l . o f e t h a n o l ; m e m b r a n e
filters must n o t be w a s h e d with ethanol. Without
r e l e a s i n g v a c u u m c o m p l e t e l y , transfer filters with
f o r c e p s i n t o c o u n t e r t u b e s , a n d d r y for 2 5 - 3 0 m i n . at
80 °C.
Scintillator solution (V) 5 ml.
Measure radioactivity in counter.
Thirty-fifty s p e c i m e n s c a n b e h a n d l e d in o n e e x p e r i m e n t w i t h o u t difficulty. T r i c h l o r o a c e t i c a c i d
is a d d e d t o all t h e t u b e s i m m e d i a t e l y after i n c u b a t i o n . T h e y are t h e n t r e a t e d further in g r o u p s
of, e. g. six (see " E q u i p m e n t " , N o . 4 ) . W i t h t h i s n u m b e r o f s a m p l e s , t h e t i m e r e q u i r e d f r o m t h e
a d d i t i o n o f t r i c h l o r o a c e t i c a c i d u n t i l d r y i n g o f t h e filter b e g i n s is 3 0 - 6 0 m i n . I d e n t i c a l s a m p l e s
g i v e t h e s a m e v a l u e s w h e t h e r filtered i m m e d i a t e l y after t h e a d d i t i o n o f t r i c h l o r o a c e t i c a c i d o r
a l l o w e d t o s t a n d in t h e ice b a t h w i t h t r i c h l o r o a c e t i c a c i d for 9 0 m i n . b e f o r e filtration.
Calculations
T h e equilibrium of the amino-acylation reaction (eq. 1) is displaced very strongly to the right. T h e result is
obtained as cpm per sample (e. g. 1500/min. after subtraction of the value for the blank). To find the result
in p m o l e / O D 2 6 0 unit this value is divided by the specific radioactivity of the a m i n o acid in Ci/mole e.g.
20), the O D 2 6 0 units of t R N A used (e.g. 0.5), and the counting efficiency in c p m per pCi (e.g. 1.73 for a
14
C - a m i n o acid). T h e acceptor activity in the example is 86 p m o l e of the a m i n o acid in q u e s t i o n / O D 2 6 0
unit of t R N A .
To determine the counting efficiency, d r o p a n accurately k n o w n quantity (e.g. 1.0 nCi) of a standardized
a m i n o acid (e. g. StanStar [ C ] - a m i n o acid from Schwarz Bioresearch, N e w York, or special sample of an
14
[ H ] - a m i n o acid from R a d i o c h e m i c a l C e n t r e , A m e r s h a m ) in solution on a glass filter, dry the filter in a

3
counter vessel, a n d determine the radioactivity as described above. In the example one obtains 1730
cpm per nCi of [ C ] - a m i n o acid. In the case of [ H ] - a m i n o acids, where the depression of the counting ef
14 3
ficiency by accompanying substances m a y play a part, the solution to be dried on the filter contains not
only the a m i n o acid b u t also the quantity of t R N A a n d a m i n o - a c y l - t R N A synthetase present in an in
cubation mixture.
T h e acceptor activity is therefore
pmole of a m i n o a c i d / O D 2 6 0 unit of t R N A = — cpm _

spec, radioactivity x O D 2 6 0 units x counting efficiency
The specific radioactivity of the a m i n o acid used is expressed in Ci/mole, a n d the counting efficiency in
c p m per pCi.
The values are reproducible to within ± 7 % . Within a single series of experiments, the agreement between
the results of duplicate d e t e r m i n a t i o n s is often better t h a n ±5%.
N o r m a l Values
The acceptor activities of t R N A Y e a s t and t R N A C o l i for most a m i n o acids are between 50 and 150 p m o l e /
OD 2 6 0 unit.
T h e incorporation of radioactive a m i n o acids into t R N A m a y be decreased by the presence of excess salt in

the incubation solution (originating e. g. from the t R N A p r e p a r a t i o n ) , as well as by nucleases, proteases,
and ATP-hydrolysing enzymes o r by non-radioactive a m i n o acids, which m a y be formed by proteolysis if
old, unfractionated a m i n o - a c y l - t R N A synthetase p r e p a r a t i o n s are used. W i t h synthetase p r e p a r a t i o n s
m o r e t h a n 4 - 6 m o n t h s old, decreased i n c o r p o r a t i o n of a m i n o acids into t R N A is occasionally observed,
though according to incorporation experiments in which the quantity of enzyme is limiting, the enzymatic
activity of the p r e p a r a t i o n s is at m o s t only slightly lower t h a n in fresh p r e p a r a t i o n s . Indications of the
causes of the various errors are provided by c o m p a r i s o n experiments with o t h e r t R N A or synthetase
preparations and by mixing experiments.
The a m i n o - a c y l - t R N A synthetases are very largely specific with regard to the a m i n o acid a n d are to some
extent species-specific. U n d e r the conditions indicated, the m e t h o d is specific to within the accuracy
t R N A : A c c e p t o r Activity for A m i n o Acids 1899
of the measurements. Deviations from specificity are o b s e r v e d e.g. with certain a m i n o acid analogues,
3
as well as on addition of organic solvents t o the incubation mixture a n d o n charging of t R N A with synthe
tases from a n o t h e r species.
Remarks
Radioactive Amino Acids
C o n t r a r y to the m a n u f a c t u r e r s ' d a t a , a m i n o acid p r e p a r a t i o n s were occasionally found to contain u p to

1 0 % of other radioactive a m i n o acids that could be i n c o r p o r a t e d into t R N A . Testing for p u r i t y : the incor
p o r a t i o n of a radioactive a m i n o acid m u s t be decreased by the calculated a m o u n t o n addition of measured
quantities of the non-radioactive form of the same a m i n o acid; the i n c o r p o r a t i o n should n o t be decreased
on addition of the 19 other (non-radioactive) a m i n o acids. In b o t h series of experiments, it is necessary to
take into account possible impurities in the non-radioactive a m i n o acids a n d salt effects on the incorporation
(on addition of large quantities of a m i n o acid hydrochlorides).
The d a t a on the specific radioactivity of the a m i n o acids are n o t always accurate. W h e n different batches of
the same a m i n o acid were c o m p a r e d in the same i n c o r p o r a t i o n experiment a n d the indicated specific
radioactivity used in the calculations, deviations of u p to 1 8 % were found in the results. F o r accurate
acceptor activity determinations, therefore, standardized a m i n o acids (see a b o v e u n d e r " C a l c u l a t i o n s " ) or
p r e p a r a t i o n s that have been c o m p a r e d with these are used in t h e t R N A loading experiment.
When [ C ] - and [ H ] - a m i n o acids with high specific radioactivities were stored in dilute a q u e o u s solutions,
14 3
the radioactivity in the solution occasionally decreased even u n d e r conditions such that the growth of bacteria
was virtually ruled out. This effect, which m a y be explained by a d s o r p t i o n of the a m i n o acids on the
walls of the tubes, must also be considered for the a m i n o acids used for s t a n d a r d i z a t i o n .
Effect of C T P
The acceptor activity of t R N A p r e p a r a t i o n s (e.g. from b a k e r ' s yeast) in which, in addition to the 3 ' -
terminal A, p a r t of the subsequent C is missing can be increased by the a d d i t i o n of 10 n m o l e of C T P , 10 fig. of
pyruvate kinase, a n d 0.2 /rniole of p h o s p h o e n o l pyruvate to the 0.1 ml. incubation mixture. T h e C- a n d A-
fixing enzyme, CCA-transferase, is present in m o s t unfractionated a m i n o - a c y l - t R N A synthetase p r e p
arations. By incorporation of radioactive C T P a n d A T P , it is possible t o estimate h o w m u c h terminal
C a n d A are missing in a t R N A p r e p a r a t i o n .
Fractionated tRNA's and Amino-Acyl-tRNA Synthetases
Relatively high values are occasionally found for the acceptor activity with purified synthetases. T h e lower
values with unfractionated synthetases are attributed to the presence of nucleases, proteases, or o t h e r
inhibitors in the p r e p a r a t i o n s . T h e a m i n o acid i n c o r p o r a t i o n with purified synthetases is occasionally
increased by addition of carrier protein, such as serum a l b u m i n , as is the i n c o r p o r a t i o n into fractionated
t R N A ' s by addition of high molecular weight R N A . T h e carrier protein o r the carrier nucleic acid must
also be a d d e d to the blank. S o m e purified t R N A ' s tend to aggregate; the acceptor activity of samples
containing aggregates is increased by deaggregation before the a m i n o acid i n c o r p o r a t i o n experiment, e. g.
by heating at 80 °C for 1 m i n u t e in 0.15 M KC1, 5 m M M g C l , 0.5 m M E D T A , 0.1 M buffer, p H 7.0.
2
Other Amino Acids and t R N A ' s of Other Species
F o r a m i n o acids other t h a n those m e n t i o n e d a n d t R N A ' s from other species, the o p t i m u m conditions are
sometimes different from those indicated. Preliminary experiments are necessary in every case. T h e
1900 Metabolites: Nucleic Acids, Purines, Pyrimidines, Nucleosides, Coenzymes
o p t i m u m concentrations of t R N A , a m i n o acid, synthetase, A T P , M g + +

, K , or NH4 m a y be different.
+
Some synthetases are stimulated by m e r c a p t o e t h a n o l o r glutathione. T h e i n c o r p o r a t i o n of cysteine into

t R N A e a s t is carried out in the presence of 7 m M m e r c a p t o e t h a n o l t o reduce cystine to cysteine. T h e
Y
9
o p t i m u m incubation t e m p e r a t u r e a n d the course of the a m i n o acid i n c o r p o r a t i o n as a function of time

must be determined; where the incubation time is t o o long, the value found for the acceptor activity may be
t o o low if the synthetase involved a n d the ester linkage in the a m i n o - a c y l - t R N A are labile. A wide range of
stabilities have been found b o t h for the synthetases a n d for the ester linkages with various a m i n o acids.
A n unfractionated a m i n o - a c y l - t R N A synthetase p r e p a r a t i o n from E. coli has been described e.g. by
Munch and Berg .10
If the incorporation of a m i n o acids into t R N A is to be distinguished from the i n c o r p o r a t i o n into protein or

cell wall c o m p o n e n t s in crude extracts, it is possible t o m a k e use of the alkali-lability of the amino-acyl-
t R N A linkage a n d the RNase-sensitivity of the loading reaction.
Variants of the Method for Determining Acceptor Activity
All the m e t h o d s are based on the a b o v e principle. In addition to the m e t h o d described here and its n u m e r o u s
variants, three other groups of m e t h o d s are used. In one, which usually requires s o m e w h a t greater quantities
of t R N A , the a m i n o - a c y l - t R N A a n d the synthetase are precipitated with acid after the incubation,
centrifuged off, washed repeatedly with acid, dissolved, a n d used for the radioactivity determination
(cited in ) . In this case, a gas-flow c o u n t e r m a y be used instead of a scintillation counter. In the second
9
m e t h o d , portions of the incubated mixture are applied to filters, which are then washed with acid. In the
11
third m e t h o d , the incubation is carried out on filters as well as the washing. This m e t h o d is r e c o m m e n d e d
12
for the rapid determination of the a p p r o x i m a t e acceptor activities of a large n u m b e r of t R N A specimens

containing salt.
References
1 T h e Genetic Code, 3 1 . Cold Spring H a r b o r S y m p o s i u m , 1966, Cold Spring H a r b o r L a b o r a t o r y ,

L. I. N e w Y o r k , 1967.
2 FEBS-Symposium " S t r u c t u r e a n d F u n c t i o n of Transfer R N A a n d 5 S R N A " , Oslo 1967, Universitets-
forlaget, Oslo.
3 G. D. Novelli in A n n u a l Rev. Biochem. 36, P a r t II, 449 [1967].
4 R. B. Loftfield&E. A. Eigner, A c t a C h e m . Scand. 17, Suppl. 1, 117 [1963].
5 T. Lindahl, A. Adams & /. R. Fresco, P r o c . N a t . A c a d . Sci. (Wash.) 55, 941 [1966].
6 / . F. Scott in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c Press, N e w Y o r k &
L o n d o n , 1968, Ed. by L. G r o s s m a n & K. M o l d a v e , Vol. X I I B, p . 173.
7 R. Thiebe & H. G. Zachau, E u r o p . J. Biochem., 5, 546 [1968].
8 P. Leder & C. J. Byrne, Analyt. Biochem. 9, 384 [1964].
9 R. Thiebe & H. G. Zachau, Biochim. Biophys. A c t a 103, 568 [1965] a n d cited literature.
10 K.H. Muench & P. Berg in G. L. Cantoni & D. R. Davies: P r o c e d u r e in Nucleic Acid Research, Harper
& Row, N e w Y o r k & L o n d o n 1966, p . 375.
11 W.E. Burnett & K. B. Jacobson, Proc. N a t . Acad. Sci. (Wash.) 57, 642 [1964].
12 J. D. Cherayil & R. M. Bock, Biochemistry 4, 1174 [1965].
Polyribonucleotides (Messenger-RNA):
Determination of the Activity
in the Peptide-Synthesizing System
Heinrich Matthaei
Polyribonucleotides can act as cofactors in the cell-free polypeptide synthesis on ribosomes, their nucleo
tide sequence determining the sequence of the a m i n o acids to be linked. W h e r e a s n a t u r a l messenger
ribonucleic acids contain special nucleotide sequences for the initiation a n d t e r m i n a t i o n of the t r a n s
lation, control patterns of this type are lacking in most synthetic polynucleotides with either r a n d o m or
regularly repeating nucleotide sequences. W i t h a n increase in the M g 2 +
c o n c e n t r a t i o n , however, artificial
initiation of the polypeptide synthesis also occurs in the presence of these polynucleotides. T h e measured
template activity of polynucleotides also d e p e n d s o n their chain length a n d secondary structure, as well
as on the availability of the amino-acyl transfer ribonucleic acids required for the translation of the
various triplets.
If the synthesized polypeptides are t o o soluble in the precipitant used for detection, the values found for
the template activity m a y be t o o low. Purer systems and differential tests for the investigation of the
individual steps in the template-controlled polypeptide synthesis have been d e s c r i b e d . A system obtained 1
from Escherichia coli is r e c o m m e n d e d here. A n o p t i m u m h u m a n system is also a v a i l a b l e . 2
Application of Method: In the biochemistry of protein biosynthesis, molecular genetics, and possibly
clinical biochemistry.
Principle
(1) A m i n o acids* + A T P — a m i n o
~ a c y l
~ t R N A
*y«*'**** * A m i n o - a c y l * - A M P
+
t PP ;
(2) Amino-acyl*-AMP + t R N A , »™"°-«*'-'»n a

synthetases t A mino-acyl*-tRNA + AMP
I
* (Initiation factors)
(3) Amino-acyl*-tRNA + G T P p o l y n u
; 1
i
e
b ° ^
o
t
m e
(
b
m R N A )
• Polypeptide* + t R N A + G D P + P,
Factors T and G
(Termination factors)
The resulting polypeptide, which has been radioactively labelled by the a m i n o acid(s), is precipitated in
the usual m a n n e r with 1 0 % trichloroacetic acid, washed, a n d d e t e r m i n e d by m e a s u r e m e n t in a liquid
scintillation spectrometer.
T h e initiation difficulties t h a t n o r m a l l y arise with synthetic polynucleotides lead t o an unphysiologically

high M g requirement, a n d are partly responsible for a lag phase in the a m i n o acid i n c o r p o r a t i o n
2 +
kinetics. T h e M g o p t i m u m should be determined for the enzyme p r e p a r a t i o n used. It is between 14

2 +
and 19 m M in E. coli systems a n d between 8 a n d 12 m M in m a m m a l i a n s y s t e m s . A m a x i m u m synthesis

1 2
rate is measured after the lag p h a s e ( ^ 5 min. at 37 °C). T h e m a x i m u m i n c o r p o r a t i o n depends inter alia
+
N o E. C. system n u m b e r as yet.
* Radioactively labelled.
1902 M e t a b o l i t e s : Nucleic Acids, Purines, Pyrimidines, Nucleosides, C o e n z y m e s
on the particular enzyme p r e p a r a t i o n a n d the q u a n t i t y used. A table of the genetic code should be used
to assist the choice of the radioactively labelled a m i n o acid a n d of any o t h e r a m i n o acids required for
the translation of a template. If all the a m i n o acids coded by a polynucleotide are to be determined simul
taneously, the r e c o m m e n d e d substrate is a mixture in which all 20 [ C ] - a m i n o acids are present in the
14
same molarity a n d specific activity.
Equipment
1. p H M e t e r w i t h g l a s s e l e c t r o d e f o r a d j u s t m e n t o f b u f f e r s o l u t i o n s
2. P r e p a r a t i v e ultracentrifuge
3. U V s p e c t r o p h o t o m e t e r f o r m e a s u r e m e n t o f t h e n u c l e i c a c i d e x t i n c t i o n a t 2 6 0 n m .
4. Liquid scintillation s p e c t r o m e t e r for m e a s u r e m e n t of [ H ] or [ C ] 3 1 4
A l s o a n ice b o x , a w a t e r b a t h ( 3 7 ° C ) , m i c r o p i p e t t e s , h a r d - g l a s s r e a c t i o n t u b e s 10 x 1 0 0 m m . ,
c o u n t i n g v i a l s . W e r i n s e g l a s s in a s o l u t i o n o f s o d i u m h y d r o x i d e in e t h a n o l , d i l u t e H C 1 , a n d
d e i o n i z e d a n d d o u b l y q u a r t z - d i s t i l l e d w a t e r . D r y i n g f o r 2 h r . a t 180 ° C . W h e n t h e s a m p l e s a r e
p r e c i p i t a t e d o n g l a s s fibre filters, it is r e c o m m e n d e d t h a t t h e s u b s e q u e n t h y d r o l y s i s a t 9 0 ° C b e
c a r r i e d o u t in a T e f l o n c o n t a i n e r w i t h s l o t s t o h o l d t h e i n d i v i d u a l filter d i s c s * .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , t r i s , 14. T r a n s f e r - R N A
A.R. soluble RNA from E. coli B; commercial
2. A m m o n i u m c h l o r i d e , N H C 1 , A . R .
4 p r e p a r a t i o n s , see p . 553.
3. P o t a s s i u m c h l o r i d e , A . R . 15. P y r u v a t e k i n a s e , P K
4. P o t a s s i u m h y d r o x i d e s o l u t i o n , A. R., 1 N from rabbit skeletal muscle, crystalline sus
5. H y d r o c h l o r i c a c i d , A . R . , c o n e . pension in 3.2 M a m m o n i u m sulphate solution;
6. M a g n e s i u m a c e t a t e , M g ( C H C O O ) * 3 2 ^ 1 5 0 U / m g . (25 ° C ) ; commercial p r e p a r a t i o n ,
4H 0,2 A.R. see p . 509.
7. M a g n e s i u m c h l o r i d e , M g C l - 6 H 0 ,
2 2 16. D e o x y r i b o n u c l e a s e
A.R. crystallized from bovine p a n c r e a s ; ^ 2 0 0 0 U/
8. 2 - M e r c a p t o e t h a n o l , h i g h e s t p u r i t y mg. (25 °C); c o m m e r c i a l p r e p a r a t i o n s , see p .
9. A l u m i n i u m o x i d e p o w d e r 447.
Alcoa 305 A, Serva or W o l m , "for T L C " 17. P o l y u r i d y l i c a c i d
10. T r i c h l o r o a c e t i c a c i d , A . R . , 5 % ( w / v ) p o t a s s i u m salt; commercial p r e p a r a t i o n s , see
and 10% (w/v) p. 549.
11. P h o s p h o e n o l p y r u v a t e 18. [ H ] - o r [ C ] - L - P h e n y l a l a n i n e
3 1 4
crystalline m o n o p o t a s s i u m salt, P E P - K ; c o m e. g. from the R a d i o c h e m i c a l Centre, A m e r s h a m ,

mercial p r e p a r a t i o n s , see p . 548. England.
12. A d e n o s i n e t r i p h o s p h a t e , A T P 19. [ C ] - L - A m i n o a c i d m i x t u r e
1 4

2 2 2 of 20 a m i n o acids, 10 Ci/mole, 1 /miole/ml. each,
preparations, see p . 527. mixed from 14 [ C]-labelled
14
amino acids
13. G u a n o s i n e t r i p h o s p h a t e , GTP ( C F B 152) a n d 6 [ C]-labelled a m i n o acids
14
trilithium salt, G T P - L i ; commercial p r e p a r a

3 ( C F B 163) o b t a i n e d from the Radiochemical
tions, see p . 542. Centre, A m e r s h a m , England.
* Obtained from Schleicher a n d Schull, 3354 Dassel, Kr. Einbeck, W . - G e r m a n y .

m R N A : Activity in the Peptide-synthesizing System 1903
20. S-134 fraction 22. Ethanol

from E. coli, for p r e p a r a t i o n , see A p p e n d i x p . m a y be d e n a t u r e d
1907. 23. Ether
21. Ribosomes 24. Toluene
from E. coli, for isolation, see A p p e n d i x p . technical, filtered through aluminium oxide.
1907. 25. 2 , 5 - D i p h e n y l o x a z o l e , P P O
Scintillation Grade, Packard.
Purity of Reagents
If the m e t h o d is used often, it is r e c o m m e n d e d that a complete set of the solutions that have given g o o d
results, including a s t a n d a r d polynucleotide, a n S-134 p r e p a r a t i o n , a n d ribosomes, be kept available
in a freezer at — 30 to — 50 °C, so t h a t in cases of excessively low i n c o r p o r a t i o n , an unsuitable solution
a m o n g those used can be quickly detected.
P r e p a r e all s o l u t i o n s w i t h w a t e r freshly d i s t i l l e d t w i c e f r o m q u a r t z a p p a r a t u s , a n d k e e p in a
freezer at - 3 0 ° C o r c o l d e r . T h e p H is a d j u s t e d at 2 0 ° C w i t h a g l a s s e l e c t r o d e .
D i s s o l v e 121 g. tris in 7 0 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7.8 w i t h c o n e . H C 1 , r o u g h l y
at first a n d t h e n a c c u r a t e l y after c o o l i n g t o 2 0 ° C ; m a k e u p t o 1 0 0 0 m l . w i t h distilled
water.
II. Tris buffer ( 1 0 m M ; p H 7 . 2 ) :
D i s s o l v e 121 g. tris in 7 0 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7 . 2 w i t h c o n e . H C 1 at 2 0 ° C ,
m a k e u p to 1000 ml. with distilled water. Dilute the resulting 1 M solution with water by a
f a c t o r o f 100 as r e q u i r e d .
III. K C 1 ( 1 M ) :
D i s s o l v e 7 4 . 6 g. K C 1 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
I V . A m m o n i u m c h l o r i d e (1 M ) :
D i s s o l v e 5 3 . 5 g. N H C 1 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
4
V a . M a g n e s i u m a c e t a t e (1 M ) :
D i s s o l v e 2 1 4 g. M g ( C H C O O ) - 4 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
3 2 2
Vb. M g C l 2 (1 M ) :
D i s s o l v e 2 0 3 g. M g C l - 6 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2 2
VI. ATP-K(lOOmM):
D i s s o l v e 150 m g . A T P - N a H - 3 H 0 in 1 m l . d i s t i l l e d w a t e r , r e m o v e c a t i o n s w i t h w a t e r
2 2 2
u s i n g D o w e x 5 0 x 2 (1 c m . x 2 c m . ; H +
f o r m ) , c o l l e c t e l u a t e s b e l o w p H 2, adjust t o
p H 7.5 w i t h 1 N K O H , a n d d i l u t e w i t h w a t e r t o 1 5 4 0 O D 2 5 9 units/ml.
VII. GTP-K(20mM):
U s i n g t h e G T P l i t h i u m salt, p r o c e e d a s d e s c r i b e d u n d e r V I , b u t adjust t o 2 7 5 O D 2 5 2
units/ml.
VIII. PEP-K(80mM):
D i s s o l v e 165 m g . P E P - K in d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
IX. Pyruvate kinase (10 mg. o f protein/ml.):
Dilute stock suspension as required with 3.2 M a m m o n i u m sulphate solution.
X. t R N A (10 m g . / m l . ) :
D i s s o l v e 10 m g . t R N A in 1 m l . d i s t i l l e d w a t e r .
X I . [ H ] - or [ C ] - P h e n y l a l a n i n e ( 0 . 5 m M ; 10 t o 100 / / C i / ^ m o l e ) ;
3 14
A d j u s t t h e s u b s t a n c e s w i t h w a t e r as r e q u i r e d .
XII. Polyuridylic acid (0.6 m M ) :
D i s s o l v e 10 m g . o f p o l y - ( U ) in 10 m l . 10 m M tris buffer, p H 7.2 a n d d i l u t e t o 6.0 O D 2 6 2
units/ml.
XIII. Ribosomes (approx. 5 mg. protein/ml.:
T h a w a l i q u o t in ice w a t e r i m m e d i a t e l y b e f o r e u s e , a n d u s e u n d i l u t e d .
X I V . S-134 fraction (approx. 6 - 8 mg. p r o t e i n / m l . ) :
T h a w a l i q u o t in ice w a t e r i m m e d i a t e l y b e f o r e u s e , a n d u s e u n d i l u t e d .
X V . Scintillator s o l u t i o n :
D i s s o l v e 0 . 4 g. o f P P O in t o l u e n e a n d m a k e u p t o 100 m l .
Stocks of the cheaper salt solutions can be stored for m o n t h s in the frozen state (approx. — 20 °C), aliquots
being kept in the refrigerator for use at short notice. Solutions VI to VIII a n d X to XII are stored in a
freezer at ^ — 30 °C in small containers, a n d are stable in this condition for a n u m b e r of years. The P K
suspension is stable for longer t h a n 1 year at 0 to + 4 °C. T h e S-134 fraction a n d the ribosomes can be
stored at —30 °C for 6 to 12 m o n t h s without serious loss of activity.
Procedure
Treatment and Stability of Sample
B e f o r e a n y n e c e s s a r y d i l u t i o n , d i s s o l v e t h e p o l y n u c l e o t i d e s t o b e t e s t e d in 10 m M KC1 o r 10 m M
tris buffer p H 7.2 in a c o n c e n t r a t i o n o f 1 m g . / m l . W i t h s o m e n u c l e o t i d e c o m p o s i t i o n s , s o l u t i o n
o c c u r s o n l y after brief w a r m i n g at 5 0 t o 6 0 ° C . T h e s e s o l u t i o n s m u s t in s o m e c a s e s b e k e p t at
2 0 °C until n e e d e d . T h e y are s t o r e d in s m a l l p o r t i o n s at ^ — 3 0 ° C . In n u c l e a s e - f r e e s o l u t i o n s ,
the t e m p l a t e a c t i v i t y p e r s i s t s for s e v e r a l y e a r s .
Assay System
Incubation
P r e p a r e r e a g e n t m i x t u r e for 14 a s s a y s a n d k e e p in i c e :
1 M tris buffer, p H 7.8 (I) 2 9 4 fil 100 m M
1 M KC1 s o l u t i o n (III) 2 0 4 /A. 80 m M
1 M MgCl 2 solution (IV) 51 ill 19 m M
1 M 2-mereaptoethanol,
diluted 1 : 1 0 with distilled water 15 til 7 mM
100 m M A T P - K s o l u t i o n (VI) 3 0 fil 1 mM
20 m M G T P - K solution (VII) 3 0 ill 0.2 m M
80 m M P E P - K s o l u t i o n (VIII) 3 0 0 ill 8 mM
10 m g . / m l . p y r u v a t e k i n a s e
suspension (IX) 6 fil 20 pg./ml
10 m g . / m l . t R N A s o l u t i o n (X) 2 2 5 fil 7 5 0 jug./ml.
0.5 m M [ H ] - o r [ C ] - p h e n y l a l a n i n e
3 1 4
solution 3 0 0 fil 0.05 m M

Water ( t o 2 1 0 0 /il) 645 ill
I n c u b a t i o n v o l u m e : 2 0 0 fil; 37 ° C ( c o n s t a n t - t e m p e r a t u r e w a t e r b a t h ) ; i n c u b a t i o n time:
5 t o 180 m i n .
I n t r o d u c e i n t o i c e - c o o l e d test t u b e s
(10 m m x 100 m m )
Reagent mixture 1 4 0 ill see a b o v e
Poly-(U) solution (XII) 0 , 1 , 2 , 3 , 5, 0 t o 4 5 /iM
10, 1 5 ^ 1 .
Water t o 2 0 0 /A.
I m m e d i a t e l y b e f o r e p l a c i n g in 37 ° C w a t e r b a t h ,
p i p e t t e t h e f o l l o w i n g i n t o e a c h test t u b e a n d m i x b y
s w i r l i n g . Insert t h e p i p e t t e far e n o u g h i n t o t h e t u b e s
to avoid wetting the walls:
Ribosome suspension (XIII) 2 0 ill a p p r o x . 0.5 n M

S-l 34 fraction (XIV) 2 0 fil a p p r o x . 0.7 m g . p r o t e i n / m l .
A f t e r 5, 15, 3 0 , 6 0 , 1 2 0 , a n d 180 m i n . , t a k e 25 pi.

a l i q u o t s for p r e c i p i t a t i o n a n d h y d r o l y s i s .
Precipitation, AA-tRNA-Hydrolysis and Determination of the Radioactivity Incorporated
P i p e t t e 25 /il a l i q u o t o n t o a g l a s s fibre filter ( W h a t m a n G F / A , 2 5 m m . ) , a n d p l a c e this i m m e

d i a t e l y in i c e - c o l d 1 0 % t r i c h l o r o a c e t i c a c i d . To p r e v e n t d i s i n t e g r a t i o n o f t h e filter d u r i n g t h e
s u b s e q u e n t h y d r o l y s i s o f t h e a m i n o a c i d ester w i t h t h e t R N A , it is r e c o m m e n d e d t h a t a Teflon
h o l d e r w i t h s l o t s t o a c c e p t t h e i n d i v i d u a l filter d i s c s ( s e e a b o v e ) b e u s e d . H y d r o l y s e t h e filter w i t h
the p r e c i p i t a t e in a b e a k e r w i t h 5 % t r i c h l o r o a c e t i c a c i d for 10 m i n . at 9 0 ° C . T h e n w a s h for
1 m i n . in e a c h o f t h e f o l l o w i n g s o l u t i o n s at r o o m t e m p e r a t u r e : 5 % t r i c h l o r o a c e t i c a c i d (2 x),
e t h a n o l - e t h e r 1 : 1 (2 x), ether. D r y t h e filter u n d e r a n I R l a m p , p l a c e in c o u n t i n g vial w i t h

2 m l . o f s c i n t i l l a t o r s o l u t i o n ( X V ) , a n d m e a s u r e in a l i q u i d s c i n t i l l a t i o n c o u n t e r .
In t h e t e s t i n g o f p o l y n u c l e o t i d e s , o n e will b e i n t e r e s t e d either in t h e a m i n o a c i d specificity
o r t h e t e m p l a t e a c t i v i t y f o r a g i v e n a m i n o a c i d . If it is d e s i r e d t o d e t e r m i n e b o t h t h e m a x i m u m
rate a n d the t o t a l a m i n o a c i d i n c o r p o r a t i o n , it is a d v i s a b l e t o p l o t t h e i n c o r p o r a t i o n ( o r d i n a t e )
a g a i n s t t i m e ( a b s c i s s a ) for all t h e s u g g e s t e d p o l y n u c l e o t i d e c o n c e n t r a t i o n s . S i n c e t h e a b s o l u t e
incorporation depends not only o n the polynucleotide used but also o n other c o m p o n e n t s
o f t h e s y s t e m , p a r t i c u l a r l y t h e r i b o s o m e p r e p a r a t i o n a n d t h e S - 1 3 4 f r a c t i o n , a similar p o l y
n u c l e o t i d e h a v i n g a h i g h e r t e m p l a t e a c t i v i t y s h o u l d b e t e s t e d for c o m p a r i s o n if p o s s i b l e .
To simplify s u c h c o m p a r i s o n s , it c o u l d b e a s s u m e d for p o l y u r i d y l i c a c i d c o d e d E. coli s y s t e m s
that the rate o f i n c o r p o r a t i o n for p h e n y l a l a n i n e is c o n s t a n t b e t w e e n 5 a n d 3 0 m i n . a n d t h a t
2 /d. o f 0.6 m M p o l y u r i d y l i c a c i d s o l u t i o n is a linearly l i m i t i n g q u a n t i t y in o u r test, w h i l e
10 pi. is s a t u r a t i n g .
Calculations
The quantities of a m i n o acid i n c o r p o r a t e d per ml. of reaction mixture are found in p m o l e by multiplication
of the count in counts/min. by the factor:
100 x 1000
f =
C o u n t i n g efficiency (%) x 2.22 x Spec, radioactivity x 25
This factor can be b r o k e n d o w n into the p o r t i o n s for conversion from counts/min.

C o u n t i n g efficiency (%)
into disintegrations/min.,—-— for conversion from disintegrations/min. into pCurie,— ^ ——
&
2.22
1
Spec, radioactivity
for conversion from p C u r i e into p m o l e , a n d for conversion from i n c o r p o r a t i o n in the 25 ul
aliquot into the i n c o r p o r a t i o n that would occur in 1 ml. T h e reaction mixture contains a b o u t 500 p m o l e
of ribosomes per ml. T h e p m o l e of a m i n o acid incorporated per p m o l e of ribosomes per minute in the
linear part of the kinetic curve give an i n c o r p o r a t i o n rate that can be used for evaluation of the quality
of the system, and in a c o m p a r i s o n for the characterization of the activities of the polynucleotides tested.
O n the other h a n d , the n u m b e r of a m i n o acid residues incorporated per nucleotide of the polynucleotide
at the end of the reaction shows o n average h o w often the nucleotide has been translated. Since 3 nucleo
tides function as a c o d o n , this n u m b e r must be multiplied by 3. T h u s (pmole of a m i n o acid/ml. : p m o l e
of nucleotide/ml.) x 3 = frequency with which the individual nucleotide has coded. W i t h the limiting
quantity of 2 /d. of 0.6 m M polyuridylic acid solution per 200 fil of reaction mixture, 1 ml. of reaction
mixture contains 6000 p m o l e uridylic acid.
Normal Values
The incorporation yield d e p e n d s n o t only on the properties of the polynucleotide p r e p a r a t i o n s but also
on the quality of the enzyme fraction a n d of the r i b o s o m e p r e p a r a t i o n . Polyuridylic acid can code 1 - 3
moles of phenylalanine per mole of uridylic acid a d d e d at the limit. T h e individual nucleotide then codes
3 to 9 times on average (cf. " C a l c u l a t i o n s " ) . T h e m a x i m u m i n c o r p o r a t i o n rates are found in this system
with 1 mole of phenylalanine per mole of ribosomes per minute. However, this rate is reached only if
the other 19 a m i n o acids (which are unnecessary in this polymerization) are absent from the system.
T h e system indicated operates with a (linear) limiting concentration of t R N A (750 /zg./ml.).
A s t a n d a r d deviation of ^ 3 % can be expected in the c o m p a r i s o n of activities in parallel experiments.

Owing to the complexity of the system, m u c h greater fluctuations can occur in the absolute results o b
tained on different days in experiments with the same or with different enzyme p r e p a r a t i o n s .
Errors sometimes occur t h r o u g h unidentified impurities in the substances u s e d ; these errors are most
easily determined empirically with the aid of the reference substances m e n t i o n e d u n d e r " P u r i t y of
Reagents".
Template activity is also exhibited by n a t u r a l messenger R N A if the cell-free system used comes from
the same or sufficiently closely related cells a n d so also contains the initiation factors required for the
start of the translation. There are also various ways in which the need for the specific initiation processes
can be avoided, e.g. in the translation of polyuridylic acid a n d most o t h e r synthetic polynucleotides,
which contain n o initiation sequence. High M g 2 +
concentrations p r o m o t e this d i s t u r b a n c e (which m a y
be desirable in model experiments) of the n a t u r a l specificity of initiation. S o m e ribonucleic acids, on the
o t h e r h a n d , exhibit practically n o template activity, examples being transfer R N A , p u r e ribosomal R N A ,
or messenger R N A from rat liver when tested in a complete E. coli system with 1 0 - 1 5 m M M g 2 +
.
Appendix
Preparation of a 134000 g Supernatant (S-134 Fraction) and Purified Ribosomes from E. coli.
Culture, Collection, and Washing of the Cells " 4 5
Inoculate eight 20 litre flasks containing 15 1. each of 0 . 8 % Difco N u t r i e n t B r o t h a n d 0 . 5 % of glucose

(extra sterilized), with 450 ml. each of an overnight culture of E. coli (e. g. strain A 19 R N a s e I~) at inter
vals of 30 min. at 33 °C. A d d a few ml. of silicone antifoam emulsion S L E (Wacker-Chemie) as re
quired. Blow in air t h r o u g h a sintered glass tube. By exponentially increased limiting aeration, logarithmic
growth of the culture is achieved (generation time 4 0 - 5 0 min.), a n d the m o s t active extracts are o b t a i n e d
in this way. If the culture contains a b o u t 2 x l 0 8
cells/ml. ( E 5 0 0 = 0.7), centrifuge with a Sharpies
continuous-flow centrifuge at 40000 to 45000 r p m . Approximately 80 g. of cells are obtained.
Slurry the cells in 1000 ml. of 10 m M tris buffer ( p H 7.8 with 10 m M M g acetate and 60 m M KC1) in
a beaker a n d resuspend. Centrifuge for 20 min. at 10000 g (Sorvall R o t o r G S A ) , decant oft s u p e r n a t a n t
fluid, and take only the clean p a r t of the sediment with a spatula. If the sediment is very dirty, repeat
this step.
Preparation of a 134000 g Supernatant Fraction
H o m o g e n i z e 2 0 - 4 0 g. of cells with twice their weight of A 1 0 (Alcoa 305 A ) in a glass m o r t a r with a

2 3
P V C pestle at a b o u t + 3 °C in a cold r o o m . K n e a d the A 1 0 p o w d e r in p o r t i o n s into the mass, which

2 3
repeatedly liquifies to give a viscous, stringy d o u g h , within 8 - 1 5 min. Avoid an excessively sudden ad
dition of A 1 0 . Elute by slow mixing of s t a n d a r d buffer (10 m M tris buffer p H 7.8 containing 10 m M
2 3
M g acetate, 60 m M KC1, 6 m M m e r c a p t o e t h a n o l (420 /d./l.)) in the p r o p o r t i o n of 2.5 times the weight

of cells.
Centrifuge for 20 min. at 20000 g (Sorvall R o t o r SS 34 with brake). A 1 0 2 3 a n d cell debris sediment.
D e c a n t s u p e r n a t a n t fluid (crude S-20 fraction) into a measuring cylinder without the cloudy dregs a n d
a d d 2 ^g./ml. of D N a s e (dissolve 1 m g . of D N a s e in 1 ml. of w a t e r ; d o n o t shake, since the D N a s e is
otherwise d e n a t u r e d a n d flocculates out). M o v e the S-20 fraction gently with the D N a s e so that it again
becomes optically h o m o g e n e o u s ; only then introduce into fresh centrifuge tubes. Centrifuge for 20 min.
at 30000 g (with brake), decant s u p e r n a t a n t fluid (S-30 fraction). Centrifuge for 30 min. at 30000 g (with
out brake). R e m o v e s u p e r n a t a n t fluid with pipette fitted with r u b b e r b u l b , without sucking u p the cloudy
sediment.
Centrifuge S-30 fraction for 2 h. at 134000 g (45000 r p m in the Spinco R o t o r 50 Ti). Siphon off the clear
t o p 3/4 of the s u p e r n a t a n t fluid (S-134 fraction) a n d dialyse overnight against s t a n d a r d buffer. K e e p
frozen at —35 °C in 2 ml. p o r t i o n s . F u r t h e r purify sediment (ribosomes).
Purification of Ribosomes 6
Purify the ribosome pellets to remove m o r e t h a n 9 9 % of their ribonucleolytic activity ( R N a s e II) by

three repetitions of the following washing o p e r a t i o n s :
a) After surface washing with s t a n d a r d buffer containing n o m e r c a p t o e t h a n o l , suspend the pellets to
480 E 2 6 0 / m l . (approx. 30 mg./ml.) in s t a n d a r d buffer without m e r c a p t o e t h a n o l by occasional gentle
stirring with a glass r o d in the course of a b o u t 2 h o u r s o r by standing overnight.
b) A d d an equal v o l u m e of ice-cold " w a s h s o l u t i o n " (1 M N H C 1 , 10 m M tris buffer p H 7.8, 10 m M
4
M g acetate, 60 m M K C 1 ; mix t h o r o u g h l y after the addition of each c o m p o n e n t ) to the suspension in

the Pyrex tube (Sorvall). K e e p the suspension in ice for a further 30 min.
c) Centrifuge for 10 min. at 10000 g with b r a k e (Sorvall R o t o r SS 34), a n d remove s u p e r n a t a n t fluid with
pipette and r u b b e r bulb.
d) Centrifuge for 3 hr. at 134000 g with b r a k e (45000 r p m in the Spinco R o t o r 50 Ti) at + 4 °C, a n d
suspend the pellets as u n d e r a).
Repeat these washes twice. T h e n suspend the ribosome pellets in s t a n d a r d buffer w i t h o u t m e r c a p t o e t h a n o l ,
adjust to 240 E 2 6 0 units/ml., a n d keep frozen in 1 ml. aliquots at —30 to —50 °C.
References
1 F. Gros & H. Matthaei: Practical Molecular Genetics. Springer-Verlag, Berlin-Heidelberg-New Y o r k

1973, in press.
2 E. Bermek & H. Matthaei, H o p p e Seyler's Z. Physiol. C h e m . 351, ttll [1970].
3 T h e Genetic C o d e , 31st. Cold Spring H a r b o r Symposium, 1966. Cold Spring H a r b o r L a b o r a t o r y , L. I.
New Y o r k 1967, p . 25.
4 H Matthaei & M. W. Nirenberg, P r o c . N a t l . Acad. Sci. ( U . S.), 47, 1580 [1961].
5 H. Matthaei, G. Heller, H. P. Voigt, R. Neth, G. Schoch & H. Kubler in D. Shugar: Genetic Elements.
Academic Press, L o n d o n a n d N e w Y o r k 1967, p . 2 3 3 - 2 5 0 .
6 H. P. Voigt & H Matthaei, Hoppe-Seyler's Z. Physiol. C h e m . 349, 54 [1968].
Adenine and Guanine
Gotthilf Naher
G u a n i n e (2-amino-6-hydroxypurine) a n d adenine (6-aminopurine) occur in all life forms as c o m p o n e n t s

of nucleic acids, nucleosides, nucleotides, a n d coenzymes, in which they are b o u n d by glycoside linkages.
As d e g r a d a t i o n p r o d u c t s of these c o m p o u n d s , guanine occurs in m e a t a n d in considerable c o n c e n t r a t i o n s
in the scales a n d skin of fish. F r e e adenine is found in tea, sugar beets, yeast, a n d various a n i m a l organs.
G u a n i n e can be specifically d e a m i n a t e d to xanthine by guanase ( G u a n i n e a m i n o h y d r o l a s e , E C 3.5.4.3).
X a n t h i n e is oxidized t o uric acid by xanthine oxidase ( X a n t h i n e : oxygen oxidoreductase, E C 1.2.3.2).
These reactions form the basis of the following p r o c e d u r e , which is based o n d a t a r e p o r t e d by Kalckar .
1
Adenine is oxidized by x a n t h i n e oxidase to form 2,8-dihydroxyadenine , with an extinction m a x i m u m at

2
305 n m . According to Klenow 3

this reaction can be used for the enzymatic d e t e r m i n a t i o n of adenine.
However, the catalytic activity of x a n t h i n e oxidase for adenine is extremely low, so t h a t this m e t h o d is
unsuitable for routine d e t e r m i n a t i o n s . We therefore d e a m i n a t e adenine with n i t r o u s acid to o b t a i n h y p o -
xanthine, which can be determined rapidly a n d accurately with x a n t h i n e oxidase. G u a n i n e , xanthine, a n d
h y p o x a n t h i n e are also determined by this m e t h o d , but these c o m p o u n d s c a n be d e t e r m i n e d separately by
specific m e t h o d s , a n d the adenine value can then be found with sufficient accuracy from the difference.
Application of Methods: In biochemistry a n d in food chemistry.
Principle
(1) Guanine + H 0 . ,
2
g u a n a s e
- Xanthine + N H 3
i J
(2) Xanthine + H 0 + 0 2 2 , x a n t h i n e
Uric acid + H 0 2 2
oxidase
(la) Adenine + H N 0 2 • Hypoxanthine + N 2 + H 0

2
, I
i
(2a) Hypoxanthine + H 0 + 0 2 2 ^ = = * Xanthine + H 0 2 2
i '
(3a) Xanthine + H 0 + 0 2 2 ., x a n t h i n e
- Uric acid + H 0 2 2
T h e increase in the uric acid content, as measured by the change in extinction at 293 n m , is p r o p o r t i o n a l
to the quantity of guanine o r of adenine present.
T h e complete oxidation of xanthine or h y p o x a n t h i n e with xanthine oxidase is possible only if the buffer
is sufficiently saturated with oxygen. W h e n tris buffer is used, the oxygen required for the oxidation is
normally contained in the solution. Oxygen shortage is to be expected only if the buffer has been stored
at r o o m t e m p e r a t u r e for a long time.
T h e d e a m i n a t i o n of adenine t o h y p o x a n t h i n e is strongly d e p e n d e n t o n t h e acid a n d nitrite c o n c e n t r a t i o n
a n d on the reaction time a n d t e m p e r a t u r e . U n d e r the conditions described here, the reaction proceeds
completely to h y p o x a n t h i n e .
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at 2 9 3 n m ; o x y g e n o r c o m p r e s s e d air
c o n n e c t i o n o r r u b b e r b u l b w i t h a d r a w n - o u t g l a s s c a p i l l a r y ; 37 ° C w a t e r b a t h or t h e r m o b l o c k ;
laboratory centrifuge.
Reagents
1. Tris-hydroxymethyl-aminomethane, 4. H y d r o c h l o r i c a c i d , 1.0 N
tris 5. P e r c h l o r i c a c i d , A . R., 7 0 % ( w / v ) ,
2. Guanase s p . gr. 1.67
from rabbit liver, suspension containing 10 mg. 6. P o t a s s i u m h y d r o x i d e s o l u t i o n , 1.0 N
enzyme protein/ml. in 3.2 M a m m o n i u m sul 7. S o d i u m nitrite, A . R.
p h a t e solution; j> 0.06 U / m g . (25 °C); c o m m e r 8. S u l p h u r i c a c i d , 9 5 - 9 8 % , h i g h e s t p u r i t y
cial p r e p a r a t i o n s , see p . 4 7 1 . 9. S u l p h u r i c a c i d , 1.0 N
3. X a n t h i n e o x i d a s e , X O D 10. S o d i u m h y d r o x i d e s o l u t i o n , A . R., 2 0 %
from milk, suspension containing 10 mg.
enzyme protein/ml. in 3.2 M a m m o n i u m sul
p h a t e solution (10 m M E D T A ) ; ^ 0 . 4 U / m g .
(25 ° C ) ; commercial p r e p a r a t i o n s , see p. 521.
Purity of Reagents
X a n t h i n e oxidase should c o n t a i n not m o r e t h a n 0.01 % each of guanase, uricase, and nucleoside phosphoryl-
ase. All reagents must be free from inorganic p h o s p h a t e . If the d e t e r m i n a t i o n is carried out in a p h o s p h a t e -
free medium, c o n t a m i n a t i o n of the g u a n a s e by nucleoside phosphorylase does not affect the result.
P r e p a r e all s o l u t i o n s w i t h freshly p r e p a r e d , d o u b l y - d i s t i l l e d w a t e r . Sterilize t h e v e s s e l s t o

prevent the growth o f micro-organisms.
I. Tris buffer (0.1 M ; p H 8 ; s a t u r a t e d w i t h o x y g e n ) :
D i s s o l v e 1.2 g. tris in 8 0 m l . d i s t i l l e d w a t e r , adjust t o p H 8.0 w i t h 1 N H C 1 , a n d m a k e u p
t o 100 m l . w i t h w a t e r . I m m e d i a t e l y b e f o r e u s e , e n r i c h t h e buffer w i t h o x y g e n b y b u b b l i n g
in p u r e o x y g e n o r c l e a n c o m p r e s s e d air for a b o u t 2 m i n . o r b y b l o w i n g in air b y m e a n s
of a rubber bulb and a glass capillary.
II. S o d i u m nitrite, 2 0 % ( w / v ) :
D i s s o l v e 2 0 g. o f N a N 0 2 in distilled w a t e r a n d m a k e u p t o 100 m l .
III. P e r c h l o r i c a c i d ( 1 . 0 N ) :
D i l u t e 8.2 m l . o f 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h d i s t i l l e d w a t e r .
IV. X a n t h i n e o x i d a s e , X O D ( 1 0 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s o l u t i o n a s r e q u i r e d w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V. G u a n a s e (10 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s o l u t i o n a s r e q u i r e d w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n .
Adenine and Guanine 1911
Store the buffer solution a n d the enzyme suspensions in stoppered containers in a refrigerator at 0 - 4 °C.
T h e buffer solution is stable as long as n o micro-organisms grow in it. T h e enzyme suspensions keep for
at least 6 m o n t h s .
Procedure
Treatment of ribonucleic acids and polynucleotides
A d d 30 m l . 1 N H S 0 2 4 t o a q u a n t i t y o f the test m a t e r i a l c o r r e s p o n d i n g t o 1 0 0 - 2 0 0 m g .
r i b o n u c l e i c a c i d , a n d h e a t for o n e h o u r in a b o i l i n g w a t e r b a t h w i t h o c c a s i o n a l s h a k i n g . C o o l ,
m a k e u p t o 100 m l . w i t h d o u b l y - d i s t i l l e d w a t e r , c e n t r i f u g e off a n y p r e c i p i t a t e t h a t h a s f o r m e d ,
a n d u s e t h e s u p e r n a t a n t fluid for t h e a s s a y . 4
Deproteinization of tissues
H o m o g e n i z e m e a t , fish s k i n , w h o l e b l o o d , etc. w i t h a n e q u a l v o l u m e o f 1 N H C 1 0 4 for 10 m i n .

at 0 ° C ; c e n t r i f u g e for 10 m i n . at 3 0 0 0 r p m . A d j u s t a n a l i q u o t o f t h e s u p e r n a t a n t fluid t o p H 10
with 1 N K O H . Establish the quantity o f K O H required to determine the dilution factor.
C e n t r i f u g e off t h e p e r c h l o r a t e p r e c i p i t a t e after s t a n d i n g in ice for 15 m i n . U s e t h e s u p e r n a t a n t
for t h e a s s a y .
Stability of sample
G u a n i n e a n d a d e n i n e are s t a b l e in a c i d i c a n d a l k a l i n e m e d i a . O n s t a n d i n g for s e v e r a l h o u r s in
s t r o n g l y a c i d i c s o l u t i o n o r o n h e a t i n g in a c i d i c s o l u t i o n , s u b s e q u e n t f o r m a t i o n o f t h e s e p u r i n e
bases from the corresponding nucleic acid c o m p o n e n t s m u s t also be expected.
G u a n i n e is p r a c t i c a l l y i n s o l u b l e in n e u t r a l m e d i a , w h i l e a d e n i n e is s p a r i n g l y s o l u b l e . F i l t r a t i o n
or c e n t r i f u g a t i o n o f t h e s a m p l e a n d p r o l o n g e d s t a n d i n g at n e u t r a l p H s h o u l d t h e r e f o r e b e
a v o i d e d . O n t h e o t h e r h a n d , g u a n i n e a n d a d e n i n e are sufficiently s o l u b l e in m i n e r a l a c i d s ,
i n c l u d i n g p e r c h l o r i c a c i d , a n d in s o d i u m h y d r o x i d e o r p o t a s s i u m h y d r o x i d e s o l u t i o n .
Assay System
W a v e l e n g t h : 2 9 3 n m ; light p a t h : 1 c m . ; r o o m t e m p e r a t u r e ; m e a s u r e a g a i n s t air.
Guanine final v o l u m e : 3.08 ml.
Tris buffer (I) 3.00 ml. a p p r o x . 0.1 M tris

Sample 0.03 ml. u p t o a p p r o x . 5 0 ,uM g u a n i n e
M i x , read e x t i n c t i o n E . x
X O D suspension (IV) 0.01 m l . 13 m U X O D / m l .
F o l l o w a n y c h a n g e in e x t i n c t i o n u n t i l a c o n s t a n t v a l u e
is r e a c h e d ( x a n t h i n e , h y p o x a n t h i n e ) , r e a d e x t i n c t i o n
E . E
2 2 — E t = A E . S u b t r a c t t h e c h a n g e in e x t i n c
x
t i o n t h a t o c c u r s o n further a d d i t i o n o f X O D f r o m E . 2
X O D suspension (IV) 0.01 m l . 26 m U X O D / m l .
M i x , read extinction E . 3
Guanase suspension (V) 0.02 ml. 4 m U guanase/ml.
M i x , f o l l o w c h a n g e in e x t i n c t i o n until a c o n s t a n t v a l u e
is r e a c h e d . R e a d e x t i n c t i o n E . E 4 4 — E 3 = AE G is
u s e d in t h e c a l c u l a t i o n s .
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n o n a d d i t i o n o f g u a n a s e s u s p e n s i o n b y a d d i n g a further
0 . 0 2 m l . o f g u a n a s e s u s p e n s i o n at t h e e n d o f the r e a c t i o n . S u b t r a c t t h e r e s u l t i n g c h a n g e in
extinction from E . 4
Adenine a n d G u a n i n e 1913
Adenine final v o l u m e : 3.04 ml.
Sample 2.00 ml. up to approx. 5 m M adenine

S o d i u m nitrite s o l u t i o n (II) 0.90 ml. a p p r o x . 0.9 M N a N 0 2
Sulphuric acid, highest purity 0.10 ml. a p p r o x . 1.2 N H S0

2 4
M i x w e l l a n d k e e p at 37 ° C for 6 0 m i n .
S o d i u m hydroxide solution, 20 % 1.00 m l .
M i x well ( = assay solution);
Tris buffer (I) 3.00 ml. 0.1 M tris

Assay solution 0.03 ml.
M i x , read extinction E . x u p t o a p p r o x . 50 ^ M h y p o x a n t h i n e
X O D suspension (IV) 0.01 m l .
M i x , f o l l o w c h a n g e in e x t i n c t i o n u n t i l c o n s t a n t v a l u e 13 m U X O D / m l .
is r e a c h e d , t h e n r e a d e x t i n c t i o n E . E - E
2 2 l = AE A is
u s e d in the c a l c u l a t i o n .
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f X O D s u s p e n s i o n by a d d i n g a further
0.01 m l . X O D s u s p e n s i o n at t h e e n d o f t h e r e a c t i o n . S u b t r a c t t h e r e s u l t i n g c h a n g e in e x t i n c t i o n
from E . 2
Calculations
U n d e r the conditions indicated, the reaction proceeds completely to uric a c i d ; A E is p r o p o r t i o n a l to the

quantity of purine bases transformed by X O D .
Guanine
The m o l a r extinction coefficient at 293 n m , p H 8.0 is 2.74 c m . / / i m o l e for g u a n i n e a n d 12.20 c m . / / m i o l e

2 2
for uric acid. U n d e r the conditions indicated, the change in extinction from guanine to uric acid is
A e = 9.46 c m . / ^ m o l e .
2
T h e following relationships are therefore valid
AE x 3 07
Assay m i x t u r e : c = G
= AE G x 0.325 [//mole/ml.]
9.46
AE x 3 07
Sample solution: c = ° ——— = z l E G x 10.8 [/imole/ml.]
9.46 x 0.03
T h e weight of guanine in ug. is found by multiplication by t h e molecular weight of guanine (151.1).

If the sample has been subjected to previous t r e a t m e n t (deproteinization, acid hydrolysis), the result must
be multiplied by the dilution factor.
Adenine
T h e m o l a r extinction coefficient at 293 n m , p H 8.0, is 0.01 c m . / / i m o l e for the h y p o x a n t h i n e formed by 2
deamination of adenine a n d 12.20 c m / / i m o l e for uric acid. U n d e r the conditions indicated, the change in
2
extinction from h y p o x a n t h i n e to uric acid is Ae = 12.19 c m . / / m i o l e . 2
T h e following relationships are therefore valid:
Assay m i x t u r e : c =-^-^^~^- = AE A x 0.25 [^mole/ml.]
Sample solution: c = x 16.7 [/jmole/ml.l

x 0.03 ^ = AE
X X
A
H
12.19 A
Multiplication by the molecular weight of adenine (135.13) gives the weight of adenine in jug.
If guanine, adenine, h y p o x a n t h i n e , a n d x a n t h i n e are present together, t h e c h a n g e in extinction d u e t o
hypoxanthine, xanthine, a n d g u a n i n e must be subtracted from A E A for the d e t e r m i n a t i o n of adenine.
It must be remembered here t h a t guanine is converted into xanthine on d e a m i n a t i o n . U n d e r the conditions
indicated, the change in extinction from xanthine to uric acid is As = 9.72 c m . / / / m o l e , a n d AE 2
G must
therefore be multiplied by the factor 1.03.
This gives the following relationships:
(2 AE A — AE G x 1.03 - AE ) X x 3.04 . , . , .
Assay m i x t u r e : c =— — [iimole/ml.l
3
2 x 11.9 L
^ 1 J
Sample solution: c — (2 AE A — AE G x 1.03 - AE ) X x 8.5 [^mole/ml.]
Multiplication by the molecular weight of adenine (135.13) gives t h e weight of adenine in pg.
Xanthine or Hypoxanthine
T h e change in extinction A E is p r o p o r t i o n a l to the sum of xanthine a n d h y p o x a n t h i n e . If only one of

x
the two purine bases is present, the calculation is as follows:
AE x 3 04
Assay m i x t u r e : c = x
^ ^ = AE X x O.313[/zmole/ml.]
Sample solution:
AE
c = ——r
x zr3 04 = AE x 10.4 [umole/ml.l
F
9.72 x 0.03 xx
Multiplication by the molecular weight of xanthine (152.1) gives the weight of x a n t h i n e in pg.;
or
Assay m i x t u r e : c = AE \219~ 3.04

X x =
^ x E x
0-25 [/xmole/ml.]
AE x 3 04
Sample solution: c ^ 19 x 0 03 = A E x x 8 , 3
[/^mole/ml.]
Multiplication by the molecular weight of h y p o x a n t h i n e (136.1) gives the weight of h y p o x a n t h i n e in /zg.

If b o t h xanthine a n d h y p o x a n t h i n e are present, these c o m p o u n d s can be determined in the presence of
each other as described by J^rgensen (see p . 1941).
Adenine a n d G u a n i n e 1915
Guanine: F o r values a r o u n d 1 mg./ml., a s t a n d a r d deviation of 0.013 m g . / m l . was found. T h e coefficient

of variation is 1.4%.
Adenine: F o r values a r o u n d 1 mg./ml., a s t a n d a r d deviation of 0.027 m g . / m l . was found. T h e coefficient

of variation is 2 . 7 % .
Adenine, Guanine, and Xanthine: F o r a mixture containing a p p r o x i m a t e l y 0.4 mg. of each of these purine
bases/ml., the following s t a n d a r d deviations were f o u n d : guanine 0.013 m g . / m l . ; adenine 0.027 m g . / m l . ;
xanthine 0.024 mg./ml. T h e coefficient of variation is C V a d e n i n e = 7%; C V g u a n i n e =4%; CV x a n t h i n e = 7%.
N o r m a l Values
W h e n guanine a n d adenine in ribonucleic acid are determined by the m e t h o d described here, the results
agree with the values found when ribonucleic acid is hydrolysed by alkali to 3'(2')-mononucleotides
followed by c h r o m a t o g r a p h i c separation of the latter o n an anion exchange resin.
Approximately 7 mg. of guanine was found in 1 g. of skin in fresh-water fish (Coregonus wartmanni).
Effects of drugs and other therapeutic measures: X a n t h i n e oxidase is specifically inhibited by allopurinol.
(Urosin®, Zyloric®).
Interference in assay technique: W h e n nucleoside p h o s p h o r y l a s e is present in the g u a n a s e as an impurity,

guanosine, inosine, a n d xanthosine are determined at the same time as g u a n i n e if p h o s p h a t e is also present.
If the assay mixture contains only traces of p h o s p h a t e , a slow, c o n t i n u o u s increase in extinction ( " c r e e p " )
is observed in the presence of these nucleosides. T h e slow change in extinction is superimposed on the
main reaction, so that it is necessary to extrapolate graphically to the time at which the reaction started,
see p . 308.
X a n t h i n e oxidase catalyses the oxidation of h y p o x a n t h i n e , xanthine, a n d a large n u m b e r of other aldehydes,

ketones, and p u r i n e s 5 - 7
. In the analysis of biological material a n d of nucleic acid hydrolysates, a possible
reaction with adenine, which is oxidized to 2,8-dihydroxyadenine with an extinction m a x i m u m at 305 n m ,
must be taken into account. However, since the rate of oxidation of a d e n i n e is only a b o u t 2 % of the
rate of oxidation of x a n t h i n e , at most only a " c r e e p " occurs o n addition of xanthine oxidase. T h e re
3
sulting slow, c o n t i n u o u s increase in extinction can be extrapolated graphically to the time at which the
reaction started, see p . 308.
References
1 H. M. Kalckar, J. biol. C h e m . 167, 461 [1947].

2 A. Bendich, G. B. Brown, F. S. Philips & J. B. Thiersch, J. biol. C h e m . 183, 367 [1950].
3 H. Klenow, Biochem. J. 50, 404 [1952].
4 H. S. Loring, J. L. Fairly & H. L. Seagran, J. biol. C h e m . 197, 823 [1952].
5 V. H. Booth, Biochem. J. 32, 494 [1938].
6 D. E. Duggan & E. Titus, J. biol. C h e m . 234, 2100 [1959],
7 H. R. Silberman & /. B. Wyngaarden, Biochim. Biophys. A c t a 47, 178 [1961].
Cytosine
A u g u s t W. H o l l d o r f
Cytosine is a precursor of cytosine nucleotides and therefore of ribo- and deoxyribonucleic acids. In most
organisms the free form is not an intermediate in the biosynthesis or catabolism of nucleotides and nucleic
acids. Consequently, so far the free form has either n o t been found or only detected in very small a m o u n t s
in biological material. Cytosine is, however, obtained in the chemical or enzymatic d e g r a d a t i o n of nucleic
acids. Identification and determination is usually based on the U V a b s o r p t i o n spectrum. Cytosine is
deaminated to uracil by cytosine d e a m i n a s e (Cytosine a m i n o h y d r o l a s e , E C 3.5.4.1). This reaction is the
1
basis for the specific determination of cytosine.
Application of Method: In biochemistry and cell physiology.
Principle
(1) Cytosine + H 0 2 Uracil + NH 3
The reaction is accompanied by a decrease of extinction at 280 n m . T h e decrease in extinction is p r o

2
portional to the a m o u n t of cytosine reacting.
The equilibrium of reaction (1) lies completely on the side of uracil formation. T h e enzyme from y e a s t 3
has a p H o p t i m u m between 7.0 and 7.4; it requires n o cofactors.
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for m e a s u r e m e n t s in the U V r a n g e ( 2 8 0 n m ) ; b e n c h c e n t r i f u g e a n d
p H meter.
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 5. R e a g e n t s for the d e p r o t e i n i z a t i o n o f n u c l e i c

2. HC1, A . R . , 1 N a c i d h y d r o l y s a t e s a n d for t h e p u r i f i c a t i o n
3. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) ; s p . g r . o f c y t o s i n e f r o m very d i l u t e s o l u t i o n s , see
1.67 "Determination of Cytidine and Deoxy-
4. C y t o s i n e d e a m i n a s e c y t i d i n e " (see p. 1 9 2 3 ) .
from b a k e r s ' yeast; enzyme suspension contain
ing 2 - 4 mg. p r o t e i n / m l . ; ^0.2-0.4 U/mg.
(25 °C); see A p p e n d i x , p . 1918.
Purity of Reagents
Cytosine deaminase must not c o n t a i n detectable a m o u n t s of cytidine d e a m i n a s e or deoxythymidine a n d

uridine phosphorylase. T h e enzyme p r e p a r a t i o n used here fulfils these requirements.
Cytosine 1917
I. Tris buffer ( 0 . 2 M ; p H 7.4):

D i s s o l v e 2 . 4 2 g. tris in c a . 5 0 m l . distilled w a t e r , a d j u s t t o p H 7 . 4 w i t h 1 N HC1 ( g l a s s
e l e c t r o d e ) a n d d i l u t e t o 100 m l . w i t h distilled w a t e r ; c h e c k t h e p H a g a i n .
II. P e r c h l o r i c a c i d ( 1 . 0 N ) :
D i l u t e 8.3 m l . 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h distilled w a t e r .
III. C y t o s i n e d e a m i n a s e :
U s e the e n z y m e solution undiluted.
I V . S o l u t i o n for d e p r o t e i n i z a t i o n o f e n z y m a t i c h y d r o l y s a t e s a n d for t h e p u r i f i c a t i o n o f c y t o s i n e
f r o m very d i l u t e s o l u t i o n s , see " D e t e r m i n a t i o n o f C y t i d i n e a n d D e o x y c y t i d i n e " ( p . 1 9 2 4 ) .
Solution I is stable for several weeks, stoppered, in a refrigerator. T h e e n z y m e solution (III) is stable for
several m o n t h s at - 1 5 °C. Solution II is stable indefinitely at r o o m t e m p e r a t u r e .
Procedure
T h e a s s a y s h a v e s o far o n l y b e e n carried o u t o n a q u e o u s s o l u t i o n s , e s p e c i a l l y D N A h y d r o
l y s a t e s . T h i s t y p e o f s a m p l e is s t a b l e for several d a y s at 0 - 4 ° C .
4
Assay System
W a v e l e n g t h : 2 8 0 n m ; q u a r t z c u v e t t e s , 1.00 c m . light p a t h ; final v o l u m e : 3 . 0 0 m l . ; i n c u b a t i o n at

37 ° C . R e a d a g a i n s t a c o n t r o l .
Pipette into centrifuge tubes: Experimental Control
assay mixture
Sample (neutralized) 1.00 m l . 1.00 m l . 1 5 - 3 0 0 ^ M cytosine

Tris buffer (I) 1.00 m l . 1.00 m l . 67 m M
H C 1 0 solution
4 (II) 0.50 ml. 167 m M
Enzyme solution (III) 0.50 ml. 0.50 ml.
M i x a n d i n c u b a t e w i t h g e n t l e s h a k i n g for 30 m i n . c a . 0.3 m g . / m l . = 0.1 U / m g .
HC10 4 solution (II) 0.50 ml. —

C e n t r i f u g e for 15 m i n . at 5 0 0 0 g , p o u r t h e s u p e r n a t a n t fluids i n t o 167 m M
t w o quartz cuvettes. R e a d experimental against control cuvette.
T h e e x t i n c t i o n d i f f e r e n c e is u s e d for t h e c a l c u l a t i o n s .
If t h e e x t i n c t i o n in b o t h c u v e t t e s is t o o h i g h , t h e s a m p l e a n d b l a n k c a n b e d i l u t e d t o t h e s a m e
e x t e n t w i t h 1 N HC1.
Calculations
U n d e r the a b o v e conditions the reaction proceeds stoichiometrically. T h e e 2 8 0 n m * 3.20 x 1 0 c m . / m o l e

s 6 2
at p H 7 . 4 ; e
2
2 8 0 nm l s
5 4 1 x 1 0 c m . / m o l e at p H 1. A c c o r d i n g to e q u a t i o n (2) on p . 312 the c o n c e n t r a t i o n
6 2
of the sample is given by
c = AE x 0.55 [^mole/ml.]
This value must be multiplied by the a p p r o p r i a t e dilution factors.
In a D N A hydrolysate repeated analysis of the cytosine content gave a value of 6.24 ± 0 . 1 3 /imole/ml.
T h e coefficient of variation is 2.1 %.
Cytosine d e a m i n a s e also reacts with 5-methylcytosine, which occurs in small a m o u n t s in several nucleic
acids, as well as with cytosine derivatives substituted with halogens in the 5 position (5-F-, C1-, Br- or I-
cytosine) . 2
Appendix
Cytosine Deaminase
T h e cytosine d e a m i n a s e required for the assay can be p r e p a r e d 20-fold purified from b a k e r s ' yeast in
2 - 3 days in 7 5 % yield . T h e purification m e t h o d includes: disintegration of cells by grinding with a l u m i n i u m
2
oxide, precipitation of inactive protein at p H 4 . 3 ; precipitation of nucleic acids with p r o t a m i n e sulphate,

fractionation with a m m o n i u m sulphate a n d dialysis.
References
1 E. Chargaff& J. Kream, J. biol. C h e m . 175, 993 [1948]; J. Kream & E. Chargaff, J. A m e r . C h e m . Soc.
74, 4274 [1952].
2 A. W. Holldorf & K. Resch, u n p u b l i s h e d experiments.
3 K. /tesc/*,unpublished experiments.
4 E. Vischer & E. Chargaff, J. biol. C h e m . 176, 715 [1948].
Adenosine
Hans Mdllering and Hans Ulrich Bergmeyer
Adenosine, 9-jS-D-ribofuranosyl adenine, is a c o m p o n e n t of coenzymes a n d occurs as a nucleoside hydrolysis

p r o d u c t of nucleic acids. It occurs in small a m o u n t s in h u m a n urine. T h e s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n
of purine derivatives with specific enzymes was described in detail in 1947 by Kalckar . 12
T h e quantitative
enzymatic d e t e r m i n a t i o n of adenosine with adenosine deaminase (Adenosine a m i n o h y d r o l a s e , E C 3.5.4.4)
from intestinal m u c o s a has superseded other m e t h o d s because of its great specificity.
Principle
(1) Adenosine + H 0 -£^£> 2 Inosine + NH 3
The m a x i m u m of the a b s o r p t i o n spectrum for adenosine is at 265 n m a n d for inosine is at 247 n m . T h e

decrease of extinction at 265 n m is m e a s u r e d .
T h e equilibrium of reaction (1) lies virtually completely on the right. T h e reaction is complete within a few
minutes in trie tha nolamine buffer ( p H 7.4) at r o o m t e m p e r a t u r e . T h e a b s o r p t i o n of adenosine is the same
between p H 7.2 a n d 8.9, of inosine between p H 7.2 a n d 8.0. W i t h inosine there is a displacement of the
m a x i m u m t o w a r d s higher wavelengths a b o v e p H 8.0; consequently the extinction difference E a d e n o s i n e —
Ejnosine is c o n s t a n t only below p H 8.0 . 3
Equipment
S p e c t r o p h o t o m e t e r suitable for precise m e a s u r e m e n t s at 265 n m ; bench centrifuge.
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e , c r y s t a l l i n e 3. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , s p .
2. A d e n o s i n e d e a m i n a s e , A D A gr. 1.67
from calf intestine, suspension in 3.2 M a m 4. P o t a s s i u m c a r b o n a t e , K C 0 , A . R .
2 3
monium sulphate solution; ^ 200 U/mg.

5. S o d i u m h y d r o x i d e s o l u t i o n , 1 N
(25 ° C ) ; commercial p r e p a r a t i o n , see p . 426.
Purity of Reagents
Adenosine de a minase m u s t be free from A - 5 - M P deaminase a n d p h o s p h o d i e s t e r a s e ; alkaline p h o s p h a t a s e ,

A T P a s e , nucleoside p h o s p h o r y l a s e a n d xanthine oxidase should be below 0 . 0 1 % (relative to the specific
activity of adenosine deaminase).
D i s s o l v e 1.86 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in 8 0 m l . d i s t i l l e d w a t e r , a d j u s t w i t h 1 N
N a O H t o p H 7.4 a n d m a k e u p t o 100 m l .
II. A d e n o s i n e d e a m i n a s e (0.1 m g . p r o t e i n / m l . ) :
III. P e r c h l o r i c a c i d (1 M ) :
D i l u t e 9 m l . 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h distilled w a t e r .
I V . P o t a s s i u m c a r b o n a t e (ca. 5 M ) :
D i s s o l v e 6 9 g. K C 0
2 3 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
Store solutions and the enzyme suspension, stoppered, in a refrigerator at 0 - 4 °C. Adenosine deaminase
is stable for ca. 1 year a n d the reagent solutions are stable indefinitely.
Procedure
Collection:
U s e t h e f r e e z e - s t o p t e c h n i q u e ( s e e p . 4 0 0 ) for cell a n d t i s s u e e x t r a c t s . S a m p l e s l o w in p r o t e i n
need not be deproteinized. These include hydrolysates o f nucleic acids, mixtures o f nucleotides
a n d n u c l e o s i d e s , p u r e a d e n o s i n e a n d p u r e p r e p a r a t i o n s in w h i c h t h e a d e n o s i n e c o n t a m i n a t i o n
is t o b e d e t e r m i n e d .
Deproteinization:
Deproteinize samples with a high protein content (e.g. yeast concentrates) with perchloric
a c i d ' . T r i c h l o r o a c e t i c a c i d is u n s u i t a b l e b e c a u s e it a b s o r b s at 2 6 5 n m . In a 10 m l . c e n t r i f u g e
1 2
tube mix t h o r o u g h l y 2.00 ml. s a m p l e (e.g. yeast concentrate) and 2.00 ml. ice-cold perchloric
a c i d ( s o l u t i o n III) w i t h a t h i n g l a s s r o d a n d c e n t r i f u g e for 15 m i n . at c a . 1 0 0 0 g. N e u t r a l i z e
1 m l . o f s u p e r n a t a n t fluid w i t h 0 . 0 5 m l . K C 0 2 3 s o l u t i o n ( I V ) , a l l o w t o s t a n d for 15 m i n . in a n
ice b a t h , filter a n d u s e 0.5 m l . o f filtrate f o r t h e a s s a y .
T i s s u e s w h i c h c o n t a i n a d e n o s i n e d e a m i n a s e d e a m i n a t e a d e n o s i n e t o i n o s i n e . In n e u t r a l i z e d
e x t r a c t s after d e p r o t e i n i z a t i o n a d e n o s i n e is s t a b l e for at least 2 4 hr. at 2 5 ° C . A l t h o u g h a d e n o s i n e
is o n l y s l o w l y h y d r o l y s e d t o a d e n i n e a n d r i b o s e at 100 ° C a n d p H 1.0, a c i d e x t r a c t s s h o u l d b e
n e u t r a l i z e d as s o o n a s p o s s i b l e .
Adenosine 1921
Assay System
If t h e s a m p l e c o n t a i n s l a r g e a m o u n t s o f o t h e r n u c l e o s i d e s o r n u c l e o t i d e s w h o s e h i g h e x t i n c t i o n
at 2 6 5 n m affects t h e a c c u r a t e d e t e r m i n a t i o n o f a d e n o s i n e , it is b e s t t o d e t e r m i n e t h e a p p r o x i m
ate e x t i n c t i o n c h a n g e d u e t o a d e n o s i n e in a p r e l i m i n a r y e x p e r i m e n t . T h e n i n t h e a c t u a l a s s a y
t h e m e a s u r e m e n t s are m a d e a g a i n s t a b l a n k c o n t a i n i n g sufficient s a m p l e s o t h a t at least 7 5 %
o f t h e e x t i n c t i o n at 2 6 5 n m n o t d u e t o a d e n o s i n e is c o m p e n s a t e d .
W a v e l e n g t h : 2 6 5 n m ; silica c u v e t t e s , l i g h t p a t h : 1 c m . ; final v o l u m e : 3 . 0 2 m l . ; r o o m t e m p e r
a t u r e ; r e a d a g a i n s t buffer s o l u t i o n (I) o r buffer s o l u t i o n + sample.
Buffer s o l u t i o n (I) 2.50 ml. 83 m M t r i e t h a n o l a m i n e

Sample (deproteinized, neutralized) 0.50 ml. u p t o c a . 15 ^ g . a d e n o s i n e / m l .
M i x thoroughly with a plastic spatula, read extinction

EJL (should n o t be m o r e than ca. 0.500).
Adenosine deaminase (II) 0.02 ml. 0 . 6 7 /xg./ml. = 1 3 4 m U / m l .
M i x a n d after c a . 5 m i n . r e a d final e x t i n c t i o n E .
2
Ei — E 2 = A E is u s e d f o r t h e c a l c u l a t i o n s .
D e t e r m i n e t h e e x t i n c t i o n i n c r e a s e d u e t o a d d i t i o n o f d e a m i n a s e s u s p e n s i o n (II) a l o n e b y t h e
a d d i t i o n o f a further 0 . 0 2 m l . s u s p e n s i o n II at t h e e n d o f t h e r e a c t i o n . A d d t h e e x t i n c t i o n
change to A E.
Calculations
S t a n d a r d curves are linear a n d cut the co-ordinates t h r o u g h zero. W i t h absolutely p u r e adenosine we

found an extinction difference A E of 0.303 for 10 ug. adenosine/ml. cuvette c o n t e n t s (37.4 uM) instead of
the published value o f 0 . 2 6 3 . Therefore the extinction coefficient for 265 n m is 8.1 c m . / / m i o l e . T h e calcula
2 2
tion formula (2) on p . 312 applies a n d the results are o b t a i n e d as ^ m o l e adenosine per ml. sample.
This value m u s t be multiplied by a factor if the sample has been deproteinized, neutralized or diluted in
any way. F o r this m e t h o d the c o n c e n t r a t i o n of samples which have n o t been deproteinized are calculated
as follows:
c = AE x 0.747 |>mole/ml.]
c = AE x 198 [/ig./ml.]
If a range of the p h o t o m e t e r scale is chosen so t h a t a A E of 0.010 can be d e t e r m i n e d with sufficient accuracy

as little as 2 fig. adenosine/ml. sample can be determined.
In a nucleotide m i x t u r e a m e a n of 11.02 ug. per ml. s a m p l e w a s found (2 s = 0.782). T h e coefficient ^f

variation is 3 . 5 % .
If p h o s p h a t a s e , p y r o p h o s p h a t a s e and phosphodiesterase are a d d e d successively to the assay system, all

c o m p o u n d s which contain adenosine p h o s p h a t e s are determined (e.g. A M P , A - 3 . 5 - M P , A D P , A T P , C o A ,
N A D and N A D P ) a n d the differentiation of adenosine, its p h o s p h a t e s a n d their derivatives is p o s s i b l e ' 2 4 - 6
.
Interference in the assay technique: Insufficient purity of the reagents (see p . 1919), in particular the
deaminase, results in t o o high adenosine values.
In rare cases tissue extracts m a y c o n t a i n inhibitors of adenosine deaminase. If the d e a m i n a t i o n proceeds
slowly or not at all, a d d m o r e enzyme. If this is n o t successful, check the correct functioning of the assay
system by the addition of ca. 5 pg. adenosine. T h e recovery of a d d e d adenosine within a few minutes
indicates the absence of inhibitors. Otherwise the sample m u s t be purified with a n ion exchange resin
(e. g. Amberlite I R 4 B or D o w e x 1 x 1 0 ) .
Turbidity of any type causes a high extinction at 265 n m ; it must be avoided.
The enzyme deaminates adenosine, 2'-deoxyadenosine, 3'-deoxy adenosine, xylosyladenine, 4'-thio-
adenosine, arabinosyl-adenine, 3'-amino-3',3-dideoxyadenosine, 2,6-diaminopurine riboside, 2',3'-dide-
oxyadenosine a n d other analogues of a d e n o s i n e " . 6 - C h l o r o p u r i n e ribonucleoside is hydrolysed to inosine
7 10
and chloride ions . Adenosine d e a m i n a s e does not react with riboguanosine, ribocytidine, A - 5 - M P ,
11
A - 3 - M P or A - 2 - M P , A-3 : 5 - M P , A T P or a d e n i n e 6 1 2
.
References
1 H. M. Kalckar,]. biol. C h e m . 167, 429 [1947].

3 G. Michal, unpublished.
4 T. P. Wang & N. O. Kaplan, J. biol. C h e m . 206, 299 [1954].
5 L. Shuster, N. O. Kaplan & F. E. Stolzenbach, J. biol. C h e m . 215, 195 [1955].
6 N. O. Kaplan in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c Press N e w Y o r k ,
1955, Vol. II, p . 473.
7 S. Frederiksen, Arch. Biochem. a n d Biophys. 113, 383 [1966].
8 A. Coddington, Biochim. Biophys. A c t a 99, 442 [1965].
9 K. K. Ogilrie, L. Slotin & P. Rheault, Biochem. Biophys. Res. C o m m . 45, 297 [1971].
10 R. Wolfenden, J. Kaufman & J. B. Macon, Biochemistry 8, 2412 [1969].
11 J. G. Cory & R. J. Suhadolnik, Biochemistry 4, 1733 [1965].
12 G. Schmidt in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c Press, N e w Y o r k
1957, Vol. I l l , p . 7 8 1 .
Cytidine and Deoxyeytidine
Edith Forster and August W. Holldorf
The pyrimidine nucleosides cytidine and deoxyeytidine are intermediate p r o d u c t s oi the d e g r a d a t i o n ol

ribonucleic and deoxyribonucleic acids a n d of cytosine nucleotides. They occur free in organisms in
very low concentrations. Their detection a n d determination is generally possible only after c o n c e n t r a t i o n
and partial isolation. C h r o m a t o g r a p h i c a n d s p e c t r o p h o t o m e t r i c m e t h o d s are u s e d ~ . Cytidine and deoxy
1 3
eytidine can be d e a m i n a t e d enzymatically to uridine and deoxyuridine respectively. This reaction is cataly
sed by cytidine d e a m i n a s e (Cytidine a m i n o h y d r o l a s e , E C 3.5.4.5), a n d can be used for the enzymatic
4
determination of b o t h substances.
Application of Method: In biochemistry a n d possibly in clinical chemistry.
Principle
(1) Cytidine + H 0 2
c y t i d i n e d e a m i n a s e
> Uridine + NH 3
(2) Deoxyeytidine + H Q 2
c y l i d i n e A
™ min
™ , Deoxyuridine + NH 2
(3) Deoxyuridine p h o ~ - ^ L o 4 > Uracil + D e o x y n b o s e
Since the d e a m i n a t i o n of b o t h nucleosides is catalysed by the same enzyme, they c a n n o t be determined in

the presence of each other in a mixture. However, deoxyeytidine can be d e t e r m i n e d separately by further
reaction of the resulting deoxyuridine with deoxythymidine p h o s p h o r y l a s e ( E C 2.4.2.4; refer to p. 1935).
T h e d e a m i n a t i o n of cytidine a n d deoxyeytidine is a c c o m p a n i e d by a decrease in extinction at 282 n m . O n a
m o l a r basis the decrease for b o t h substrates is the same, a n d is p r o p o r t i o n a l t o the q u a n t i t y of substrate
transformed.
Reactions ( l ) - ( 3 ) proceed practically irreversibly. T h e p H o p t i m u m for reaction (1) a n d (2) is 9.2, while t h a t
for reaction (3) is between 6.0 a n d 7.0.
Equipment
S p e c t r o p h o t o m e t e r suitable for m e a s u r e m e n t s in the U V range ( 2 8 0 - 3 0 0 n m ) ; laboratory

centrifuge; p H meter and rotary evaporator
Reagents
1. Diethanolamine 6. A m m o n i a s o l u t i o n , 1 N
2. M a g n e s i u m c h l o r i d e , M g C l - 6 H 0 ,
2 2 7. A m m o n i a s o l u t i o n , A . R . ( 2 5 % ) ,
A.R. s p . gr. 0 . 9 1 0
3. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , 8. E t h a n o l , A . R . , a p p r o x . 9 5 % ( w / w )
s p . gr. 1.67 9. A c t i v e c h a r c o a l " N o r i t e A "
4. P o t a s s i u m h y d r o x i d e s o l u t i o n , A . R., 2 N 10. C y t i d i n e d e a m i n a s e
5. H y d r o c h l o r i c a c i d , A . R., 1 N from E. coli; a p p r o x . 1.5-2.5 U / m g . (25 °C).
F o r isolation, see A p p e n d i x p . 1927.
Purity of Reagents
Cytidine deaminase should n o t contain detectable quantities of cytosine d e a m i n a s e a n d / o r thymidine

phosphorylase. This condition is generally satisfied by the enzyme p r e p a r a t i o n indicated.
Preparation of Active Charcoal
Stir 100 g. Norite A in 2000 ml. 1 N HC1 for 60 min. at 5 0 - 6 0 °C a n d then at r o o m temperature in the
same volume of 1 N a m m o n i a . Wash with water until p H is 7 . 0 - 7 . 2 . D r y the charcoal for 18 h. at 110 °C a n d
finely powder in a m o r t a r .
P r e p a r e all s o l u t i o n s w i t h d i s t i l l e d w a t e r .
I. D i e t h a n o l a m i n e buffer ( 0 . 2 M ; p H 9 . 2 ) :
D i s s o l v e 2 1 . 0 2 g. d i e t h a n o l a m i n e in a b o u t 5 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 9.2 w i t h
1 N H C 1 u s i n g a g l a s s e l e c t r o d e , a n d m a k e u p t o 100 m l . w i t h d i s t i l l e d w a t e r . C h e c k p H .
II. M a g n e s i u m c h l o r i d e (0.1 M ) :
D i s s o l v e 2 . 0 3 g. M g C l - 6 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
2 2
D i l u t e 8.7 m l . 7 0 % H C 1 0 4 t o 1 0 0 m l . w i t h distilled w a t e r .
I V . E t h a n o l - a m m o n i a - w a t e r m i x t u r e ( r e q u i r e d o n l y if n u c l e o s i d e s are t o b e c o n c e n t r a t e d
with active charcoal):
A d d 2.0 ml. cone, a m m o n i a a n d 25 ml. distilled water t o 100 ml. ethanol.
V. Cytidine deaminase ( 0 . 2 - 0 . 5 mg. protein/ml.):
U s e e n z y m e solution undiluted. Slight turbidity that occurs o n t h a w i n g can be eliminated
w i t h o u t l o s s o f a c t i v i t y b y c e n t r i f u g a t i o n for 5 m i n . at 5 0 0 0 g (0 t o + 4 ° C ) .
Solutions II to IV keep indefinitely in stoppered containers at r o o m t e m p e r a t u r e , and solution I is stable

at r o o m temperature for a few weeks. T h e enzyme solution V can be kept for at least 6 m o n t h s at — 15 °C.
Slight loss of activity occurs on repeated thawing.
Procedure
F o r h u m a n s u b j e c t s , c o l l e c t b l o o d in t h e u s u a l m a n n e r f r o m t h e c u b i t a l v e i n a n d a d d h e p a r i n
t o p r e v e n t c o a g u l a t i o n . C o l l e c t b l o o d f r o m rats o r m i c e b y c a r d i a c p u n c t u r e . C o l l e c t t i s s u e
s a m p l e s w i t h t h e " q u i c k - f r e e z e " t o n g s (see p. 4 0 0 ) .
Deproteinization :
W h o l e b l o o d : T h o r o u g h l y m i x 10 m l . o f b l o o d in a n ice b a t h w i t h 5 m l . c o l d p e r c h l o r i c a c i d s o
l u t i o n (III). C e n t r i f u g e for 10 m i n . at 5 0 0 0 g. C a r e f u l l y adjust t h e s u p e r n a t a n t t o p H 9 . 0 - 9 . 2
Cytidine a n d Deoxyeytidine 1925
w i t h K O H s o l u t i o n a n d a l l o w t o s t a n d for 3 0 m i n . in t h e ice b a t h . C e n t r i f u g e off p r e c i p i t a t e d

p o t a s s i u m p e r c h l o r a t e . B r i n g 1.0 o r 2 . 0 m l . o f t h e s u p e r n a t a n t fluid t o r o o m t e m p e r a t u r e a n d
u s e in t h e a s s a y . W h e n t h e n u c l e o s i d e c o n c e n t r a t i o n s are l o w , p r i o r c o n c e n t r a t i o n w i t h c h a r c o a l
is n e c e s s a r y (see b e l o w ) .
T i s s u e (liver, k i d n e y , o r s p l e e n ) : H o m o g e n i z e 1 p a r t o f t i s s u e w i t h 3 p a r t s o f p e r c h l o r i c a c i d
s o l u t i o n (III), a n d t h e n p r o c e e d a s in t h e c a s e o f w h o l e b l o o d .
N u c l e o s i d e m i x t u r e s f r o m e n z y m a t i c s y s t e m s (e. g. D N A o r R N A h y d r o l y s a t e s ) : If d e p r o t e i n i
z a t i o n is n e c e s s a r y , treat as for w h o l e b l o o d .
U r i n e : H i g h c o n c e n t r a t i o n s o f p u r i n e s (uric a c i d ) w i t h v e r y s t r o n g a b s o r p t i o n in t h e m e a s u r e
m e n t r a n g e p r e v e n t t h e u s e o f t h e m e t h o d for t h e d e t e r m i n a t i o n o f t h e n u c l e o s i d e s in u r i n e .
In this c a s e , t h e c y t i d i n e o r d e o x y e y t i d i n e m u s t b e s e p a r a t e d b y a d s o r p t i o n o n a c t i v e c h a r c o a l
and paper c h r o m a t o g r a p h y 1 , 7
' 8
.
Concentration with active charcoal:
A d d 0.5 m l . 1 N H C 1 a n d 2 0 0 - 2 5 0 m g . a c t i v e c h a r c o a l ( N o r i t e A ) t o 10 m l . o f a s a m p l e h a v i n g
a l o w n u c l e o s i d e c o n t e n t , a n d stir for 15 m i n . at r o o m t e m p e r a t u r e . C e n t r i f u g e for 15 m i n .
at 2 5 0 0 0 g. E l u t e t h e n u c l e o s i d e s f r o m c a r b o n s e d i m e n t w i t h 2 0 m l . o f e t h a n o l - a m m o n i a -
w a t e r m i x t u r e ( I V ) b y stirring for 15 m i n . at r o o m t e m p e r a t u r e a n d t h e n c e n t r i f u g i n g t h e car
b o n s u s p e n s i o n for 15 m i n . at 2 5 0 0 0 g. E v a p o r a t e t h e s u p e r n a t a n t fluid ( c o n t a i n i n g m o r e t h a n
9 5 % o f t h e n u c l e o s i d e s ) t o d r y n e s s in a r o t a r y e v a p o r a t o r ( b a t h t e m p e r a t u r e 4 0 ° C ) a n d d i s s o l v e
in 1.2 m l . o f H 0 . E l i m i n a t e s l i g h t t u r b i d i t y b y c e n t r i f u g a t i o n f o r 5 m i n . U s e 1.0 m l . o f t h e s u p e r
2
n a t a n t fluid i n t h e a s s a y .
Stability of samples:
T h e s a m p l e s c a n b e k e p t f o r s e v e r a l d a y s in a refrigerator at all s t a g e s o f t h e t r e a t m e n t .
Assay System
W a v e l e n g t h : 2 8 2 n m ; q u a r t z c u v e t t e s , light p a t h 1.00 c m . ; a s s a y v o l u m e : 3 . 0 0 m l . ; m e a s u r e
at r o o m t e m p e r a t u r e a g a i n s t air.
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o a d d i t i o n o f t h e e n z y m e s o l u t i o n ( V ) b y a d d i t i o n
o f a further 0 . 1 0 m l . o f e n z y m e s o l u t i o n ( V ) after r e a d i n g E . S u b t r a c t t h e r e s u l t i n g c h a n g e
2
in e x t i n c t i o n f r o m E . If d e o x y c y t i d i n e is t o b e d e t e r m i n e d s e p a r a t e l y , t a k e 2 . 0 0 m l . o f t h e a s s a y
2
m i x t u r e after r e a c t i o n h a s o c c u r r e d , a n d u s e for t h e d e t e r m i n a t i o n o f t h e d e o x y u r i d i n e f o r m e d
in a c c o r d a n c e w i t h p . 1 9 3 5 .
Sample (deproteinized, p H 9 . 0 - 9 . 2 ) 2.00 ml. 1 0 - 1 5 0 /zM c y t i d i n e

and/or deoxycytidine
D i e t h a n o l a m i n e buffer (I) 0.60 ml. 40 m M
MgCl 2 solution (II) 0.30 ml. 10 m M
M i x , r e a d e x t i n c t i o n at i n t e r v a l s o f 1 m i n . T h e e x t i n c t
ion should remain constant. Read E . t
Cytidine deaminase (V) 0.10 ml. 5-15jig./ml. = 10-30 mU/ml.
M i x , read e x t i n c t i o n after 10, 2 0 a n d 3 0 m i n . W h e n n o

further c h a n g e in e x t i n c t i o n o c c u r s , r e a d final v a l u e
E . E
2 x — E 2 = A E is u s e d in t h e c a l c u l a t i o n .
Calculations
The nucleoside content in ^ m o l e / m l . of sample is obtained with the aid of formula (2) on p . 312. This
value must be multiplied by factors connected with the treatment of the sample (deproteinization, neutral
ization, dilution, concentration). T h e extinction coefficients for either substrate at 282 n m are as f o l l o w s ' : 5 6
s = 3.62 x 10 c m . / m o l e at p H 7.4; e = 4.40 x 10 c m . / m o l e at p H 9.2; s = 5.15 x 10 c m . / m o l e at

6 2 6 2 6 2
p H 14.
U n d e r the assay conditions indicated, the cytidine (deoxycytidine) c o n c e n t r a t i o n in the sample is c =
AE x 0.341 [^mole/ml.].
On average, 100 g. of rat blood contains 1.27 + 0.16 mg. of deoxycytidine. The coefficient of variation
is 12.5%.
N o r m a l Values
The average concentrations per 100 g. of tissue in r a t s 1 - 3

' 6
a r e : liver 1.25 mg. of deoxycytidine; spleen
8.8 mg. of deoxycytidine. H u m a n whole b l o o d contains 2 - 3 mg. of deoxycytidine per 100 g. . Deoxy 6
cytidine occurs in concentrations of 1-2 mg. per 100 g. of tissue in various m o u s e tissues . After treatment 1
with X rays, the concentration of deoxycytidine in the whole blood increases by a factor of two to t h r e e 6
and the excretion of deoxycytidine in the urine increases by a factor of a b o u t five . Cytidine has not yet 8
been detected in any tissue without first concentrating the extract.

Cytidine a n d Deoxyeytidine 1927
Interference in the assay technique: H i g h a b s o r p t i o n of the samples at the wavelength used, d u e mainly to
purine nucleotides, nucleosides, o r bases, m a y m a k e the m e a s u r e m e n t impossible. This difficulty can be
overcome in the case of samples with high cytidine o r deoxyeytidine c o n t e n t s . If this is insufficient, p r i o r
concentration of the nucleosides by t r e a t m e n t with active charcoal a n d s e p a r a t i o n by p a p e r c h r o m a t o g r a
phy is necessary.
Specificity
In addition to cytidine a n d deoxyeytidine, the u n u s u a l 5-methyldeoxycytidine a n d synthetically p r e p a r e d

halogenated cytidine a n d deoxyeytidine derivatives (5-Cl-deoxycytidine, 5-I-deoxycytidine, 5-Br-deoxy-
cytidine, etc.) also react with cytidine d e a m i n a s e . 5,6
Appendix
Cytidine Deaminase
Isolation of cytidine deaminase from E. coli . 5,6

Cells of E. coli are grown on a glycerol-salt m e d i u m with
1 m M cytidine as inducer. T h e enzyme can be c o n c e n t r a t e d from these cells by a factor of a b o u t 50 in
a yield of a p p r o x . 2 5 % by a p r o c e d u r e t h a t takes 3 - 4 days a n d involves precipitation of nucleic acids with
M n C l , precipitation of inactive proteins at p H 4, fractionation with a m m o n i u m sulphate a n d c h r o m a t o
2
graphy on DEAE-cellulose. This p r e p a r a t i o n is satisfactory for the analyses described a b o v e .
References
1 W. C Schneider, J. biol. C h e m . 216, 287 [1955].

2 W. C Schneider & L. W. Brownwell, J. N a t l . Cancer Inst. 18, 579 [1957].
3 W. C Schneider, J. N a t l . C a n c e r Inst. 18, 569 [1957].
4 S. S. Cohen & H. D. Burner, J. biol. C h e m . 226, 631 [1957].
5 E. Forster, Dissertation, F r e i b u r g 1966.
6 E. Forster & A. W. Holldorf, E u r o p e a n J. Biochem. [1973], in press.
7 J. Rotherham & W. C Schneider, Biochim. Biophys. Acta 41, 344 [I960].
8 T. Arient, Z . Dienstbier & J. Skoda, N a t u r e 182, 721 [1958].
Guanosine
Alan Coddington
T h e m e t h o d described here was developed by Kalckar . 1

In the presence of inorganic p h o s p h a t e , guanosine
is converted to guanine a n d ribose-1-phosphate by nucleoside p h o s p h o r y l a s e (Purine nucleoside: o r t h o -
p h o s p h a t e ribosyltransferase, E C 2.4.2.1). G u a n a s e ( G u a n i n e a m i n o h y d r o l a s e , E C 3.5.4.3) converts guanine
to xanthine, and xanthine oxidase ( X a n t h i n e : oxygen oxidoreductase, E C 1.2.3.2) converts the latter to
uric acid. The extinction at 293 n m increases. After addition of uricase ( U r a t e : oxygen oxidoreductase,
E C 1.7.3.3) uric acid is converted to allantoin; the extinction at 293 n m decreases accordingly.
Principle
(1) Guanosine + Phosphate t

n u c l e o s i d e
» Guanine 4- Ribose-1-phosphate
phosphorylase
I
(2) Guanine + H 0 — 2
8 u a n a s e
> Xanthine + N H 3
I
(3) Xanthine + 0 2 + H 0 2 ™.^ n
e
c
> Uric acid + H 0 2 2
I
(4) Uric acid + 0 2 + H 0 — — • A l l a n t o i n + C0
2 2 + H 0
2 2
T h e decrease of the uric acid concentration, as measured by the decrease in extinction at 293 n m , is a mea
sure of the a m o u n t of guanosine in the assay system.
In tris buffer with 5 m M p h o s p h a t e a n d p H 7.9 the equilibrium of reaction (1) is completely to the right.
Equipment
S p e c t r o p h o t o m e t e r suitable for m e a s u r e m e n t s at 293 n m .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , t r i s , 6. X a n t h i n e o x i d a s e , X O D ,
A. R. from milk, suspension in 3.2 M ammonium
2. H y d r o c h l o r i c a c i d , 0.2 N , A . R . sulphate s o l u t i o n ; ^ 0.3 U / m g . (25 °C); com
3. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , mercial p r e p a r a t i o n , see p . 521.
KH P0 2 4 7. N u c l e o s i d e p h o s p h o r y l a s e
4. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA from calf spleen, suspension in 3.2 M a m m o n i u m
disodium salt, E D T A - N a H • 2 H 0 , A . R .
2 2 2
sulphate s o l u t i o n ; ^ 25 U / m g . (25 °C); com
5. Guanosine. mercial p r e p a r a t i o n , see p . 490.
Commercial p r e p a r a t i o n , see p . 542.
Guanosine 1929
8. Guanase 9. Uricase
from rabbit liver, suspension in 3.2 M am from hog liver, solution in 5 0 % glycerol, p H 10;
m o n i u m sulphate s o l u t i o n ; ^ 60 m U / m g . ^ 4 . 5 U / m g . (25 ° C ) ; commercial p r e p a r a t i o n ,
(25 ° C ) ; commercial p r e p a r a t i o n , see p . 4 7 1 . see p . 518.
Purity of Reagents
T h e enzymes should be completely free from alkaline p h o s p h a t a s e a n d a d e n o s i n e d e a m i n a s e .
U s e fresh, d o u b l y d i s t i l l e d w a t e r .
I. Tris buffer ( 5 0 m M ; p H 7 . 9 ; 5 m M p h o s p h a t e ; 1 m M E D T A ) :
D i s s o l v e 1.21 g. tris, 7 4 m g . E D T A - N a H • 2 H 0 a n d 136 m g . K H P 0
2 2 2 2 4 in 1 5 0 m l . d i s t i l l e d
w a t e r , a d j u s t t o p H 7.9 w i t h ca. 3 0 m l . 0.2 N H C 1 a n d d i l u t e t o 2 0 0 m l . w i t h d i s t i l l e d w a t e r .
II. G u a n o s i n e s t a n d a r d s o l u t i o n (5 m M ) :
D i s s o l v e 2 8 . 3 m g . g u a n o s i n e in d i s t i l l e d w a t e r w i t h w a r m i n g a n d m a k e u p t o 2 0 m l .
III. N u c l e o s i d e p h o s p h o r y l a s e ( 2 . 5 U / m l . ) :
D i l u t e t h e s t o c k s u s p e n s i o n w i t h 2.5 M a m m o n i u m s u l p h a t e s o l u t i o n a c c o r d i n g l y .
IV. X a n t h i n e oxidase, X O D (2.5 U / m l . ) :
D i l u t e t h e s t o c k s u s p e n s i o n w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n ( p H 8; 10 m M
EDTA).
V. G u a n a s e (0.5 U / m l . ) :
VI. Uricase (2.5 U / m l . ) :
Dilute the stock suspension with 50% glycerol ( p H 10); dissolve freeze-dried preparations
in d i s t i l l e d w a t e r .
Store solution I at 0 - 4 °C a n d m a k e u p freshly every m o n t h . Store the enzyme stock solutions in a deep
freeze.
Procedure
T h e m e t h o d h a s s o far o n l y b e e n u s e d for t h e d e t e r m i n a t i o n o f g u a n o s i n e in p u r e a q u e o u s
solutions.
Assay System
W a v e l e n g t h : 2 9 3 n m ; q u a r t z c u v e t t e s , light p a t h : 1 c m . ; final v o l u m e : 3 . 0 0 m l . ; r o o m t e m
p e r a t u r e ; read a g a i n s t r e f e r e n c e c u v e t t e c o n t a i n i n g buffer ( s o l u t i o n I) i n s t e a d o f s a m p l e .
assay mixture
Buffer (I) 2.58 ml. 4 3 m M tris, 0 . 8 6 m M

E D T A , 0.43 m M phosphate
S a m p l e or standard solution (II) 0.02 ml. u p t o 33 juM g u a n o s i n e
X O D suspension (III) 0.05 m l . ca. 4 0 m U / m l .
Nucleoside phosphorylase
suspension (IV) 0.05 m l . ca. 4 0 m U / m l .
Guanase suspension (V) 0.25 m l . ca. 4 0 m U / m l .
M i x b y g e n t l e a e r a t i o n , f o l l o w e x t i n c t i o n until c o n s t a n t
a n d read E.1
Uricase solution (VI) 0.05 m l . ca. 4 0 m U / m l .
M i x w i t h a s t r e a m o f air, f o l l o w e x t i n c t i o n until
constant read E . 2 E x — E 2 = A E is u s e d for the
calculations.
D e t e r m i n e t h e e x t i n c t i o n c h a n g e d u e t o a d d i t i o n o f t h e u r i c a s e s o l u t i o n b y a further a d d i t i o n
o f 0.05 ml. s o l u t i o n V I at t h e e n d o f t h e r e a c t i o n ; a d d the i n c r e a s e in e x t i n c t i o n t o E.
x
Calculations
U n d e r the above conditions the reaction is stoichiometric. T h e extinction coefficient for uric acid is
12.1 cm. //imole at 293 n m . T h e guanosine concentration of the sample is therefore obtained as follows:
2
— x
^— = AE x 12.4 [wmole/ml.]
x
c =
12.1 0.02
This depends on the accuracy of the pipetting a n d the p r e p a r a t i o n of the solutions. N o values are available
for the s t a n d a r d deviation or coefficient of variation.
T h e main source of error is impurities in the enzymes used. T h e commercially available enzymes are of the
required purity.
Guanosine 1931
Nucleoside phosphorylase is n o t specific for g u a n o s i n e ; it also hydrolyses inosine, adenosine a n d other

derivatives, e. g. the c o r r e s p o n d i n g deoxy derivatives. X a n t h i n e oxidase oxidizes x a n t h i n e , h y p o x a n t h i n e
a n d purine. In contrast, g u a n a s e a n d uricase are very specific for g u a n i n e a n d uric acid respectively.
G u a n o s i n e can be determined in the presence of inosine by a difference m e t h o d . T h e assay is also possible
in the presence of guanylic acid, providing the system does not contain alkaline p h o s p h a t a s e .
References

Inosine
Alan Coddington
The m e t h o d described here was developed by Kalckar . 1

In the presence of inorganic p h o s p h a t e and nucleo
side phosphorylase (Purine nucleoside: o r t h o p h o s p h a t e ribosyltransferase, E C 2.4.2.1) inosine is converted
to hypoxanthine a n d ribose-1-phosphate. H y p o x a n t h i n e is oxidized by xanthine oxidase ( X a n t h i n e : oxygen
oxidoreductase, E C 1.2.3.2) t o uric acid.
Principle
(1) Inosine + P h o s p h a t e ^ n u c l e o s i d e
> Hypoxanthine + Ribose-1-phosphate
phosphorylase .
* Z
(2) Hypoxanthine + 2 H 0 + 2 0 2 2
x
™£™ > Uric acid + 2H 0 2 2
The increase in the uric acid concentration, as measured by the increase in the extinction at 293 n m ,
is p r o p o r t i o n a l to the a m o u n t of inosine present.
With excess of inorganic p h o s p h a t e and p H 7.4 reaction (1) proceeds quantitatively from left to right.
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e f o r m e a s u r e m e n t s at 2 9 3 n m .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 5. X a n t h i n e o x i d a s e , X O D
KH P0 2 4
from milk, suspension in 3.2 M a m m o n i u m sul
2. D i s o d i u m h y d r o g e n p h o s p h a t e , p h a t e s o l u t i o n ; ^ 0.3 U / m g . (25 ° C ) ; commercial
p r e p a r a t i o n , see p . 521.
Na HP0
2 4 -7H 0 2
3. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA 6. N u c l e o s i d e p h o s p h o r y l a s e
disodium salt, E D T A - N a H -2 H 0 2 2 2
from calf spleen, suspension in 3.2 M a m m o n i u m
4. Inosine sulphate solution; ^ 2 5 U / m g . (25 °C); com

mercial p r e p a r a t i o n , see p. 490.
Purity of Reagents
The enzymes must be completely free from alkaline p h o s p h a t a s e , adenosine deaminase, guanase, adenine
deaminase a n d uricase.
Inosine 1933
U s e o n l y fresh d e s t i l l e d w a t e r .
I. P h o s p h a t e buffer ( 5 0 m M ; p H 7 . 4 ; 1 m M E D T A ) :
a) D i s s o l v e 2 7 . 2 g. K H P 0 2 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
b) D i s s o l v e 5 3 . 6 g. N a H P 0
2 4 • 7 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2
c) D i s s o l v e 0 . 3 7 2 g. E D T A - N a H 2 2 • 2 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
2
M i x 1 9 . 0 m l . s o l u t i o n a ) , 8 1 . 0 m l . s o l u t i o n b) a n d 4 0 m l . s o l u t i o n c) a n d d i l u t e t o 4 0 0 m l .
II. I n o s i n e s t a n d a r d s o l u t i o n (5 m M ) :
D i s s o l v e 2 6 . 8 m g . i n o s i n e in d i s t i l l e d w a t e r a n d m a k e u p t o 2 0 m l .
III. N u c l e o s i d e p h o s p h o r y l a s e ( 2 . 5 U / m l . ) :
IV. X a n t h i n e oxidase, X O D (2.5 U / m l . ) :
D i l u t e t h e s t o c k s u s p e n s i o n a c c o r d i n g l y w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n ( p H 8 ;
10 m M E D T A ) .
Store solutions I a n d II at 0 - 4 °C, a n d p r e p a r e freshly each m o n t h . Store the enzyme stock solutions
deep-frozen.
Procedure
T h i s m e t h o d h a s s o far o n l y b e e n u s e d for t h e d e t e r m i n a t i o n o f i n o s i n e in a q u e o u s s o l u t i o n s .
Assay System
W a v e l e n g t h : 2 9 3 n m ; light p a t h : 1 c m . , q u a r t z c u v e t t e s ; final v o l u m e : 3 . 0 m l . ; r o o m t e m p e r a
t u r e ; r e a d a g a i n s t a r e f e r e n c e c u v e t t e c o n t a i n i n g buffer s o l u t i o n (I) i n s t e a d o f s a m p l e .
Buffer s o l u t i o n (I) 2.78 ml. ca. 50 m M p h o s p h a t e ;

1 mM EDTA
Sample or standard solution (II) 0.02 ml. u p t o 33 ^ M i n o s i n e
X O D suspension (IV) 0.05 ml. ca. 40 m U / m l .
M i x w i t h a s t r e a m o f air. F o l l o w e x t i n c t i o n until
constant and then read extinction E . t
Nucleoside phosphorylase
suspension (III) 0.05 ml. ca. 40 m U / m l .
M i x w i t h a s t r e a m o f air. F o l l o w e x t i n c t i o n until
constant and then read E . E 2 2 — E x = A E is u s e d
for the calculations.
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n o n a d d i t i o n o f n u c l e o s i d e p h o s p h o r y l a s e suspension
a l o n e b y t h e further a d d i t i o n o f 0 . 0 5 m l . s u s p e n s i o n III at t h e e n d o f t h e r e a c t i o n . S u b t r a c t t h e
c h a n g e in e x t i n c t i o n f r o m E .2
Calculations
U n d e r the above conditions the reaction proceeds stoichiometrically. T h e extinction coefficient for uric
acid under these conditions is 12 c m . / ^ m o l e at 293 nm. Therefore the inosine c o n c e n t r a t i o n of the sample
2
is calculated as follows:
AE x 3
c = = zlE x 12.5 [umole/ml.l
12 x 0.02
The reproducibility depends on the accuracy of the pipetting and the p r e p a r a t i o n of the solutions. N o
information is available on the s t a n d a r d deviation and coefficient of variation to be expected.
The main source of error is c o n t a m i n a t i o n of the enzymes used. However, the commercial preparations
at present available fulfil the requirements.
Nucleoside phosphorylase is not specific for inosine; it also hydrolyses adenosine, guanosine a n d their
derivatives. Similarly, xanthine oxidase oxidizes purines, xanthine a n d h y p o x a n t h i n e . However, it is
possible to determine inosine in the presence of adenosine and guanosine, provided the enzymes used are
free from adenine deaminase, adenosine deaminase and guanase. In the same way inosine can be determined
in the presence of inosinic acid if the assay system does n o t contain any alkaline p h o s p h a t a s e .
References

Deoxythymidine and Deoxyuridine
Ursula Friebe and August W. Holldorf
D e o x y t h y m i d i n e is an intermediate in t h e d e g r a d a t i o n of t h y m i n e deoxynucleotides a n d of deoxyribonucleic

acid ( D N A ) . In m o s t o r g a n i s m s d e o x y t h y m i d i n e is of m i n o r i m p o r t a n c e for the biosynthesis of t h y m
ine deoxynucleotides a n d therefore for D N A synthesis. D e o x y u r i d i n e is formed as an intermediate in the de
gradation of deoxyuridine p h o s p h a t e s a n d by d e a m i n a t i o n of d e o x y c y t i d i n e . B o t h nucleosides occur only
1
in very low c o n c e n t r a t i o n in biological m a t e r i a l ' . 2 3
D e o x y t h y m i d i n e is formed in large a m o u n t s in the chemical or enzymatic hydrolysis of D N A . T h e two

nucleosides are usually characterized a n d determined by c h r o m a t o g r a p h y a n d by their U V a b s o r p t i o n
spectra . Deoxythymidine phosphorylase '
4 5 6
( T h y m i d i n e : o r t h o p h o s p h a t e deoxyribosyltransferase, EC
2.4.2.4) catalyses a p h o s p h o r o l y t i c cleavage of b o t h nucleosides t o give d e o x y r i b o s e - 1 - p h o s p h a t e a n d the
c o r r e s p o n d i n g free base.
Application of Method: In biochemistry a n d possibly in clinical chemistry.
Principle
(1) Deoxythymidine + H A s Q J ^ ^ ^
3 4
1
Thymine + Deoxyribose-1-arsenate
(2) Deoxyuridine + H A s 0 3 4 / e o x y t h y m i d i n e
v Uracil + Deoxyribose-1-arsenate
v 7
phosphorylase
T h e s p o n t a n e o u s hydrolysis of deoxyribose-1-arsenate proceeds at a rapid rate a n d therefore the whole

process is irreversible. T h e progress of the reaction in alkaline conditions can be m e a s u r e d by the increase
in extinction at 300 or 290 n m . A t p H 14 e 3 0 0 n m for the conversion of d e o x y t h y m i d i n e is 3.61 x 1 0 c m . /6 2
mole a n d e 290 n m for the conversion of deoxyuridine is 3.60 x 1 0 c m . / m o l e . 6 2 6
A second possibility is t o determine the deoxyribose with the t h i o b a r b i t u r i c acid reaction of Saslaw a n d
Waravdekar . 1
This reaction is five times m o r e sensitive t h a n the s p e c t r o p h o t o m e t r i c assay, but it is less
specific (see "Specificity of M e t h o d " ) .
D e o x y t h y m i d i n e p h o s p h o r y l a s e has a wide p H o p t i m u m between p H 5.8 a n d 6.8. T h e enzyme is sensitive

to heavy metals a n d therefore all assays should be carried o u t in the presence of 2 - 5 m M m e r c a p t o e t h a n o l .
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e f o r m e a s u r e m e n t s in t h e U V r a n g e ( 2 8 0 - 3 0 0 n m ) ; p H m e t e r a n d
laboratory centrifuge.
Reagents
1. 2-Mercaptoethanol 4. S o d i u m h y d r o x i d e , A . R . , 1 N
2. S u c c i n i c a c i d , A . R . 5. P o t a s s i u m h y d r o x i d e , A . R . , 2 N
3. D i s o d i u m h y d r o g e n a r s e n a t e , 6. P o t a s s i u m h y d r o x i d e , A . R .
Na HAs0 -7H 0,
2 4 2 A.R. 7. H y d r o c h l o r i c a c i d , A . R . , 1 N
1936 Metabolites: Nucleic Acids, Purines, Pyrimidines, Nucleosides,Coenzymes
8. S u l p h u r i c a c i d , A . R . , 1 N 12. 2 - T h i o b a r b i t u r i c a c i d , 1.5 H 0
2
9. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) ; sp. 13. D e o x y t h y m i d i n e p h o s p h o r y l a s e
gr. 1.67. from E. coli.; 1 - 2 mg. p r o t e i n / m l . ; ^ 50 U / m g .
10. S o d i u m m e t a - p e r i o d a t e , N a I 0 4
(25 °C). F o r isolation, see A p p e n d i x p . 1939.
11. S o d i u m m e t a - a r s e n i t e , N a A s 0 , A . R .
2
Purity of Reagents
Deoxythymidine phosphorylase must not contain any detectable purine nucleoside phos
p h o r y l a s e , uridine p h o s p h o r y l a s e
8 9
or cytidine d e a m i n a s e activity.
P r e p a r e all s o l u t i o n w i t h distilled or d e i o n i z e d w a t e r .
I. S u c c i n a t e - a r s e n a t e buffer (0.1 M a r s e n a t e ; 0.1 M s u c c i n a t e ; p H 6 . 0 ) :

A d d 1.18 g. s u c c i n i c a c i d a n d 3 . 1 2 g. N a H A s 0 - 7 H 0 t o c a . 50 m l . d i s t i l l e d w a t e r a n d
2 4 2
a d j u s t t o p H 6.0 w i t h 1 N N a O H ( g l a s s e l e c t r o d e ) ; d i l u t e w i t h d i s t i l l e d w a t e r t o 100 m l .
and check p H with glass electrode.
II. M e r c a p t o e t h a n o l (0.1 M ) :
D i l u t e 0 . 7 0 m l . m e r c a p t o e t h a n o l w i t h distilled w a t e r t o 1 0 0 m l .
D i l u t e 8.3 m l . 7 0 % p e r c h l o r i c a c i d w i t h distilled w a t e r t o 100 m l .
IV. D e o x y t h y m i d i n e p h o s p h o r y l a s e ( 1 - 2 m g . protein/ml.):
U s e the e n z y m e solution undiluted.
V. S o d i u m a r s e n i t e ( 2 % in 0.5 N H C 1 ) :
D i s s o l v e 2.0 g. s o d i u m a r s e n i t e in 50 m l . 1 N H C 1 a n d d i l u t e w i t h distilled w a t e r t o 100 m l .
V I . P e r i o d i c a c i d ( 0 . 0 2 5 N in 0 . 1 2 5 N H S0 ):
2 4
A d d 532 mg. N a I 0 4 t o 12.5 m l . 1 N H S 0 2 4 a n d d i l u t e w i t h distilled w a t e r t o 100 m l .

VII. Thiobarbituric acid ( 0 . 6 % ; p H 2 ) :
D i s s o l v e 7 1 0 m g . t h i o b a r b i t u r i c a c i d in 9 0 m l . distilled w a t e r w i t h stirring a n d w a r m i n g
t o 75 ° C . A d d 0 . 7 0 m l . 1 N N a O H a n d d i l u t e w i t h distilled w a t e r t o 100 m l . R e m o v e a n y
turbidity by filtration.
VIII. Potassium hydroxide (about 7 N ) :
C a r e f u l l y d i s s o l v e 39 g. K O H in 7 0 m l . d i s t i l l e d w a t e r . A f t e r c o o l i n g , d i l u t e t o 100 m l .
w i t h distilled w a t e r .
Solutions I and II are stable for several weeks in a refrigerator, solutions VI and VII at r o o m temperature.
T h e enzyme solution (IV) is stable for several m o n t h s in a refrigerator at 0 - 4 °C.
D e o x y t h y m i d i n e a n d Deoxyuridine 1937
Procedure
A s s a y s h a v e s o far o n l y b e e n carried o u t o n a q u e o u s s o l u t i o n s o f n u c l e o s i d e s , e n z y m a t i c h y d r o -
l y s a t e s o f D N A a n d liver e x t r a c t s .
R e m o v e liver o f e x p e r i m e n t a l a n i m a l s w i t h f r e e z e - c l a m p s o r a d d v e r y q u i c k l y t o i c e - c o l d
perchloric acid (see "Cell a n d Tissue Disintegration", p. 400).
Deproteinization :
L i v e r : P r e p a r e a n h o m o g e n a t e o f t h e t i s s u e w i t h 3 v o l u m e s 1 N p e r c h l o r i c a c i d (III). C e n t r i f u g e
for 10 m i n . at 1 0 0 0 0 x g. C a r e f u l l y d e c a n t t h e s u p e r n a t a n t fluid a n d a d j u s t t o p H 8 . 5 - 9 . 0
w i t h 2 N K O H . L e a v e t h e s a m p l e in a n i c e b a t h f o r 3 0 m i n . C e n t r i f u g e o r filter off t h e p r e c i p i t a t e
o f p o t a s s i u m p e r c h l o r a t e ( 1 0 m i n . at 5 0 0 0 x g ) a n d a d j u s t s u p e r n a t a n t fluid o r filtrate t o p H
6 - 7 w i t h 1 N H C 1 . N o t e t h e v o l u m e s at all s t a g e s o f t h e o p e r a t i o n s a n d u s e t h e s e f o r t h e c a l c u l
ations.
DNA hydrolysate: A d d 1 v o l u m e 1 N p e r c h l o r i c a c i d (III) t o 3 v o l u m e s h y d r o l y s a t e . C e n t r i f u g e

for 10 m i n . at 1 0 0 0 0 x g. Treat s u p e r n a t a n t fluid a s d e s c r i b e d a b o v e f o r t h e d e p r o t e i n i z a t i o n
o f liver.
Very d i l u t e s o l u t i o n s o f d e o x y t h y m i d i n e o r d e o x y u r i d i n e c a n b e c o n c e n t r a t e d b y a d s o r p t i o n
o n a c t i v a t e d c h a r c o a l . T h e p r o c e d u r e is a s f o r t h e p u r i f i c a t i o n o f c y t i d i n e a n d d e o x y c y t i d i n e
(see p . 1 9 2 5 ) .
S a m p l e s a r e s t a b l e f o r s e v e r a l d a y s in a refrigerator at 0 - 4 ° C .
Assay System
1. Spectrophotometric measurements
W a v e l e n g t h : 3 0 0 n m f o r d e o x y t h y m i d i n e , 2 9 0 n m f o r d e o x y u r i d i n e ; silica c u v e t t e s ; light p a t h :
1.00 c m . ; i n c u b a t i o n v o l u m e 2 . 4 0 m l . ; final v o l u m e : 3 . 0 0 m l . ; i n c u b a t i o n at 37 ° C . R e a d
against a control cuvette.
1938 M e t a b o l i t e s : Nucleic Acids, Purines, Pyrimidines, N u c l e o s i d e s , C o e n z y m e s
Pipette into cuvettes: Test Control
assay mixture
Sample (pH 6 - 7 ) 1.00 m l . 1.00 m l . 5 - 1 5 0 pM nucleoside

Buffer (I) 1.20 m l . 1.20 m l . 50 m M e a c h o f a r s e n a t e
and succinate
Mercaptoethanol (II) 0.10 ml. 0.10 ml. 4.2 m M
N a O H (1 N ) — 0.60 ml.
Enzyme solution (IV) 0.10 ml. 0.10 ml. ca. 60 / i g . / m l .
I n c u b a t e f o r 3 0 m i n . at 37 ° C .
N a O H (1 N ) 0.60 ml. — 0.2 N
M i x and read extinction against control cuvette.
If t h e e x t i n c t i o n is t o o h i g h , c o n t e n t s o f test a n d c o n t r o l c u v e t t e s c a n b e d i l u t e d t o t h e s a m e
extent with 1 N N a O H .
2. Colorimetric measurements
W a v e l e n g t h : 5 4 3 n m ; l i g h t p a t h : 1 c m . ; i n c u b a t i o n v o l u m e : 3.1 m l . a n d 4 . 0 m l . ; final v o l u m e :
3.1 m l . ; i n c u b a t i o n at 37 ° C . R e a d a g a i n s t a c o n t r o l .
P i p e t t e i n t o test t u b e s : Test Control
assay mixture
Sample (pH 6 - 7 ) 2.00 ml. 2.00 ml. 1 - 5 0 / / M nucleoside

Buffer (I) 0.90 ml. 0.90 ml. 29 m M each of arsenate
and succinate
Mercaptoethanol (II) 0.10 ml. 0.10 ml. ca. 3 m M
Perchloric acid (III) 0.40 ml.
Enzyme solution (IV) 0.10 ml. 0.10 ml. ca. 50 jUg./ml.
I n c u b a t e for 3 0 m i n . at 3 7 ° C .
Perchloric acid (III) 0.40 ml. 114 m M

Periodic acid solution (VI) 0.50 ml. 0.50 ml. c a . 3.1 m N
I n c u b a t e f o r 2 0 m i n . at 37 ° C .
Arsenite solution (V) 1.00 m l . 1.00 m l . 0.4%
M i x and a l l o w to stand for 2 - 3 m i n .

P i p e t t e i n t o fresh test t u b e s :
Incubation mixture 1.00 m l . 1.00 m l .

Thiobarbiturate solution (VII) 2.00 ml. 2.00 ml. ca. 0 . 3 9 %
M i x t h o r o u g h l y a n d stopper with glass marbles. H e a t for 20 m i n .

in a b o i l i n g w a t e r b a t h a n d i m m e d i a t e l y p i p e t t e :
7 N K O H (soln. VIII) 0.10 ml. 0.10 ml. ca. 0.23 N
M i x and allow to cool. Read extinction against control.

Deoxythymidine and Deoxyuridine 1939
Calculations
1. Spectrophotometric method: U n d e r the conditions described above the reaction proceeds stoichiometric-
ally a n d therefore the calculation formula ( 2 ) o n p . 312 a n d the extinction coefficients given on p . 1935
are used. T h e results are o b t a i n e d in //mole d e o x y t h y m i d i n e or deoxyuridine/ml. sample. This value m u s t
be multiplied by the a p p r o p r i a t e dilution factors.
Wavelength: 300 nm 290 nm

Deoxythymidine c = AE x 0.831 — [//mole/ml.]
Deoxyuridine c = — AE x 0.833 [jumole/ml.]
2. Colorimetric method: T h e extinction coefficient for the pink dye is e 5 4 3 n m = 153 x 1 0 6

cm. /mole.
2
Taking into a c c o u n t the dilution the following relationship applies u n d e r the above c o n d i t i o n s :
D e o x y t h y m i d i n e (deoxyuridine) c = AE x 0.051 [pmole/ml.]
This value m u s t be multiplied by the dilution factors arising from the p r e p a r a t i o n of the sample.
F o r rat liver a m e a n c o n c e n t r a t i o n of 4.5 + 0.9 /miole/100 g. fresh wt. was found with the s p e c t r o p h o t o
m e t r i c ; the coefficient of variation is 2 0 % .
N o r m a l Values
In rat liver the c o n c e n t r a t i o n of d e o x y t h y m i d i n e a n d deoxyuridine was found to be 4.5 + 0.9 jUmole/100 g.

fresh wt. W i t h o t h e r m e t h o d s the d e o x y t h y m i d i n e c o n t e n t of cells of E. coli was found t o be 10 - 1 2 nmole/g.
wet w t . .
3
T h e only physiological substrates for d e o x y t h y m i d i n e p h o s p h o r y l a s e are deoxythymidine a n d deoxyurid

ine. Separate d e t e r m i n a t i o n of these t w o nucleosides is therefore n o t possible w i t h o u t previous c h r o m a t o
graphic separation. T h e enzyme can also react with the anti-metabolites, 5-fluoro-, 5-chloro, 5-bromo-
a n d 5-iododeoxyuridine. Deoxycytidine does n o t react. However, it can be d e t e r m i n e d as deoxyuridine after
d e a m i n a t i o n (see p . 1923). If the t h i o b a r b i t u r i c acid reaction is used to determine the free deoxyribose
formed, it m u s t be n o t e d that the p u r i n e deoxyribonucleosides (deoxyadenosine, deoxyguanosine, etc.)
react in the same way as d e o x y r i b o s e . Consequently this m e t h o d is n o t applicable in the presence of purine
7
deoxyribonucleosides.
Appendix
Deoxythymidine Phosphorylase
D e o x y t h y m i d i n e p h o s p h o r y l a s e can be purified a b o u t 100-fold a n d in a 5 0 % yield from cells of E. coli by

precipitation of inactive protein at the isoelectric point, precipitation of nucleic acids with p r o t a m i n e
sulphate a n d c h r o m a t o g r a p h y on D o w e x l 1 0
. It is r e c o m m e n d e d t h a t cells already c o n t a i n i n g a high
activity of the enzyme or cells in which the enzyme can be induced by deoxythymidine d u r i n g growth are
used. N u m e r o u s thymine-deficient m u t a n t s of E. coli, e.g. A T C C 9 7 2 3 h have high initial activity of the
enzyme.
1940 M e t a b o l i t e s : Nucleic Acids, Purines, Pyrimidines, Nucleosides,Coenzymes
References
1 E. Harbers: Die Nucleinsauren, G e o r g T h i e m e Verlag, Stuttgart 1964.

2 W. C. Schneider, J. biol. C h e m . 216, 287 [1955].
3 A. W. Holldorf, unpublished experiments.
4 G. H. Beaven, E. R. Holiday & E. A. Johnson in E. Chargaff & J. N. Davidson: T h e Nucleic Acids,
vol. 1, p. 4 9 3 ; A c a d e m i c Press, N e w Y o r k , 1955.
5 M. Friedkin & D. Roberts, J. biol. C h e m . 207, 245 & 257 [1954].
6 W. E. Razzell & H. G. Khorana, Biochim. Biophys. A c t a 28, 562 [1958].
7 L. D. Saslaw & V. S. Waravdekar in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, vol. 12a,
p . 108, A c a d e m i c Press, N e w Y o r k 1967.
8 L. A. Manson & /. O. Lampen, J. biol. C h e m . 193, 539 [1951].
9 L. M. Paege & F. Schlenk, Arch. Biochem. Biophys. 40, 42 [1952].
10 W.E. Razzell in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, vol. 12a, p . 118, Academic
Press N e w Y o r k 1967.
Hypoxanthine and Xanthine
UV-Assay
Soren Jorgensen
T h e m e t h o d described here was developed by Plesner a n d Kalckar 1

a n d a d a p t e d by Petersen, Jerni a n d
Jergensen for m e a s u r e m e n t s on biological material.
2
Principle
(1) Hypoxanthine + H 0 + 0 2 2 Xanthine + H 0
2 2
(2) Xanthine + H 0 + 02 2 Uric acid + H 0

2 2
T h e sum of h y p o x a n t h i n e + xanthine is measured by the increase in extinction at 293 n m d u e to the

formation of uric acid (Fig. 1). If the sample contains m o r e t h a n d o u b l e the a m o u n t of uric acid as of
h y p o x a n t h i n e a n d xanthine, the uric acid m u s t be removed with uricase before the assay (see p. 1951),
followed by destruction of the uricase by addition of alkali to give p H l l . 3
0.250
Uric acid
0.200
0.150
0.100
0.050
Fig. 1. Spectra of h y p o x a n t h i n e , xanthine a n d uric acid in

60 m M glycylglycine buffer, p H 8.2. C o n c e n t r a t i o n : 20 pM.
M e a s u r e m e n t s against buffer. 240 250 260 270 280 290 300 nm
H y p o x a n t h i n e can be determined in the presence of xanthine if the extinction changes are measured at
t w o wavelengths: increase of extinction at 280 n m (due to the conversion of h y p o x a n t h i n e to uric acid)
a n d increase of extinction at 293 n m (due t o the conversion of h y p o x a n t h i n e + xanthine to uric acid).
See Fig. 1.
* X a n t h i n e : o x y g e n oxidoreductase, E C 1.2.3.2
M e a s u r e m e n t s at wavelengths lower t h a n 2 7 0 - 2 8 0 n m are often impossible, because biological material

absorbs t o o strongly in this range.
T h e enzymatic reactions (1) a n d (2) proceed best at p H 8.2; the assay is therefore carried out between
p H 8.0 a n d 8.5. Sufficient X O D should be used so t h a t the reaction is complete in less t h a n 20 min.
Equipment
Spectrophotometer s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at 2 8 0 a n d 2 9 3 n m ; laboratory

centrifuge.
Reagents
1. G l y c i n e 6. X a n t h i n e o x i d a s e , X O D
2. Glycylglycine isolated according t o , ^ 1 1 U / m g . (25 °C) or
4
3. S o d i u m h y d r o x i d e , A . R . , 1.6 N commercial p r e p a r a t i o n , see p . 521, e.g. as a

4. H y d r o c h l o r i c a c i d , A . R., 1.6 N suspension in 3.2 M ammonium sulphate
5. T r i c h l o r o a c e t i c a c i d solution.
7. Uricase
isolated according t o , ^ 4 U / m g . (25 °C) or
5
commercial p r e p a r a t i o n , see p . 5 1 8 , e.g. as a

solution in 5 0 % glycerol.
Purity of Reagents
In general, the commercially available uricase p r e p a r a t i o n s satisfy the requirements. X O D should contain
less t h a n 0.01 % (relative to the X O D activity) uricase, adenosine deaminase, guanase a n d nucleoside
phosphorylase, a n d less t h a n 0.1 % alkaline p h o s p h a t a s e .
U s e only glass distilled water; check p H with glass electrode.
I. G l y c i n e buffer ( 0 . 6 M ; p H 9 . 3 ) :
D i s s o l v e 4 . 4 8 g. g l y c i n e in c a . 4 0 m l . distilled w a t e r , a d d 11.7 m l . 1 N N a O H , d i l u t e t o
100 m l . w i t h distilled w a t e r a n d m i x .
II. G l y c y l g l y c i n e buffer (0.6 M ; p H 8 . 2 ) :

D i s s o l v e 3.17 g. g l y c y l g l y c i n e in ca. 10 m l . 1 N N a O H a n d d i l u t e w i t h distilled w a t e r t o 4 0 m l .
III. T r i c h l o r o a c e t i c a c i d ( 0 . 9 8 M ) :
D i s s o l v e 16.0 g. t r i c h l o r o a c e t i c a c i d in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
IV. X a n t h i n e oxidase, X O D (ca. 30 m U / m l . ) :

D i l u t e X O D s o l u t i o n s o r s u s p e n s i o n s a c c o r d i n g l y w i t h 0 . 2 M p h o s p h a t e buffer, p H 7.4.
V . U r i c a s e ( c a . 0.5 U / m l . ) :
D i s s o l v e t h e c o m m e r c i a l p r e p a r a t i o n i n 6 0 m M g l y c i n e buffer ( s o l u t i o n I d i l u t e d 1 : 10)
or dilute other preparations accordingly.
Hypoxanthine and Xanthine 1943
T h e buffer solutions I a n d II, with addition of 0.5 ml. toluene/100 ml., are stable for 6 - 8 weeks at 4 °C.
Store the X O D solution in the frozen state in small p o r t i o n s c o r r e s p o n d i n g t o the daily requirements.
K e e p the solution at 4 °C d u r i n g a series of m e a s u r e m e n t s . T h e uricase suspension is stable for 2 - 3 weeks
at 4 °C.
Procedure
Collection :
A l l o w 1 8 - 2 0 m l . b l o o d t o flow f r o m a n u n r e s t r i c t e d v e i n i n t o a s m a l l c e n t r i f u g e t u b e c o n t a i n
ing 1 - 2 d r o p s o f heparin solution (39 m g . heparin/ml. distilled water; 5 0 0 0 International
U n i t s / m l . ) . If p l a s m a is t o b e a n a l y s e d c e n t r i f u g e i m m e d i a t e l y for 15 m i n . at 3 0 0 0 r p m .
T h e b r e a k d o w n o f A T P c o n t a i n e d i n b l o o d c e l l s starts i m m e d i a t e l y after c o l l e c t i o n o f t h e
b l o o d a n d t h i s l e a d s t o t h e f o r m a t i o n o f h y p o x a n t h i n e b o t h in t h e c e l l s a n d in t h e p l a s m a .
6
F o r this reason s e r u m c a n n o t be analysed.
Pretreatment with uricase and deproteinization :

P l a s m a : P i p e t t e i n t o a c e n t r i f u g e t u b e 8 . 0 0 m l . p l a s m a , 0 . 0 5 m l . 1.6 N N a O H a n d 0 . 0 2 5 m l .
u r i c a s e s u s p e n s i o n ( V ) ; t h e p H s h o u l d b e 9 . 3 . I n c u b a t e f o r 1 hr. at r o o m t e m p e r a t u r e . A d d
2 . 0 0 m l . t r i c h l o r o a c e t i c a c i d ( I I I ) a n d m i x t h o r o u g h l y . C e n t r i f u g e for 15 m i n . a t 3 0 0 0 r p m . ;
t h e s u p e r n a t a n t fluid s h o u l d b e clear. P i p e t t e i n t o a m e a s u r i n g c y l i n d e r : 5 m l . s u p e r n a t a n t
fluid, 0 . 5 0 m l . 1.6 N N a O H ; a l l o w t o s t a n d 15 m i n . a n d t h e n a d d 0 . 4 0 m l . 1.6 N N a O H a n d
0 . 5 0 m l . g l y c y l g l y c i n e buffer (II) t o n e u t r a l i z e . A d j u s t t o p H 8.2 w i t h N a O H o r H C 1 a n d d i l u t e
with distilled water t o 12.0 ml.
U r i n e : D e p r o t e i n i z a t i o n is n o t n e c e s s a r y . T h e a m o u n t o f u r i n e t a k e n for a n a l y s i s d e p e n d s
o n t h e e x t e n t o f t h e d i u r e s i s . If t h e v o l u m e o f 2 4 hr. u r i n e is 1.5 litres, a d d 0 . 3 m l . o f u r i n e
t o 4 m l . d i s t i l l e d w a t e r . If v o l u m e o f t h e 2 4 hr. u r i n e d e v i a t e s f r o m 1.5 litres, u s e a n a m o u n t
o f u r i n e f o r t h e a s s a y c o r r e s p o n d i n g t o t h e r a t i o 1 5 0 0 : 0 . 3 . A d d 0 . 8 0 m l . g l y c i n e buffer (I)
a n d a d j u s t p H t o 9.3 w i t h N a O H . M i x in 0 . 0 1 - 0 . 0 2 m l . u r i c a s e s u s p e n s i o n ( V ) a n d i n c u b a t e
for 1 hr. at r o o m t e m p e r a t u r e . D e s t r o y t h e u r i c a s e b y t h e a d d i t i o n o f 0.8 m l . 1.6 N N a O H .
A l l o w t o s t a n d f o r 15 m i n . a n d a d d 0 . 8 5 m l . 1.6 N H C 1 . N e u t r a l i z e w i t h c a . 1.0 m l . g l y c y l g l y c i n e
buffer (II) ( i n d i c a t o r p a p e r ) a n d d i l u t e w i t h d i s t i l l e d w a t e r t o 10 m l .
T h e samples can be stored deep-frozen until analysed.
Assay System
W a v e l e n g t h : 2 8 0 a n d 2 9 3 n m ( t w o s e p a r a t e m e a s u r e m e n t s ) ; l i g h t p a t h : 1 c m . silica c u v e t t e s ;
final v o l u m e 3 m l . ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t t h e c o n t r o l c u v e t t e . If t h e s a m p l e c o n t a i n s
m o r e t h a n 5 - 1 0 jumole x a n t h i n e a n d / o r h y p o x a n t h i n e p e r litre, t h e e x t i n c t i o n after XOD
additions changes relatively quickly ( 0 . 0 1 0 - 0 . 0 2 0 / m i n . ) and therefore m a k e the m e a s u r e m e n t s
in t w o s e p a r a t e c u v e t t e s .
Pipette successively into C o n c e n t r a t i o n in a s s a y

Test Control
cuvettes mixture
Prepared sample 3.00 ml. 3.00 ml. u p t o c a . 2 0 pM o f e a c h

purine
T h e e x t i n c t i o n o f t h e test a n d t h e c o n t r o l at 2 8 0 n m s h o u l d b e
the same. R e a d extinction E x for t h e test c u v e t t e .
X O D suspension (IV) 0.01 t o - 3 mU/ml.

0.02 ml.
M i x w i t h a p l a s t i c s p a t u l a . R e a d t h e e x t i n c t i o n at 2 8 0 n m 4 t o
5 t i m e s w i t h i n 6 0 - 8 0 sec. P l o t t h e v a l u e s a g a i n s t t i m e a n d o b t a i n
E 2 by extrapolation to the time o f X O D addition.
R e p e a t t h e s a m e m e a s u r e m e n t w i t h a n o t h e r 3 m l . s a m p l e at 2 9 3 n m .
Calculations
N u m e r o u s m e a s u r e m e n t s of h y p o x a n t h i n e and xanthine s t a n d a r d solutions gave the following A E values:
1 pM H y p o x a n t h i n e A E 2 8 0 n m = +0.0070
AE 2 +0.0114
1 fiM X a n t h i n e AE 293nm = +0.0083
T h e p o r t i o n of the extinction change at 293 n m d u e to h y p o x a n t h i n e can be calculated from the extinction

changes of a m i c r o m o l a r solution at 280 a n d 293 n m :
+ AE% 3nm = JE«* 0 n m x - M i l l = JE«* 0 n m x 1.63
The remainder of the extinction change at 293 n m is due to xanthine. T h e h y p o x a n t h i n e concentration

of the sample is given b y :
c = ^sornn [^mole/ml.]
7 x v
where
V = final v o l u m e of sample
v = volume of undiluted sample
To calculate the value for xanthine subtract the p o r t i o n of the extinction at 293 n m d u e to hypoxanthine
from the m e a s u r e d A E 2 9 3 n m .
T h e n a n a l o g o u s to the above e q u a t i o n the xanthine concentration of the sample is given b y :
c = l 8.3l x v
AE 9 nm
X V
[/miole/ml.j
Recovery experiments with xanthine and h y p o x a n t h i n e a d d e d in physiological a m o u n t s to urine gave

8 6 - 1 3 3 % a n d 9 4 - 1 1 9 % respectively . W i t h p l a s m a the c o r r e s p o n d i n g values were 6 0 - 1 4 0 % ( a m o u n t s
2
u p t o 20 nmole/ml.).
Normal Values
In 20 p l a s m a samples from n o r m a l subjects we f o u n d < 20 //mole h y p o x a n t h i n e a n d x a n t h i n e p e r litre.

In m o r n i n g urine specimens from 5 n o r m a l male subjects the values for 6 successive days have been
averaged. O n e subject h a d the highest value of 496 ( 3 6 6 - 5 7 0 ) //mole h y p o x a n t h i n e per litre, a n o t h e r
the lowest of 71 ( 1 8 - 1 2 3 ) //mole h y p o x a n t h i n e per litre. In the same subjects t h e highest a n d lowest
values for xanthine were 246 ( 1 1 9 - 4 0 0 ) //mole/litre a n d 64 ( 2 4 - 1 0 7 ) //mole/litre. T h e daily excretion
of 6 resting (minor surgery) male subjects was between 75 a n d 121 //mole (10 a n d 16 mg.) h y p o x a n t h i n e
per 24 hr. a n d between 37 a n d 241 //mole (6 a n d 36 mg.) x a n t h i n e per 24 hr. T h e r a t i o H x / H x + X did
n o t vary for a particular subject, b u t did vary from subject t o subject.
In patients treated with allopurinol (4-hydroxyhydrazol-(3,4-d)-pyrimidine) the c o m p l e t e oxidation of

the hydroxypurines is inhibited. To be certain t h a t X O D is a d d e d in sufficient excess to the assay mixture,
when the reaction is complete a d d a k n o w n a m o u n t of x a n t h i n e o r h y p o x a n t h i n e a n d test their r e c o v e r y . 9
T h e extinctions of xanthine a n d h y p o x a n t h i n e are strongly p H - d e p e n d e n t . T h e values given u n d e r

" C a l c u l a t i o n s " are for p H 8.2.
Urine contains 8 k n o w n a n d 3 u n k n o w n p u r i n e s . Of these only 7-methylguanine occurs in similar

8
c o n c e n t r a t i o n t o x a n t h i n e , b u t it is n o t oxidized o r d e a m i n a t e d by X O D . T h e c o n c e n t r a t i o n of this p u r i n e
is only 35 % of the s u m of h y p o x a n t h i n e a n d xanthine. It is therefore certain t h a t c o m p o u n d s o t h e r t h a n
h y p o x a n t h i n e a n d x a n t h i n e d o n o t significantly c o n t r i b u t e to the extinction changes at 280 n m a n d 293 n m
after addition of X O D at p H 8.2.
References
1 P. Plesner & H. M. Kalckar in D. Glick: M e t h o d s of Biochemical Analysis. Interscience, N e w Y o r k

1956, vol. I l l , p . 103.
2 Birte Brandt Petersen, J. Jorni & S. Jergensen, Scand. J. clin. L a b . Invest. 77, 454 [1965].
3 K. Ro, Biochem. J. 14, 361 [1931].
4 77. Klenow, Arch. Biochem. Biophys. 58, 276 [1955].
5 C. G. Holmberg, Biochem. J. 33, 1901 [1939].
6 S. Jorgensen & H. E. Poulsen, A c t a p h a r m a c o l . et toxicol. 11, 287 [1955].
7 Birte Brandt Petersen, J, Jerni & S. Jergensen, Scand. J. clin. L a b . Invest. 77, 460 [1965].
8 B. Weissmann, Ph. A. Bromberg & A. Gutman, J. biol. C h e m . 224, 423 [1957].
9 7. R. Klinenberg, S. Goldfinger, K. 77. Bradley & J. E. Seegmiller, Clin. C h e m . 13, 834 [1967].
Colorimetric Assay
Rainer Fried and Lygia W. Fried*
H y p o x a n t h i n e (6-hydroxypurine) a n d xanthine (2,6-hydroxypurine) a r e p r o d u c t s of p u r i n e , p u r i n e

nucleoside o r nucleotide metabolism. They are formed from a d e n i n e o r g u a n i n e by d e a m i n a t i o n a n d from
the corresponding nucleosides a n d nucleotides by d e a m i n a t i o n a n d hydrolysis. All p u r i n e derivatives
* T h e a u t h o r s were s u p p o r t e d by the Milheim F o u n d a t i o n for C a n c e r Research a n d G . D . Searle & C o .

from nucleic acids are metabolized via h y p o x a n t h i n e a n d x a n t h i n e which are then oxidized to uric by
xanthine oxidase, X O D ( X a n t h i n e : oxygen oxidoreductase, E C 1.2.3.2). T h e oxidation of hypoxanthine
a n d x a n t h i n e by x a n t h i n e oxidase serves as a specific test for these t w o purines.
T h e enzymatic m e t h o d c a n be c o m b i n e d with radiochemical m e t h o d s 1 - 3
. This m e t h o d is usually used
when a U V s p e c t r o p h o t o m e t e r is available. Otherwise xanthine a n d h y p o x a n t h i n e can be determined
conveniently with x a n t h i n e oxidase in the presence of tetrazolium salts as electron a c c e p t o r s . 4,5
Principle
(1) H y p o x a n t h i n e + Tetrazolium salt X Q P

> Xanthine + Formazan
(2) X a n t h i n e + Tetrazolium (yellow) x o p

> Uric acid + F o r m a z a n (violet)
T h e strongly coloured reaction p r o d u c t , formazan, is measured at 5 3 0 - 5 8 0 n m a n d the purine concentra

tion can be calculated from a s t a n d a r d curve. With this m e t h o d it is not possible to determine xanthine
a n d h y p o x a n t h i n e separately.
If the nitro-BT tetrazolium salt is used, the resulting formazan has a wide extinction m a x i m u m between
530 a n d 580 n m . T h e usually insoluble formazan is held in emulsion by gelatine a n d can be measured
directly. T h e assay m e t h o d is based o n similar m e t h o d s developed for o t h e r r e d u c t a s e s ; in principle the
6,7
activities of alcohol d e h y d r o g e n a s e a n d uricase can also be d e t e r m i n e d . T h e relationship between substrate

8
a n d formazan is non-linear (Fig. 1). T h e shape of the curve c a n n o t be altered by variation of the concentra-
Fig. 1. S p e c t r o p h o t o m e t r i c determination
of purines - X = xanthine, A A HX =
h y p o x a n t h i n e , A values with heat de
n a t u r e d enzyme, p H 7.8; 38 °C. X a n t h i n e
oxidase from milk, diluted 1 : 100.
tions of the reactants or the i n c u b a t i o n t e m p e r a t u r e . A linear form c a n n o t be o b t a i n e d by semilogarithmic,

log/log, o r s q u a r e r o o t plots, b u t linearity is o b t a i n e d if a Lineweaver-Burk plot of 1/v against 1/[S]
is m a d e ; t h e line does n o t pass t h r o u g h the zero p o i n t of the co-ordinates.
H y p o x a n t h i n e gives a p p r o x i m a t e l y twice the intensity of formazan colour as a n e q u i m o l a r concentration

of x a n t h i n e ; the curves have a similar shape. T h e reason for the non-linear relationship is n o t clear.
Possibly it is d u e to allosteric activation of the enzyme by x a n t h i n e o r h y p o x a n t h i n e . 9
X a n t h i n e oxidase h a s a substrate o p t i m u m between 0.1 a n d 0.2 m M . T h e c o n c e n t r a t i o n of enzyme is

so a r r a n g e d t h a t an extinction change of ca. 0 . 0 5 - 0 . 1 0 0 / m i n . at 540 n m is o b t a i n e d . Gelatine h a s a d o u b l e
function: it keeps the insoluble formazan in solution a n d for reasons t h a t a r e n o t k n o w n , catalyses the
reaction. E D T A c a n n o t replace gelatine. Over a wide c o n c e n t r a t i o n range p h e n a z i n e m e t h o s u l p h a t e
causes a 2 0 % activation of X O D . T h e extent of this activation varies with different p r e p a r a t i o n s a n d
with the age of the enzyme, b u t this can usually be ignored.
T h e reason for the lag p h a s e of the reaction is n o t k n o w n ; it c a n n o t be eliminated by alteration of the
assay conditions.
Equipment
S p e c t r o p h o t o m e t e r o r c o l o r i m e t e r for m e a s u r e m e n t s b e t w e e n 530 a n d 580 n m . Preferably

u s e a n i n s t r u m e n t w i t h a c o v e r e d cell h o u s i n g , b e c a u s e t h e r e a c t i o n s y s t e m is l i g h t - s e n s i t i v e .
S t o p w a t c h ; w a t e r b a t h o r i n c u b a t o r ; test t u b e s m a d e of d a r k glass.
Reagents
1. D i s o d i u m h y d r o g e n p h o s p h a t e , ditetrazolium chloride, e. g. from Sigma C h e m i

Na HP02 4 • 7H 02
cal C o . , St. Louis, M o . , U S A , G r a d e III
2. G e l a t i n e ( t h e p u r e s t g r a d e available) 6. Hypoxanthine
3. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA 7. X a n t h i n e o r i t s d i s o d i u m s a l t
E D T A - N a H • 2 H 0 or E D T A - N a • H 0
2 2 2 4 2
8. S o d i u m h y d r o x i d e , 0.1 N
4. P h e n a z i n e m e t h o s u l p h a t e , PMS 9. H y d r o c h l o r i c a c i d , 0.1 N
(light sensitive) 10. X a n t h i n e o x i d a s e , X O D
5. N i t r o - B T t e t r a z o l i u m s a l t , N B T from milk according t o 1 0 1 1
or commercial
(light sensitive) 2,2'-Di-p-nitrophenyl-5,5'- p r e p a r a t i o n (see p . 521); ca. 0.4 U / m g . (25 °C).
diphenyl-3,3 '-(3,3-dimethoxy-4,4 '-diphenylene)-
Purity of Reagents
X a n t h i n e oxidase m u s t be free from uricase. T h e reagents m u s t n o t contain any aldehydes. A l l o p u r i n o l

interferes.
P r e p a r e all s o l u t i o n s w i t h d i s t i l l e d w a t e r .
I. P h o s p h a t e b u f f e r ( 0 . 1 M ; p H 7 . 8 ) :
D i s s o l v e 2 6 . 8 g. N a H P 0 2 4 • 7 H 0 i n c a . 8 0 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7.8 w i t h
2
0.1 N H C 1 a n d d i l u t e w i t h d i s t i l l e d w a t e r t o 1 0 0 0 m l .
II. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E T D A (10 m M ) :
D i s s o l v e 3.8 g. E D T A - N a 4 • H 0 i n c a . 8 0 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7.8 w i t h
2
0.1 N H C 1 a n d d i l u t e w i t h d i s t i l l e d w a t e r t o 1 0 0 0 m l .
III. Gelatine ( 1 % w/v):
D i s s o l v e 1 g. g e l a t i n e i n 100 m l . b o i l i n g p h o s p h a t e b u f f e r ( I ) . C o o l .
IV. X a n t h i n e oxidase, X O D :
D i s s o l v e c o m m e r c i a l l y a v a i l a b l e X O D f r o m m i l k in p h o s p h a t e buffer (I) a n d d i l u t e
a c c o r d i n g l y (1 : 1 0 0 - 1 : 1 0 0 0 a c c o r d i n g t o p r e l i m i n a r y tests).
V. Phenazinemethosulphate, P M S (0.2 m g . / m l . ) :
D i s s o l v e 10 m g . P M S in 5 0 m l . p h o s p h a t e buffer (I). W e i g h in s u b d u e d light a n d s t o r e
in a d a r k b o t t l e . P r e p a r e t h e s o l u t i o n a f r e s h a s s o o n a s it s h o w s a s t r o n g g r e e n c o l o u r .
VI. Nitro-BT-tetrazolium, N B T (4 m g . / m l . ) :
D i s s o l v e 2 0 0 m g . N B T in 5 0 m l . p h o s p h a t e buffer (I). W e i g h in s u b d u e d light a n d store
in a d a r k b o t t l e . P r e p a r e t h e s o l u t i o n a f r e s h a s s o o n a s a p r e c i p i t a t e a p p e a r s .
V I I . X a n t h i n e s t a n d a r d s o l u t i o n (1 m M ) :
D i s s o l v e 1 9 . 6 m g . x a n t h i n e , d i s o d i u m salt, in 2 0 m l . 0.01 N N a O H ( s t o c k s o l u t i o n
5 m M ) . D i l u t e t h e s t o c k s o l u t i o n 1 : 5 w i t h p h o s p h a t e buffer (I) ( w o r k i n g s t a n d a r d
1 m M ) . P r e p a r e t h e w o r k i n g s t a n d a r d s o l u t i o n freshly b e f o r e e a c h series o f m e a s u r e
ments.
V I I I . H y p o x a n t h i n e s t a n d a r d s o l u t i o n (1 m M ) :
D i s s o l v e 1 3 . 6 m g . h y p o x a n t h i n e in 2 0 m l . 0.01 N N a O H ( s t o c k s o l u t i o n 5 m M ) . D i l u t e
t h e s t o c k s o l u t i o n 1 :5 w i t h p h o s p h a t e b u f f e r (I) ( w o r k i n g s t a n d a r d s o l u t i o n , 1 m M ) .
P r e p a r e t h e w o r k i n g s t a n d a r d s o l u t i o n freshly b e f o r e e a c h series o f m e a s u r e m e n t s .
Store all solutions in a refrigerator at 0 - 4 °C. Store the P M S and N B T solutions in dark bottles; their
stability is limited (see above). The recommended preparation of N B T is completely soluble; filter solutions
prepared from other sources before storage. Prepare the xanthine and hypoxanthine stock solutions
freshly every two weeks and store frozen; prepare the working standard solutions freshly each day.
Prepare the xanthine oxidase solution each day from the commercially available suspensions. The X O D
stock suspension loses activity on storage; the activity should be determined before the reagent mixture
is prepared.
Procedure
D i a l y s e p l a s m a ( 4 - 1 2 m l . ) for 6 hr. at r o o m t e m p e r a t u r e a g a i n s t 5 v o l u m e s distilled w a t e r .

L y o p h i l i z e t h e d i a l y s i s fluid ( o u t s i d e fluid) a n d d i s s o l v e t h e l y o p h i l i z a t e in p h o s p h a t e buffer
( s o l u t i o n I ) . It is n o t n e c e s s a r y t o d e s t r o y t h e uric a c i d w i t h u r i c a s e .
3 3
D e p r o t e i n i z a t i o n is o f t e n n o t n e c e s s a r y (e. g. u r i n e ) . If t h e s a m p l e c o n t a i n s o x i d i z i n g s u b s t a n c e s
or c o m p o u n d s which reduce tetrazolium, boil the solutions or deproteinize.
Stability of sample: X a n t h i n e a n d h y p o x a n t h i n e are s t a b l e in s e r u m a n d u r i n e for several

d a y s at 0 - 4 ° C . T h e p u r i n e c o n c e n t r a t i o n in w h o l e b l o o d a n d e r y t h r o c y t e s i n c r e a s e s 5 0 - t o
1 0 0 - f o l d if t h e s a m p l e s are a l l o w e d t o s t a n d ; it is n o t k n o w n w h e t h e r t h e a m o u n t s in u r i n e
1 2
change o n standing.
Assay System
W a v e l e n g t h : 5 3 0 - 5 8 0 n m , p r e f e r a b l y 5 4 0 n m ; o r f o r a m e r c u r y filter p h o t o m e t e r , 5 4 6 a n d
5 7 8 n m ; light p a t h : 1 c m . ; final v o l u m e : 6 m l . ; i n c u b a t i o n t e m p e r a t u r e : 38 ° C ; c o l o r i m e t r i c
m e a s u r e m e n t s at r o o m t e m p e r a t u r e . If p o s s i b l e p r e p a r e t r i p l i c a t e t u b e s ; r e a d a g a i n s t a i r ;
p r e p a r e a b l a n k c o n t a i n i n g w a t e r i n s t e a d o f X O D s o l u t i o n f o r e a c h series o f m e a s u r e m e n t s .
Just b e f o r e t h e a s s a y p r e p a r e t h e f o l l o w i n g m i x t u r e in b r o w n test t u b e s ( s u b d u e d l i g h t ) :
P h o s p h a t e buffer (I) 4 parts

E D T A solution (II) 6 „
G e l a t i n e s o l u t i o n (III) 5 „
N B T solution (V) 3 „
P M S solution (VI) 2 „
20 parts
P i p e t t e i n t o d a r k test t u b e s : C o n c e n t r a t i o n in a s s a y m i x t u r e
Reagent mixture 2.60 ml. 100 m M p h o s p h a t e *

0.87 m M EDTA
2.2 m g . gelatine/ml.
173 fig. NBT/ml.
5.8 fig. PMS/ml.
X O D solution (IV) 0.40 ml.
S a m p l e + distilled water 3.00 ml. up to 0.2 m M xanthine**
u p t o 0.1 m M hypoxanthine**
M i x , i n c u b a t e for 15 m i n . at 38 ° C , p o u r i n t o c u v e t t e s
and read extinctions.
Calculations
Subtract the extinction of the blank from the extinctions of the samples. Using the A E values read off
the p u r i n e concentrations from s t a n d a r d curves for x a n t h i n e a n d h y p o x a n t h i n e . T h e s u m of h y p o x a n t h i n e
+ xanthine can be expressed as xanthine o r h y p o x a n t h i n e 3 , 1 2
.
Standard curves: Take 0.20 to 1.20 m l . x a n t h i n e s t a n d a r d solution (VII), equivalent t o 0.20 to 1.20 //mole,
a n d half of these volumes of h y p o x a n t h i n e s t a n d a r d solution (VIII) instead of sample (preferably in parallel
with the samples). Plot the AE values (ordinate) against the /rniole x a n t h i n e or h y p o x a n t h i n e (abscissa);
see Fig. 1.
N - 9 ; x = 0.537 (0.522 to 0.550); s = 0.010; coefficient of variation = 1.86%.
Normal Values
T h e total a m o u n t of x a n t h i n e a n d h y p o x a n t h i n e in fresh b l o o d o r p l a s m a is 1 - 3 / / g . / m l . . 12
T h e total a m o u n t of the h y d r o x y p u r i n e s in urine of 6 subjects was between 4 a n d 19 mg./24 hr. calculated

as h y p o x a n t h i n e o r 5 a n d 12 mg./24 hr. calculated as x a n t h i n e . 3
In cases of x a n t h i n u r e a a x a n t h i n e excretion of 176 mg./24 hr. h a s been r e p o r t e d ; the excretion of hydroxy-

purines decreases in polycythemia vera, leukemia o r intermittent g o u t . 1 3
* All reagents are dissolved in p h o s p h a t e buffer.

** T h e range of the m e t h o d d e p e n d s o n the p h o t o m e t e r . We use a Gilford 300 s p e c t r o p h o t o m e t e r which
can m e a s u r e accurately u p to 2.0 extinction units.
Effects of drugs and other therapeutic measures: A l l o p u r i n o l inhibits the s p e c t r o p h o t o m e t r i c m e t h o d for

the assay of xanthine. A similar inhibition occurs when the tetrazolium m e t h o d described here is used
(R. Fried ).
15
T h e inhibition caused by other inhibitors depends on the type of assay system used; some
c o m p o u n d s which cause an inhibition in the s p e c t r o p h o t o m e t r i c assay do not inhibit the reduction of
tetrazolium 8,14
.
A substance has been isolated from rat liver h o m o g e n a t e s which inhibits the reduction of t e t r a z o l i u m 15
a n d has been identified as superoxide d i s m u t a s e . 17
Interference in the assay technique: If the X O D p r e p a r a t i o n c o n t a i n s uricase, erroneously high values

are obtained. Similarly, the presence of aldehydes in the sample leads to high values. Deproteinization
is necessary if the sample contains reductases a n d their corresponding substrates. L o w concentrations of
these interfering substances can be corrected for by the blank.
A p a r t from x a n t h i n e a n d h y p o x a n t h i n e , aldehydes, pterins a n d certain drugs are oxidized; the rate of

oxidation is variable. This assay m e t h o d d o e s n o t differentiate between x a n t h i n e a n d h y p o x a n t h i n e . These
purines can be separated before the d e t e r m i n a t i o n , for example, by thin-layer c h r o m a t o g r a p h y . 16
Special Details
This m e t h o d is suitable for the d e t e r m i n a t i o n of hydroxypurines in turbid solutions or emulsions. T h e

degree of turbidity permissible is d e p e n d e n t on the p h o t o m e t e r available. T h e assay system must be
homogenized before m a k i n g the m e a s u r e m e n t s . Semi-quantitative assays can be m a d e by visual compari
son with s t a n d a r d s .
References

2 P. Plesner & H. M. Kalckar, M e t h . Biochem. A n a l . 3. 97, 110 [1956].
3 J. R. Klinenberg, S. Goldfinger, K. H. Bradly & J. E. Seegmiller, Clin. C h e m . 13, 834, 846 [1967].
4 R. Fried, A n a l . Biochem. 16, 427 [1966].
5 R. Fried & L. W. Fried, this b o o k , p . 644.
6 M. M. Nachlas, S. I. Margulies &A.M. Seligman, J. biol. C h e m . 235, 499 [I960].
7 M. M. Nachlas, S. I. Margulies, J. D. Goldberg & A. M. Seligman, A n a l . Biochem. 1, 317 [I960].
8 R. Fried, P r o c . Int. S y m p . o n Alcohol a n d Alcoholism, Santiago (Chile) August 1965.
9 S. Muraoka, Biochem. Biophys. A c t a 73, 17; 27 [1963].
10 D. A. Gilbert & F. Bergel, Biochem. J. 90, 350 [1964].
11 L.I. Hart & R. C. Bray, Biochim. Biophys. A c t a , 146, 611 [1967].
12 S. Jorgenson & H. E. Paulsen, A c t a P h a r m a c o l , et toxicol. 11, 287 [1955].
13 C. Long, E. J. King & W. Sperry, Biochemist H a n d b o o k , p . 929 Van N o s t r a n d , N e w Y o r k [1961].
14 R. Fried & L. W. Fried, Biochem. P h a r m a c o l . 75, 1890 [1966].
15 R. Fried, L. W. Fried & D. Babin, E u r . J. Biochem. 16, 399 [1970].
16 P. Orsulak, W. Haab & M. D. Appleton, A n a l . Biochem. 23, 156 [1968].
17 R. Fried, L. W. Fried & D. R. Babin, Eur. J. Biochem. 33, 439 [1973].
Uric Acid
Peter Scheibe, Erich Bernt a n d H a n s Ulrich Bergmeyer
Uric acid is the end p r o d u c t of p u r i n e m e t a b o l i s m in m a n . F u r t h e r d e g r a d a t i o n by the enzyme uricase

to allantoin, as in most o t h e r m a m m a l s , occurs only to a very small extent if at all in t h e h u m a n b o d y .
Enzymatic cleavage with uricase ( U r a t e : o x y g e n oxidoreductase E C 1.7.3.3) to allantoin a n d H 0 2 2 can
be used for the d e t e r m i n a t i o n of uric acid in s e r u m a n d urine. M e t h o d s have been described in which
the hydrogen peroxide formed is d e c o m p o s e d by peroxidase, P O D ( D o n o r : h y d r o g e n - p e r o x i d e oxidore
ductase, E C 1.11.1.7), a n d the oxygen liberated oxidizes a hydrogen d o n o r (e. g. o-dianisidine) to a c o l o u r e d
c o m p o u n d , . Since the samples (e.g. serum) m u s t be deproteinized a considerable loss of uric acid can
1 2
occur at this step .

M e t h o d s that d o n o t involve deproteinization a n d t h a t are therefore m o r e accurate, are those in which the
decrease in the quantity of uric acid is m e a s u r e d directly at 293 n m o r 297 n m 3
o r in which, after
Kageyama ", the hydrogen peroxide formed d u r i n g the reaction oxidizes m e t h a n o l to f o r m a l d e h y d e u n d e r
4
the influence of catalase. T h e formaldehyde can be readily determined in the presence of a m m o n i u m ions
a n d acetylacetone (Hantzsch reaction) . 10
UV-Assay with Uricase

Principle
(1) Uric a c i d + 2 H 0 + 0 2 2
u r i c a s e
> Allantoin+C0 + H 0
2 2 2
Uric acid has a characteristic a b s o r p t i o n m a x i m u m at 293 n m , whereas allantoin a n d H 0 2 2 do not absorb

in this wavelength region. T h e decrease in the uric acid c o n c e n t r a t i o n can therefore be m e a s u r e d at 293 n m
o r H g 297 n m . E r r o r s t h a t m a y result from turbidity of the s a m p l e o r dilution of the assay m i x t u r e a r e
eliminated by m e a n s of a sample b l a n k . 5
T h e p H o p t i m u m of the enzyme is p H 9 to 10. T h e t u r n o v e r n u m b e r at 38 °C is 12 000 moles of uric

acid/min/mole of e n z y m e . T h e Michaelis c o n s t a n t is K = 1 . 7 x 1 0 " M . T h e reaction proceeds fastest
6 7
M
5
in the presence of b o r a t e . T h e equilibrium of the reaction lies completely o n the right.

7
Equipment
S p e c t r o p h o t o m e t e r a n d s p e c t r a l line p h o t o m e t e r c a p a b l e o f a c c u r a t e m e a s u r e m e n t s at 2 9 3 o r
H g 297 n m .
Reagents*
1. B o r i c a c i d , A . R . 4. Glycine
2. S o d i u m c a r b o n a t e , A . R . 5. G l y c e r o l , A . R .
3. Uricase doubly distilled, a p p r o x . 8 7 % (v/v)
from pig liver, solution in 5 0 % (v/v) glycerol;
^ 4 . 0 U / m g . (25 °C), commercial p r e p a r a t i o n s ,
see p . 518.
Purity of Reagents
T h e boric acid, glycerol, a n d sodium c a r b o n a t e must be of A. R. quality.
M a k e u p all s o l u t i o n s w i t h freshly p r e p a r e d d o u b l y distilled w a t e r . S t e a m flasks t o i n h i b i t

the growth o f micro-organisms.
I. B o r a t e buffer ( 0 . 1 M ; p H 9 . 5 ) :
D i s s o l v e 3 . 1 2 g. b o r i c a c i d a n d 5 g. s o d i u m c a r b o n a t e in 5 0 0 m l . o f w a t e r . C h e c k p H
w i t h a p H m e t e r a n d a d j u s t a c c u r a t e l y if n e c e s s a r y .
II. U r i c a s e ( 2 m g . p r o t e i n / m l . ) :
Dilute stock solution with 5 0 % glycerol solution (III):
III. G l y c e r o l ( 5 0 % v / v ; 5 0 m M g l y c i n e ; 0 . 1 3 M N a C 0 ) : 2 3
D i s s o l v e 3 7 5 m g . g l y c i n e a n d 1.38 g. N a C 0 2 3 in a mixture o f 4 0 ml. water a n d 60 ml.

glycerol.
W h e n stored in stoppered containers in a refrigerator at a b o u t 4 °C, all solutions are stable for at least
6 months.
Procedure
Collection of sample: C o o l b l o o d i m m e d i a t e l y after r e m o v a l a n d o b t a i n s e r u m .
Stability of sample: N o d e c r e a s e i n t h e uric a c i d c o n c e n t r a t i o n in s e r u m o c c u r s after 5 d a y s a t

4 °C or 25 °C.
Assay System
W a v e l e n g t h : 2 9 3 o r H g 2 9 7 n m ; q u a r t z c u v e t t e s , light p a t h : 1 c m ; final v o l u m e : 3 . 5 2 m l . ;
r o o m temperature. R e a d against sample blank.
* C o m p l e t e reagent kits a r e commercially available, see p . 558.

Uric Acid 1953
F o r e a c h series o f m e a s u r e m e n t s , d e t e r m i n e t h e r e a g e n t b l a n k o n c e w i t h buffer (I) i n s t e a d o f

s a m p l e (see b e l o w ) .
M i x 0 . 2 m l . s e r u m o r 0 . 0 2 m l . u r i n e w i t h 10 m l . b o r a t e buffer (I), a l l o w t o s t a n d at r o o m
t e m p e r a t u r e f o r 15 m i n . , m i x a g a i n , a n d u s e 3 . 5 0 m l . p o r t i o n s o f this d i l u t e s o l u t i o n for t h e
d e t e r m i n a t i o n for t h e s a m p l e a n d t h e s a m p l e b l a n k .
Sample and Sample Blank
P i p e t t e o n t o t h e b o t t o m o f test t u b e s : Sample Sample blank C o n c e n t r a t i o n in t h e test
Uricase solution (ID 0.02 ml. 11.5 / / g / m l . = 4 5 m U

uricase/ml.
Solution (III) 0.02 ml. approx. 0 . 3 % glycerol
Diluted sample 3.50 ml. 3.50 ml. a p p r o x . 0.1 M b o r a t e buf
fer u p t o 25 pM uric a c i d
M i x a n d a l l o w t o s t a n d for a b o u t 10 m i n . M a r k t w o c u v e t t e s A
a n d B , set s a m p l e in A t o e x t i n c t i o n E = 0, a n d m e a s u r e e x t i n c t i o n
o f t h e b l a n k in cell B . T h e c h a n g e in e x t i n c t i o n A E due to the
s a m p l e
d e g r a d a t i o n o f uric a c i d is o b t a i n e d .
Reagent Blank
Pipette o n t o the b o t t o m
Cuvette A Cuvette B
of cuvette A and B :
Uricase solution (ID 0.02 ml. —

Glycerol solution (III) — 0.02 ml.
B o r a t e buffer (I) 3.50 ml. 3.50 ml.
M i x , set e x t i n c t i o n o f c u v e t t e B t o E = 0 , a n d m e a s u r e e x t i n c t i o n
o f c u v e t t e A (A E ) . R B
E ampie+ E
S R B = J E is u s e d in t h e c a l c u l a t i o n s .
Calculations
U n d e r the a b o v e conditions, the reaction proceeds quantitatively. F o r m u l a (2, 3) on p . 312 is therefore

valid. T h e extinction coefficient of uric acid is e = 1 2 . 6 c m . / / / m o l e at 293 n m a n d £ = 1 1 . 7 c m . / / / m o l e at
2 2
H g 297 n m .
The result is obtained in //mole uric acid per ml. sample or m g . uric acid per 100 ml. sample.
Serum
c = JEx68.4 JEX73.9 [mg./100ml.]
c = A E x 4.1 JEx4.4 [//mole/1.]
Urine
c = AExeii JEx726 [mg./100ml.]
c = zlEx40 A E x 43 [//mole/1.]
W i t h an average of 5.5 mg. uric acid/100 ml. serum, a s t a n d a r d deviation of 0.14 mg. uric acid was found.
T h e coefficient of variation is 2 . 5 % .
Normal Values
M a l e serum contains 2 . 6 - 6 . 8 mg. uric acid/100 ml. s e r u m , while female serum contains 2 . 0 - 6 . 3 mg.
8
uric acid/100 ml. s e r u m , 0 . 2 5 - 0 . 7 5 g. uric acid is found in the 24 hr. u r i n e .

8 9
Effects of drugs and other therapeutic measures: n o n e k n o w n . Uricase is inhibited by metal chelating
agents that are also reducing agents.
Interference in assay technique: Since even very slight turbidity interferes with the measurement, only
perfectly clear solutions m a y be used. To avoid c o n t a m i n a t i o n of the blank cuvette B with uricase, always
use cuvette A for samples.
In the measurement of the reagent blank, differences in the transparency of the cuvette are also eliminated.
If this difference is greater t h a n the reagent blank, the extinction of cuvette A m a y be less t h a n zero. In this
case set cuvette B to extinction E = 0 . 1 0 0 a n d m e a s u r e extinction of cuvette A. T h e measured extinction
difference must then be subtracted from A E s a m p l e .
Uricase reacts specifically with uric acid. Structural analogues such as substituted purine derivatives d o not
react, but inhibit the cleavage of uric a c i d . 7
Colorimetric Assay with Uricase and Catalase
Principle 4 1 0 1 1
(1) Uric Acid + 2 H 0 + 0 2 2

u r i c a s e
> Allantoin + C 0 + H 0
2 2 2
(2) H 0 + CH OH
2 2 3
catalase
*> H C H O + 2 H 0 2
(3) HCHO+2CH COCH COCH + NH3 2 3 3 • 3,5-Diacetyl-l,4-dihydrolutidine+3H 0 2
T h e reaction p r o d u c t H 0 oxidizes m e t h a n o l to formaldehyde. T h e latter reacts with acetylacetone a n d

2 2
a m m o n i a . T h e yellow p r o d u c t 3,5-diacetyl-l,4-dihydrolutidine has an a b s o r p t i o n m a x i m u m at 410 n m .

T h e b r o a d absorption b a n d allows the m e a s u r e m e n t t o be carried o u t in the wavelength range from 400
to 420 n m .
* Hydrogen-peroxide:hydrogen-peroxide oxidoreductase E C 1.11.1.6.

Uric Acid 1955
The p H o p t i m a of the reaction steps catalysed by uricase a n d by catalase are p H 9 - 1 0 (eqn. 1) and p H 4 - 9
(eqn. 2). Since the c o n d e n s a t i o n reaction (eqn. 3) proceeds best at p H 7.0 a n d is rate-determining u n d e r the
conditions of the d e t e r m i n a t i o n , this p H is chosen for the entire reaction. T h e colour development is
complete after 60 min at 37 °C.
Equipment
S p e c t r o p h o t o m e t e r , s p e c t r u m - l i n e p h o t o m e t e r , o r p h o t o m e t e r c a p a b l e o f m e a s u r e m e n t at
4 0 0 - 4 2 0 n m ( H g 405 n m ) ; constant-temperature water bath.
Reagents*
1. D i a m m o n i u m h y d r o g e n p h o s p h a t e , 6. L i t h i u m c a r b o n a t e , L i C 0 , A . R.
2 3
( N H ) H P 0 , A . R.
4 2 4 7. Catalase
2. o - P h o s p h o r i c a c i d , H P 0 ,
3 4 A.R. from bovine liver, crystalline suspension in
sp.gr. 1.71, a p p r o x . 8 5 % (w/w) water; ^ 5 0 . 0 0 0 U / m g . (25 °C), commercial
3. M e t h a n o l , A . R . p r e p a r a t i o n s , see p . 438.
4. A c e t y l a c e t o n e A . R . 8. Uricase
5. U r i c a c i d , p u r e from pig liver, solution in 5 0 % (v/v) glycerol;
for biochemical p u r p o s e s ^ 4 . 0 U / m g . (25 °C), commercial p r e p a r a t i o n s ,
see p . 518.
Purity of Reagents
All substances must be of p u r e or A. R. quality.
P r e p a r e all s o l u t i o n s w i t h fresh d i s t i l l e d w a t e r . Sterilize c o n t a i n e r s t o p r e v e n t t h e g r o w t h o f

micro-organisms.
I. B u f f e r / m e t h a n o l / c a t a l a s e s o l u t i o n ( 0 . 5 M ( N H ) H P 0 ; p H 7 . 0 ; 7 5 0 U
4 2 4 catalase/ml.;
1.7 M m e t h a n o l ) :
D i s s o l v e 13.8 g. ( N H ) H P 0 4 2 4 in a p p r o x . 150 m l . w a t e r a n d a d j u s t t o p H 7.0 w i t h H P 0 .3 4
A d d 13.8 m l . m e t h a n o l a n d 1.5 x 1 0 5
U catalase, m a k e up to 200 ml. with water, and
mix thoroughly.
II. A c e t y l a c e t o n e s o l u t i o n ( 0 . 4 2 M a c e t y l a c e t o n e ) :
M a k e u p 0 . 6 5 m l . a c e t y l a c e t o n e a n d 1.5 m l . m e t h a n o l t o 15 m l . w i t h w a t e r a n d m i x
carefully.
III. Uricase(5U/ml.):
D i l u t e s t o c k s o l u t i o n w i t h 5 0 % g l y c e r o l as r e q u i r e d .
* Complete reagent kits are commercially available, see p . 558.

IV. U r i c acid standard (6 m g . uric a c i d / 1 0 0 m l . ; 0.357 m M ) :

a) S t o c k s o l u t i o n : D i s s o l v e 5 0 . 0 m g . uric a c i d a n d 4 0 m g . L i C 0 2 3 in w a t e r w i t h stirring
and make up to 50.0 ml.
b) Working s o l u t i o n : D i l u t e 6.00 ml. stock solution t o 100.0 ml. with water a n d m i x well.
V . U r i c a c i d r e a g e n t ( 0 . 4 7 M ( N H ) H P 0 ; p H 7 . 0 ; 7 1 0 U c a t a l a s e / m l . ; 1.7 M m e t h a n o l ;
4 2 4
20 m M acetylacetone):
A d d 0.5 v o l u m e s o l u t i o n II t o 10 v o l u m e s s o l u t i o n I ( a m o u n t s a c c o r d i n g t o t h e n u m b e r
o f samples) a n d m i x well.
S o l u t i o n s I, II, a n d III a r e s t a b l e f o r at least 6 m o n t h s , a n d s o l u t i o n V f o r at least 2 w e e k s ,

at 4 ° C . F r e s h s o l u t i o n I V a a n d I V b m u s t b e p r e p a r e d d a i l y .
Procedure
Collection of sample: s e e p . 1 9 5 2 .
Dilution of sample: D i l u t e 0 . 5 m l . s e r u m o r uric a c i d s t a n d a r d ( I V b ) o r 0 . 0 5 m l . u r i n e w i t h

5.00 m l . uric a c i d r e a g e n t ( V ) a n d m i x w e l l . S t a b i l i t y o f s a m p l e : s e e p . 1 9 5 2 .
Assay System
W a v e l e n g t h : 4 0 0 - 4 2 0 n m ( H g 4 0 5 n m ) ; g l a s s c u v e t t e s : light p a t h 1 c m . ; final v o l u m e : 2 . 5 2 m l . ;
i n c u b a t i o n t e m p e r a t u r e : 37 ° C ; r e a d s a m p l e a n d s t a n d a r d a g a i n s t c o r r e s p o n d i n g blank
without uricase (use the remainder o f the diluted sample or diluted standard solution). O n e
s t a n d a r d d e t e r m i n a t i o n is sufficient f o r e a c h series o f m e a s u r e m e n t s .
P i p e t t e o n t o t h e b o t t o m o f test t u b e s C o n c e n t r a t i o n in a s s a y m i x t u r e
Uricase solution (III) 0.02 ml. ^ 4 0 mU/ml.

Diluted sample or diluted standard (IV b) 2.50 ml. u p t o 1 1 0 pM u r i c a c i d
ca. 0.47 M ( N H ) H P 0 4 2 4
ca. 7 0 0 U catalase/ml.
c a . 1.6 M m e t h a n o l
c a . 18 m M a c e t y l a c e t o n e
M i x well, a n d i n c u b a t e s a m p l e , s t a n d a r d , a n d b l a n k
for at least 6 0 m i n at 3 7 ° C . M e a s u r e e x t i n c t i o n s o f t h e
sample a n d o f the standard against corresponding
blanks ( E s a m p l e , E s t a n d a r d ).
Uric Acid 1957
Calculations
U p to 20 m g . uric acid/100 ml. s e r u m or 200 mg. uric acid/100 ml. urine, the extinction is p r o p o r t i o n a l
to the concentration. T h e calculation is based o n the extinction of the uric acid s t a n d a r d . T h e 0.357 m M
s t a n d a r d solution ( I V b ) c o n t a i n s 6 mg. uric acid/100 ml., a n d serum is diluted 1 + 1 0 a n d urine 1 + 100.
T h e following expressions are therefore valid for the uric acid c o n c e n t r a t i o n :
in s e r u m : c = E s a m
P l c
x0.36 [mmole/1.]
'-'standard
c =
E
sam ie P x 6 [mg./100 ml.]
^standard
in u r i n e : c = ^ s a m p l e
x3.28 [mmole/1.]
'-'standard
c ^ Esample x 55 j [ . / 1 0 0 ml.]
m g
b-standard
Recovery of uric acid a d d e d t o the assay mixture is 100%. W i t h an average of 5.5 mg. uric acid/100 ml.,
the s t a n d a r d deviation is s = 0 . 1 4 mg./lOO ml. This gives a coefficient of variation of 2 . 5 % .
Normal Values
Serum : 1 2
Males 3.4-7.0 mg./lOO ml. or
0 . 2 0 - 0 . 4 2 mmole/1.
Females 2.4 - 5 . 7 mg./lOO ml. or
0 . 1 4 - 0 . 3 4 mmole/1.
Urine : 9
0 . 2 5 - 0 . 7 5 g. uric acid in the 24 hr. urine
Effects of drugs and other therapeutic measures: none known.

Errors due to possible interference with the Hantzsch condensation are eliminated by the use of a sample
blank.
Interference in assay technique: W h e n filter p h o t o m e t e r s are used, it is necessary to ensure t h a t the radiation
is m o n o c h r o m a t i c (half width of the m e a s u r i n g radiation ^ 1 5 n m ) .
Uricase reacts specifically with uric acid. In the presence of short-chain aliphatic alcohols (particularly
methanol) together with hydrogen peroxide, catalase specifically catalyses the oxidation to the correspond
ing aldehydes.
References
1 K. Lorentz & W. Berndt, A n a l . Biochem. 18, 58 [1967].
2 C. F. Domagk & H. H. Schlicke, A n a l . Biochem. 22, 219 [1968].
3 E. Praetorius & H. Poulsen, Scand. J. clin. L a b . Invest. 3, 273 [1953].
4 N. Kageyama, Clin. C h i m . Acta. 31, 421 [1971].
5 M. Kortum & O. Kling, Arztl. L a b o r a t o r i u m 18, 33 [1971].
6 / / . R. Mahler, G. Hubscher & H. Braun, J. biol. C h e m . 216, 625 [1955].
7 / / . Braun, G. Hubscher & //. R. Mahler, Biochim. biophys. Acta. 22, 514 [1956].
8 N. Zollner, Z. klin. C h e m . 1, 178 [1963].
9 / . Henry: Clinical Chemistry, H a r p e r & R o w Publishers Inc., N e w York 1964, p . 286.
10 T. Nash, Biochem. J. 55, 416 [1953].
11 D. Keilin & E. F. Hartree, Biochem. J. 60, 310 [1955].
12 W. Thefeld, H Hoffmeister, E. W. Busch, P. U. Roller & I. Vollmar, Dtsch. med. Wschr. 98, 380 [1973].
Orotate
Determination with 0-5-MP pyrophosphorylase
Hans Mollering
Orotic acid, uracil-4-carboxylic acid, was discovered in milk p r o d u c t s ( w h e y ) . T h e physiological a n d b i o 1
chemical i m p o r t a n c e of o r o t a t e lies in its role as a g r o w t h factor in n u t r i t i o n a l deficiencies of p l a n t s a n d

animals and as a protective agent for liver (e. g. it h a s effects o n n u m e r o u s hepatic enzyme systems. T h u s in
vitamin B 1 2 deficiency a d m i n i s t r a t i o n of orotic acid results in an increase in R N A , D N A a n d n u c l e o t i d e s ) . 2
A p a r t from vitamin B 1 2 the g r o w t h factors in milk a n d milk p r o d u c t s are a t t r i b u t e d t o orotic acid. In c o n t r a s t

to sheep a n d goat milk, cow milk c o n t a i n s relatively large a m o u n t s of o r o t a t e , whereas it is virtually absent
from h u m a n m i l k . Like v i t a m i n B , o r o t a t e takes p a r t in a n u m b e r of m e t a b o l i c reactions a n d is the starting
3
1 2
point for the synthesis of pyrimidine nucleotides.

In this process c a r b a m y l a s p a r t a t e is formed from aspartic acid a n d c a r b a m y l p h o s p h a t e , then the ring is
closed with utilization of A T P to give an acid a m i d e (dihydroorotic acid). T h e d i h y d r o o r o t i c acid is oxidized
to orotic acid by a d e h y d r o g e n a s e (a flavoprotein) . 4
5 - P h o s p h o r i b o s e - d i p h o s p h a t e is then attached t o
orotic acid t o give O - 5 - M P * with the liberation of p y r o p h o s p h a t e ; the O - 5 - M P is rapidly converted t o
U - 5 - M P with a decarboxylase. O r o t a t e , together with uric acid a n d oxalic acid, plays an i m p o r t a n t role in
arthritic d i s e a s e s . 5 - F l u o r o r o t a t e a n d 5-fluorouracil have inhibitory properties for t u m o u r s . T h e enzymatic
5 6
synthesis of 5-fluorouridine-5'-monophosphate h a s been described by Dahl et a l . . 7
A p a r t from the relatively unspecific chemical m e t h o d for the d e t e r m i n a t i o n of o r o t a t e , an enzymatic 8
m e t h o d with d i h y d r o o r o t a t e d e h y d r o g e n a s e ( E C 1.3.3.1) h a s been d e s c r i b e d . T h e enzymatic assay j u s t

4
m e n t i o n e d , which involves t h e a d d i t i o n of t h e flavoenzyme together with reducing agents t o the assay

system, is only possible u n d e r special conditions (e.g. m e a s u r e m e n t s at 282 n m ; correction for the " c r e e p "
d u e to side r e a c t i o n s ; inclusion of s t a n d a r d s ; p u r e enzyme). Lieberman et a l . 9 , 1 0
were first t o describe t h e
enzymatic assay of P R P P with O - 5 - M P p y r o p h o s p h o r y l a s e a n d O - 5 - M P decarboxylase from yeast by
the decrease in extinction o n conversion of o r o t a t e t o U - 5 - M P . Rosenbloom a n d Seegmiller 11
have used
this m e t h o d t o d e t e r m i n e o r o t a t e with an excess of P R P P a n d we have modified this m e t h o d .
Application of Method: In p h a r m a c y , biochemistry, foodstuff chemistry a n d clinical chemistry.
Principle
(1) Orotate + P R P P ° ~ " 5 p p y r o p h o s p h o r y l a s e

) O - 5 - M P + PPj
(2) O-5-MP O - 5 - P decarboxylase , T J - 5 - M P + C0 2
T h e reaction is quantitative. T h e decrease in extinction at 295 n m is m e a s u r e d .
In t h e c o u p l e d r e a c t i o n t h e e q u i l i b r i u m lies o n t h e s i d e o f U - 5 - M P f o r m a t i o n . S t u d i e s o n t h e
c h o i c e o f b u f f e r s s h o w e d t h a t g l y c y l g l y c i n e w a s m o r e s u i t a b l e t h a n t r i e t h a n o l a m i n e o r tris. T h e
p H o p t i m u m o f t h e c o u p l e d r e a c t i o n is p H 8 . 0 . T h e M i c h a e l i s c o n s t a n t , K , f o r o r o t a t e is 2 0 M pM.
* Abbreviation: O - 5 - M P = o r o t i d i n e - 5 ' - m o n o p h o s p h a t e : P R P P = 5-phospho-a-D-ribose-l-diphosphate;

U-5-MP = uridine-5'-monophosphate; O-5-MP-pyrophosphorylase = Orotidine-5'-phosphate: pyro
p h o s p h a t e phosphoribosyltransferase, E C 2.4.2.10; O-5-MP-decarboxylase = O r o t i d i n e - 5 ' - p h o s p h a t e
carboxy-lyase, E C 4.1.1.23.
T h e e q u i l i b r i u m c o n s t a n t is
= [Orotate] x [PRPP]
[0-5-MP] x [PPi]
O - 5 - M P pyrophosphorylase requires 2 m M M g 2 +
for o p t i m u m a c t i v i t y .
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for p r e c i s e m e a s u r e m e n t s at 2 9 5 n m ; b e n c h c e n t r i f u g e .
Reagents
1. Glycylglycine
4 . P e r c h l o r i c a c i d , A . R . , s p . gr. 1.67; c a . 7 0 %
2. 5 - P h o s p h o - a - D - r i b o s e 1 - d i p h o s p h a t e
(w/w)
tetrasodium salt, ca. 4 5 % P R P P (determined
5. M a g n e s i u m c h l o r i d e , A . R . ; M g C l • 6 H 0
enzymatically). Commercial preparation, see 2 2
6. P o t a s s i u m c a r b o n a t e , A . R . , K C0
p. 549. 2 3
7. T r i e t h a n o l a m i n e h y d r o c h l o r i d e , T R A
3. O - 5 - M P pyrophosphorylase containing
O - 5 - M P decarboxylase from yeast
lyophilizate ^ 1 0 U O - 5 - M P pyrophosphory
lase per g. lyophilizate (25 °C). Commercial
preparation, see p. 490.
The enzyme mixture must n o t contain phosphatases or pyrophosphatases; see under "Sources of E r r o r " .
P r e p a r e all s o l u t i o n s w i t h fresh d o u b l y d i s t i l l e d w a t e r .
I. G l y c y l g l y c i n e b u f f e r / M g C l 2 (0.05 M glycylglycine, p H 8.0; 2 m M M g C l ) : 2
D i s s o l v e 0 . 6 6 g g l y c y l g l y c i n e in 8 0 m l . d i s t i l l e d w a t e r , a d d 2 m l . 0.1 M M g C l 2 solution
( 2 . 0 3 M g C l - 6 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l ) , a d j u s t t o p H 8 . 0 w i t h 1 N
2 2
N a O H a n d dilute to 100 ml. with distilled water.

II. 5 - P h o s p h o - a - D - r i b o s e 1 - d i p h o s p h a t e ( 1 0 m M P R P P ) :
D i s s o l v e 10 m g . P R P P s o d i u m salt in 1 m l . s o l u t i o n I.
III. 0 - 5 - M P - p y r o p h o s p h o r y l a s e / 0 - 5 - M P d e c a r b o x y l a s e ( 2 0 m g . l y o p h i l i z a t e / m l . ; ^ c a . 0.2 U
O-5-MP pyrophosphorylase/ml.):
D i s s o l v e 4 0 m g . l y o p h i l i z a t e in 2 m l . s o l u t i o n I. p H v a l u e o f t h e s o l u t i o n is > 7 . Any
turbidity s h o u l d be r e m o v e d by centrifugation.
I V . P e r c h l o r i c a c i d (1 M ) :
Dilute 9 ml. 7 0 % perchloric acid to 100 ml. with distilled water.
V . N e u t r a l i z a t i o n s o l u t i o n (0.21 M t r i e t h a n o l a m i n e ; 1.16 M K C0 ):
2 3
D i s s o l v e 1 g. t r i e t h a n o l a m i n e * H C 1 a n d 4 g. K C 0 2 3 in d i s t i l l e d w a t e r a n d m a k e u p t o 2 5 m l .
Buffer s o l u t i o n s I a n d V s t o r e d a t 4 ° C are s t a b l e for at least 4 w e e k s . P R P P s o l u t i o n II a n d

e n z y m e s o l u t i o n III are s t a b l e for 2 d a y s at 4 ° C . S o l u t i o n I V is s t a b l e indefinitely.
Orotate 1961
Procedure
Collection of sample: P u r e o r o t a t e s o l u t i o n s o r p r o t e i n - f r e e e x p e r i m e n t a l m a t e r i a l , e . g . s o l u b l e
p h a r m a c e u t i c a l s , d o n o t n e e d t o b e t r e a t e d further. C o l l e c t t i s s u e s a m p l e s b y f r e e z e - c l a m p i n g
( s e e " T i s s u e a n d Cell D i s i n t e g r a t i o n " , p . 4 0 0 ) . M i l k , y e a s t e x t r a c t s , s e r u m a n d o t h e r b i o l o g i c a l
material must be deproteinized.
Deproteinization: Tissues: I m m e d i a t e l y after c o l l e c t i o n h o m o g e n i z e t h e t i s s u e w i t h t w i c e t h e

a m o u n t o f ice-cold 1 M perchloric acid (solution I V ) at 0 °C in a n Ultraturrax (Jahnke a n d
K u n k e l K G , S t a u f e n i. B r s g . , G e r m a n y ) a n d t h e n c e n t r i f u g e . A d j u s t t h e s u p e r n a t a n t fluid t o
p H 7 - 8 w i t h 4 N K O H , c o o l a n d filter in t h e c o l d t o r e m o v e t h e p r e c i p i t a t e o f p e r c h l o r a t e .
Measure the v o l u m e o f the neutral extract (used for t h e calculations).
Milk or serum: M i x a 5 m l . s a m p l e w i t h 5 m l . p e r c h l o r i c a c i d ( I V ) a n d c e n t r i f u g e f o r 1 0 m i n . a t
3 0 0 0 r p m . A d d 1.0 m l . n e u t r a l i z a t i o n s o l u t i o n ( V ) t o 3 . 0 0 m l . s u p e r n a t a n t fluid t o b r i n g t h e
p H t o a b o u t p H 9. A l l o w t o s t a n d f o r 1 0 m i n . in a n i c e b a t h , filter o f f t h e p r e c i p i t a t e a n d t a k e
1.00 m l . filtrate f o r t h e a s s a y .
Stability of sample: T h e o r o t a t e c o n t e n t o f n e u t r a l i z e d e x t r a c t s d o e s n o t alter w i t h i n 2 4 hr. a t

0 ° C . S i m i l a r l y , t h e o r o t a t e c o n t e n t o f m i l k d o e s n o t s i g n i f i c a n t l y c h a n g e o n s t o r a g e at - 1 5 ° C . 3
Assay System
W a v e l e n g t h : 2 9 5 n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 2 . 0 0 m l . ; r o o m t e m p e r a t u r e . R e a d a g a i n s t
a reference q u a r t z c u v e t t e c o n t a i n i n g d i s t i l l e d w a t e r .
Pipette into quartz cuvettes: Concentration in assay mixture
G l y c y l g l y c i n e buffer (I) 0.88 m l . 22 m M glycylglycine

0.9 m M M g C l 2
Enzyme solution (III) 0.10 ml. c a . 10 m U O - 5 - M P

pyrophosphory lase/ml.
Sample (neutralized) 1.00 m l . u p t o c a . 1 0 0 pM orotate
M i x a n d o n c o m p l e t i o n o f the reaction read extinction

Ex.
P R P P solution (II) 0.02 ml. 1 0 0 pM PRPP
M i x a n d o n completion o f the reaction (ca. 2 0 m i n . )

read extinction E . 2
A E = E j - E is u s e d f o r t h e c a l c u l a t i o n s .
2
D e t e r m i n e t h e e x t i n c t i o n d u e t o t h e a d d e d P R P P s o l u t i o n in t h e b l a n k ( d i s t i l l e d w a t e r i n s t e a d
of sample) a n d m a k e a suitable correction.
Calculations
U n d e r t h e above conditions t h e reaction proceeds stoichiometrically. T h e extinction coefficient of o r o t a t e

is £
2 9 5 nm = 3.95 c m . / / m i o l e . A c c o r d i n g t o Dahl et a l . the value for 5 - f l u o r o o r o t a t e is
2 7
£ 2 9 5 n m = 3.80 cm. /
2
/zmole.
The results are obtained in //mole o r o t a t e (5-fluoroorotate) per ml. sample. This value m u s t be multiplied
by a factor if the sample has been deproteinized, neutralized or diluted in any way. In the case of whole
blood, the specific gravity (ca. 1.06) a n d the water c o n t e n t (ca. 80%) m u s t be taken into account. T h u s
for example for milk (no correction for solids a n d fat content) a factor of 2.67 is obtained after t a k i n g
into account the dilution 1 + 1 o n deproteinization a n d 3 + 1 o n precipitation of the perchlorate. T h e
following relationships therefore h o l d for the calculation of the orotic acid c o n c e n t r a t i o n of milk (mole
cular weight of orotic acid: 156.1):
Wavelength: 295 nm
c = AE x 1.35 [^mole/ml.]
c = AE x 213 [/*g./ml.]
W i t h a m e a n value of 6.2 m g . orotic acid in 100 ml. pasteurized cow's milk a s t a n d a r d deviation (n = 10)
of 0.3 mg. orotic acid/100 ml. was found. T h e coefficient of variation is 4 . 8 % . The limit of sensitivity
of the m e t h o d is a r o u n d 0.01 /rniole o r o t a t e per 2 ml. assay mixture.
N o r m a l Values
The mean o r o t a t e content of cow's milk is ca. 5 m g . / l 0 0 ml. In n o r m a l h u m a n serum a n d urine orotic
acid is not d e t e c t a b l e .
11
D e t e r m i n a t i o n o f O r o t a t e and O - 5 - M P
If a phosphatase, e.g. alkaline p h o s p h a t a s e from calf intestine, E C 3.1.3.1, is a d d e d to the sample at

p H 8 - 9 before deproteinization, b o t h o r o t a t e a n d O - 5 - M P are determined. T h e same applies to the
5-fluoro derivatives.
Interference in assay technique: Insufficient purity of the reagents, in particular the enzyme mixture
(e.g. the presence of p h o s p h a t a s e s , phosphodiesterases a n d p y r o p h o s p h a t a s e s ) can result in values which
are t o o high or t o o low. Hydrolysis of P R P P gives a t o o low content. H i g h c o n c e n t r a t i o n s of inorganic
salts inhibit the reaction, e.g. 0.2 M N a C l inhibits O - 5 - M P p h o s p h o r y l a s e ca. 3 0 % . 6-Uracilsulphonic
acid (K, == 7 uM), 6-uracilsulphonamide a n d 6-uracilmethylsulphonate are competitive i n h i b i t o r s . 12
Allopurinol ribonucleotide is a strong inhibitor of O - 5 - M P decarboxylase (K, = 0.7 / / M ) ~ . If P R P P is 1 3 1 4
present in the sample the assay m u s t be started with enzyme solution (III). In this case it is necessary t o
pipette the enzyme solution exactly because of its high extinction a n d a suitable correction must be for
this extinction change.
5-Fluoroorotate ( K M = 20 uM) reacts at twice the rate shown by orotic acid ( K M = 20 pM). The follow
ing d o not react: adenine, uracil, DL-ureidosuccinate, L-dihydroorotate, orotidine, 5-bromo-, 5-chloro-,
5-amino-, 5-nitro- a n d 5-methyl derivatives o r o r o t a t e . 5-Fluorouracil does n o t r e a c t . Reaction (1) is
12
absolutely specific for P R P P .

Orotate 1963
References
1 G. Biscaro & E. Belloni, A n n . Soc. C h i m . M i l a n o 11, 18 [1904].

2 C M. Caldarera, M. Marchetti & G. Moruzzi, Boll. soc. ital. biol. sper. 36, 1905 [I960].
3 F. Muenchberg, G. Tsompanidou & R. Leskova, Milchwissenschaft 26, 210 [1971].
4 H. C. Friedmann & G. Krakow in H. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag
Chemie, Weinheim 1970, 2nd. e d n „ p . 1888.
5 F r e n c h Patent, P. V. n o . , 99.929 N o . 6341, 23. M a r c h 1967.
6 C. Heidelberger, L. Griesbach, B. J. Montag, D. Mooren, O. Cruz, R. J. Schnitzer & E. Grunberg,
Cancer Research 18, 305 [1958].
7 / . L. Dahl, J. L. Way & R. E. Parks, J. biol. C h e m . 234, 2998 [1959].
8 A. Buckl, Diss. Techn. H o c h s c h u l e M u n i c h [1967].
9 / . Lieberman, A. Romberg & E. S. Simms, J. biol. C h e m . 215, 403 [1955].
10 A. Romberg, I. Lieberman & E. S. Simms, J. biol. C h e m . 215, 389 [1955].
11 F.M. Rosenbloom & J. E. Seegmiller, J. L a b . Clin. M e d . 63, 492 [1964].
12 J.G. Flask in S. P. Colowick & N. O. Raplan: M e t h o d s in Enzymology, A c a d e m i c Press, N e w Y o r k
1963, vol. VI, p . 148.
13 W. N. Relley, I. H. Fox, T. D. Beardmore & I. C Meade, A n n . N e w Y o r k A c a d . Sci. 179, 588 [1971].
14 A. Fyfe, R. A. Miller & T. A. Rrenitsky, J. biol. C h e m . 248, 3801 [1973].
Determination with Dihydroorotate dehydrogenase

Herbert C. Friedmann and G l a d y s K r a k o w
Orotic acid is converted by the inducible flavoprotein d i h y d r o o r o t a t e dehydrogenase (L-5,6-Dihydro-

o r o t a t e : N A D oxidoreductase, E C 1.3.1.14) t o d i h y d r o o r o t a t e . 1
Principle
(1) Orotate + N A D H + H +
dihydroorotate L _ 5_ ihydroorotate + N A D
4 ) D
+
dehydrogenase
The reaction proceeds virtually quantitatively. T h e decrease of the a b s o r p t i o n at 282 n m due to o r o t a t e is

measured.
The equilibrium of the reaction lies in favour of d i h y d r o o r o t i c a c i d ; the equilibrium c o n s t a n t , K is 2
2.4 x 10 . The turnover n u m b e r of the enzyme, based on its flavin content, is a b o u t 1 200. T h e reduction
9
of orotic acid has a rather s h a r p m a x i m u m at p H 6.5. W i t h excess N A D H the reaction proceeds sufficiently
rapidly and orotic acid is almost quantitatively reduced. U n d e r aerobic conditions, however, more N A D H
is oxidized t h a n is expected from the orotic acid c o n t e n t of the s a m p l e . Consequently the reduction of
1,3
orotic acid is determined by the decrease of a b s o r p t i o n at 282 n m . N A D a n d N A D H are isosbestic at this

1
wavelength. The reaction requires the presence of cysteine, a n d because this is slowly oxidized, resulting
in an increase in extinction at 282 n m , a control cuvette m u s t be p r e p a r e d c o n t a i n i n g all the reagents with
the exception of the d i h y d r o o r o t i c acid dehydrogenase. T h e reaction is c o m p l e t e as soon as the extinction
of the experimental cuvette starts to increase at the same rate as the extinction of the control cuvette.
1964 Metabolites: Nucleic acids, Purines, Pyrimidines, Nucleosides, Coenzymes
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at 2 8 2 n m .
Reagents
1. S o d i u m d i h y d r o g e n p h o s p h a t e , 6. R e d u c e d nicotinamide-adenine dinucle
NaH P0 H 0
2 4 2 otide, N A D H
2. D i s o d i u m h y d r o g e n p h o s p h a t e , disodium salt, N A D H - N a ; commercial p r e p
2
Na HP0 -2H 0
2 4 2 aration, see p . 545.
3. C y s t e i n e h y d r o c h l o r i d e 7. D i h y d r o o r o t i c a c i d d e h y d r o g e n a s e
4. S o d i u m h y d r o x i d e , 1 N crystalline from Zymobacterium oroticum*'**.
5. O r o t i c a c i d See A p p e n d i x , p . 1966.
twice recrystallized from w a t e r ; commercial
preparation, see p . 548.
M i x 9 2 m l . 0.2 M N a H P 0 2 4 s o l u t i o n ( 2 . 7 6 g. N a H P 0
2 4 • H 0 m a d e u p t o 100 m l . ) a n d 8 m l .
2
0.2 M N a H P 0 2 4 s o l u t i o n ( 3 . 5 6 g. N a H P 0 - 2 H 0 m a d e u p t o 100 m l . ) .
2 4 2
II. P h o s p h a t e buffer (1 M ; p H 6 . 2 ) :
M i x 81.5 ml. 1 M N a H P 0 2 4 s o l u t i o n ( 1 3 . 8 g. N a H P 0 H 0 m a d e u p t o 100 m l . ) a n d
2 4 2
18.5 m l . 1 M N a H P 0 2 4 s o l u t i o n ( 1 7 . 8 g. N a H P 0 - 2 H 0 m a d e u p t o 100 m l . ) .
2 4 2
III. C y s t e i n e h y d r o c h l o r i d e (0.1 M ) :
D i s s o l v e 7 9 m g . c y s t e i n e h y d r o c h l o r i d e i m m e d i a t e l y b e f o r e u s e in 5 m l . distilled w a t e r .
I V . S o d i u m o r o t a t e (ca. 0 . 0 2 M ) :
To a s u s p e n s i o n o f 0 . 3 1 2 g. o r o t i c a c i d in a little distilled w a t e r , a d d 4 m l . 1 N N a O H a n d
dilute t o 100 m l . w i t h d i s t i l l e d w a t e r .
V . R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 3 m M j S - N A D H ) : D i s s o l v e 8.4 m g .
NADH-Na 2 in distilled w a t e r a n d m a k e u p t o 3 m l .
VI. D i h y d r o o r o t i c acid d e h y d r o g e n a s e :
E x t r a c t t h e c r y s t a l l i n e e n z y m e w i t h i c e - c o l d 0 . 2 M p h o s p h a t e buffer ( S o l u t i o n I). F r e e t h e
extract f r o m slight a m o u n t o f i n s o l u b l e m a t e r i a l b y c e n t r i f u g a t i o n . D i l u t e t h e s u p e r n a t a n t
fluid w i t h c o l d 0.2 M p h o s p h a t e b u f f e r ( s o l u t i o n I) s o t h a t in t h e a s s a y for e n z y m e s t a n d a r d
i z a t i o n t h e e x t i n c t i o n d e c r e a s e s b y a b o u t 0 . 2 in 3 m i n . ( s e e u n d e r " P r o c e d u r e " ) .
Store solutions I a n d II at r o o m t e m p e r a t u r e , IV a n d VI at 4 °C, V at - 1 5 °C a n d p r e p a r e III just be

fore use. A dilute solution of the enzyme (VI) rapidly loses its activity a b o v e a b o u t 20 °C.
Procedure
T h e m e t h o d d e s c r i b e d h e r e for t h e d e t e r m i n a t i o n o f o r o t i c a c i d h a s o n l y b e e n u s e d for p u r e
s o d i u m o r o t a t e s o l u t i o n s . Its u s e for t h e a n a l y s i s o f o r o t i c a c i d in t i s s u e e x t r a c t s after p u r i -
Orotate 1965
fixation, e. g. c h r o m a t o g r a p h i c s e p a r a t i o n o n p a p e r , o r o n D o w e x 5 0 5
or D o w e x 2 , has not
6
been tested.
Standardization of Enzyme
W a v e l e n g t h : 3 4 0 n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 3 m l . R e a d a g a i n s t a r e f e r e n c e c u v e t t e
c o n t a i n i n g d i s t i l l e d w a t e r . P i p e t t e t h e s o l u t i o n s i n t o t h e c u v e t t e s in t h e s t a t e d o r d e r :
0 . 4 m l . buffer ( s o l u t i o n II)
0.2 m l . c y s t e i n e s o l u t i o n (III)
0.3 m l . o r o t a t e s o l u t i o n ( I V )
1.0 m l . d i s t i l l e d w a t e r
e n z y m e solution (VI) a n d distilled water t o 2.9 ml.
M i x a n d a l l o w t o s t a n d for 10 m i n . at 2 0 ° C . A d d
0.1 m l . N A D H s o l u t i o n ( V ) ,
m i x , a n d as r a p i d l y a s p o s s i b l e r e a d t h e e x t i n c t i o n at 3 0 sec. i n t e r v a l s . U s e a n a m o u n t o f e n z y m e
for t h e a s s a y w h i c h g i v e s a d e c r e a s e in e x t i n c t i o n o f a b o u t 0.2 in 3 m i n .
Assay System
W a v e l e n g t h 2 8 2 n m . ; l i g h t p a t h : 1 c m . ; final v o l u m e : 3 m l . ; r o o m t e m p e r a t u r e . R e a d a g a i n s t
reference cuvette c o n t a i n i n g distilled water.
Special care must be taken to ensure that the experimental a n d reference cuvettes c o n t a i n
e x a c t l y t h e s a m e v o l u m e s o f o r o t a t e , c y s t e i n e a n d (later) N A D H s o l u t i o n s .
C o n c e n t r a t i o n in a s s a y
Pipette into cuvettes: Experimental Control
mixture
Buffer ( s o l u t i o n II) 0.4 ml. 0.4 ml. 133 m M

Cysteine solution (III) 0.2 ml. 0.2 ml. 6.7 m M
Sample + water 1.0 m l . 1.0 m l . 0 . 0 1 - 0 . 0 4 /imole
orotate/ml.
E n z y m e solution (IV) according to
standardization + water 1.3 m l .
Distilled water 1.3 m l .
M i x a n d a l l o w t o s t a n d f o r 10 m i n .
N A D H solution (V) 0.1 m l . 0.1 m l .
M i x , read t h e e x t i n c t i o n at 3 0 s e c . o r 1 m i n . i n t e r v a l s u n t i l t h e
e x t i n c t i o n o f t h e e x p e r i m e n t a l c u v e t t e starts t o rise a s r a p i d l y a s
t h a t o f t h e c o n t r o l c u v e t t e . T h i s p o i n t is r e a c h e d w h e n t h e
extinction of the experimental cuvette ceases to decrease.
T h e r e a c t i o n s h o u l d b e c o m p l e t e in 1 0 - 1 5 m i n .
Instead of measuring the extinctions of the experimental and control cuvettes against water,
r e a d t h e e x t i n c t i o n o f t h e c o n t r o l c u v e t t e a g a i n s t t h e e x p e r i m e n t a l c u v e t t e . In t h i s c a s e , t h e
r e a c t i o n is c o m p l e t e w h e n t h e e x t i n c t i o n r e a c h e s a c o n s t a n t v a l u e .
1966 M e t a b o l i t e s : Nucleic acids, Purines, Pyrimidines, Nucleosides, Coenzymes
Calculations
If the experimental and control cuvettes are read against water the extinction change A E is the difference
(final extinction of control cuvette) - (final extinction of experimental cuvette). T h e extinction at 282 n m
of the enzyme contained in the experimental cuvette can be ignored.
U n d e r the conditions described here orotic acid has an extinction coefficient at 282 n m of 7.5 c m . / ^ m o l e .
7 2
Therefore according to e q u a t i o n (2) on p . 312 the orotic acid c o n c e n t r a t i o n of the sample is given b y :
0 4
c = A E x —'•— [^mole/ml.]
v
v = volume of sample.
With experimental mixtures containing 0.04, 0.08 and 0.12 //mole orotic acid the measured extinction
changes were 94 ± 1%, 95 + 1% or 96 + 1% of the calculated value; 0.03 //mole orotic acid gave 9 1 % of the
expected extinction change. T h e limit of sensitivity of the m e t h o d is 0.008 /rniole o r o t a t e / 3 ml. assay mixture.
S o u r c e s o f Error and Specificity
A p a r t from orotate, 5-fluoroorotate is reduced by N A D H a n d d i h y d r o o r o t i c acid dehydrogenase.

5-Fluoroorotate has the same Michaelis c o n s t a n t ( a b o u t 0.13 m M ) , whereas the m a x i m u m velocity is
greater . N o other pyrimidine substrates are k n o w n . Uracil, cytosine, 5-methylcytosine or t h y m i n e
3 1
do
not act as substrates or inhibitors. 5-Methyl o r o t a t e (2 m M ) inhibits the reduction of orotic acid a b o u t 50%.
N a C l (0.2 M ) and sodium p h o s p h a t e (0.2 M ; p H 6.5) inhibit a b o u t 5 5 % a n d 20% respectively.
Appendix
Dihydroorotic Acid Dehydrogenase
The enzyme is prepared as a flavoglobulin from cell-free extracts of Z . oroticum. T h e purification includes
the following s t e p s : repeated t r e a t m e n t with p r o t a m i n e sulphate to r e m o v e nucleic acids, interspersed
4
with a m m o n i u m sulphate fractionation and precipitation by dialysis. T h e nucleic acid-free enzyme cry
stallizes from 0.2 M N a H P 0 2 4 solution in the form of fine, stump-like, orange-yellow needles. It contains
1 mole F M N and F A D as well as 2 mole F e per 62000 g. protein. T h e yellow c o l o u r rapidly disappears on
treatment with excess N A D H or dithionite. It disappears less rapidly a n d less completely on t r e a t m e n t
with excess dihydroorotic acid; the presence of cysteine a n d absence of oxygen are necessary. F o r maxi
m u m activity a preliminary incubation of the enzyme with cysteine is necessary.
References
1 /. Lieberman & A. Kornberg, Biochim. biophys. A c t a 12, 223 [1953].

2 G. Krakow & B. Vennesland, J. biol. C h e m . 236, 142 [1961].
3 H. C. Friedmann & B. Vennesland, J. biol. C h e m . 233, 1 398 [1958].
4 H. C. Friedmann & B. Vennesland, J. biol. C h e m . 235, 1 526 [I960].
4a V. Aleman & P. Handler, J. biol. C h e m . 242, 4 0 8 7 [1967].
5 R. A. Yates & A. B. Pardee, J. biol. C h e m . 221, 743 [1956].
6 P. Reichard, J. biol. C h e m . 197, 391 [1952].
7 D. Shugar & J. 7 . Fox, Biochim. biophys. A c t a 9, 199 [1952].
Coenzyme A
Gerhard Michal and Hans Ulrich Bergmeyer
Coenzyme A is a constituent of practically every living cell. It is the coenzyme for the transfer of acetyl
groups ("activated acetic a c i d " ) a n d for lipid m e t a b o l i s m . It takes p a r t in a large n u m b e r of i m p o r t a n t
1 2
biochemical r e a c t i o n s . Some of these are suitable for the d e t e r m i n a t i o n of C o A . In the catalytic

3
assays
(cf. p . 132), C o A is reformed in the course of the reaction, so that the rate of the reaction is the m e a s u r e
of the C o A c o n t e n t 4 - 8
. These m e t h o d s are generally very sensitive. Procedures using the end-point method
(see p. 103) give absolute values. Their lower sensitivity can be increased by fluorimetric measurements . 9
Choice of M e t h o d
T h e m e t h o d s described here d o n o t give identical values, because their specificity is different.
a) T h e assay with 3-hydroxyacyl-CoA dehydrogenase (HOADH) according to the end-point m e t h o d

measures C o A S H , d e p h o s p h o - C o A , pantetheine, N-(acetyl-/?-alanyl)-cysteamine, N-acetyl-cysteamine
and other c o m p o u n d s which can act as biochemical precursors of C o A . This m e t h o d can be used as
a universal test for all potential CoA-active c o m p o u n d s . T h e assay can be so a r r a n g e d that oxidized
c o m p o u n d s (R-S-S-R) a n d acyl-CoA can also be determined.
b) The assay with phosphotransacetylase (PTA) only determines intact C o A . T h e rate of the reaction
with d e p h o s p h o - C o A is negligible. This is true for b o t h the catalytic a n d e n d - p o i n t m e t h o d s . W h e n
the P T A assay is used as an e n d - p o i n t m e t h o d interference can occur from N a , p h o s p h a t e a n d acetyl-
+
C o A ; the kinetic assay is free from this interference. It is the m e t h o d of choice for biological material
because of its high sensitivity. Differentiation of C o A a n d acetyl-CoA or C o A - S - S - C o A is possible
with the kinetic m e t h o d ; the stoichiometric assay is specific for C o A - S H .
In measurements on samples containing various C o A fragments as well as C o A , lower values are according
ly obtained by the P T A m e t h o d t h a n by the H O A D H m e t h o d . A yeast extract ( b a k e r ' s yeast, 1 part of dried
yeast + 1 2 p a r t s of water, boiled a n d centrifuged) m a y be t a k e n as a n example. F o r C o A - S H , 10.2 n m o l e / m l .
was found in the P T A assay by the end-point m e t h o d , 10.18 n m o l e / m l . by the kinetic P T A assay, a n d
15.6 nmole/ml. by the H O A D H assay.
Application of Method: In biochemistry, the sensitive kinetic m e t h o d with P T A is also used in medical
research.
Normal Values
According to o u r own m e a s u r e m e n t s , the total C o A content* found in rat liver by the H O A D H m e t h o d is

ca. 200 A*g./g. fresh weight, a n d the c o n t e n t of C o A a n d acetyl-CoA* found by the (kinetic) P T A m e t h o d
is ca. 120 //g./g. T h e c o r r e s p o n d i n g values for rat kidney are ca. 75 a n d 50 ^g./g. respectively, a n d for rat
heart a p p r o x . 55 and 35 /*g./g. respectively.
* Calculated as C o A - S H ( M W - 7 6 7 . 6 ) .
Determination of CoA-SH and CoA-S-S-CoA with

3-Hydroxyacyl-CoA Dehydrogenase
3-Hydroxyacyl-CoA dehydrogenase, H O A D H ( L - 3 - H y d r o x y a c y l - C o A : N A D oxidoreductase, E C 1.1.1.35),

catalyses the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by reduced nicotinamide-adenine
dinucleotide . 2
To determine C o A - S H , this is converted into acetoacetyl-CoA with d i k e t e n e : 1 0
CH =2 C - 0
W | | + CoA—SH^CH —CO—CH —CO—S—CoA 3 2
H C—C = 0
2
If CoA-S-S-CoA a n d acetyl-CoA are also to be determined, the sample is t r e a t e d 3

with thioglycollic
acid at p H 9.
(2) R—SH + Co A—S—S—Co A—R—S—S—Co A + Co A—SH
(3) R—S—S—Co A + R—SH—>R—S—S—R+Co A—SH
The reduction proceeds quantitatively only with a large excess of thioglycollic acid. U n d e r these conditions,
any acyl-CoA in the sample transfers its acyl residue to the thioglycollic acid, a n d is thus also converted
into C o A - S H . Reaction (1) is then carried out.
Acetoacetyl-CoA is determined in any case. It is possible to determine C o A - S H a n d the total C o A in the
same mixture.
Principle
(4) CH —CO—CH —CO—S—Co A + NADH +

3 2
+ H +
iiPA™! C H — C H O H — C H — C O — S — C o A + N A D
3 2
+
T h e decrease in the quantity of N A D H , as m e a s u r e d by the change in the extinction at 340 (334, 365) n m ,
is p r o p o r t i o n a l to the co ncentratio n of acetoacetyl-CoA a n d hence of C o A - S H a n d total C o A .
The equilibrium of reaction (4) lies almost completely o n the right at p H ^ 7.5. T h e equilibrium c o n s t a n t 11
at 25 °C is
K = [CH -CHOH-CH -CO-S-CoA3[NAD-]

3 2
[ C H — C O — C H — C O — S — C o A ] [ N A D H ] [H ]
3 2
+ 1
' J
The p H o p t i m u m 1 2
is between 6.0 a n d 7.0, but the instability of the N A D H interferes at excessively acidic
p H values ( < 6.5).
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r c a p a b l e o f a c c u r a t e m e a s u r e m e n t s at 3 4 0 ,
334, or 365 n m ; laboratory centrifuge.
Coenzyme A 1969
Reagents
1. S o d i u m p y r o p h o s p h a t e , A . R., 7. S o d i u m h y d r o g e n c a r b o n a t e , NaHC0 , 3
Na P O 10H O
4 2 7 2 l%(w/v)
2. H y d r o c h l o r i c a c i d , A . R., 1 N 8. 3 - H y d r o x y a c y l - C o A d e h y d r o g e n a s e ,
3. D i k e t e n e * , b . p . 6 8 ° C .
9 2 HOADH
T h e p r e p a r a t i o n t u r n s yellow on storage. It must from pig heart, crystalline suspension in 3.2 M
be distilled, then kept in a refrigerator a n d used ammonium sulphate solution; ^50 U/mg.
within 14 days. (25 °C). C o m m e r c i a l p r e p a r a t i o n s , see p. 474.
4. T h i o g l y c o l l i c a c i d , a p p r o x . 8 0 % , p u r e 9. P e r c h l o r i c a c i d , A . R., 7 0 % ( w / w ) , s p . g r .
5. S o d i u m h y d r o x i d e s o l u t i o n , A . R . , 2 N 1.67
6. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o 10. P o t a s s i u m c a r b o n a t e , A . R . , K C02 3
tide, N A D H
disodium salt, N A D H - N a ; commercial prep
2
arations, see p . 545.
Purity of Reagents
The enzyme p r e p a r a t i o n should contain < 0 . 2 % malate dehydrogenase a n d lactate dehydrogenase

(based on the specific activity of the H O A D H ) . C r o t o n a s e , butyryl-CoA d e h y d r o g e n a s e , a n d deacylases
should not be detectable.
M a k e u p all s o l u t i o n s w i t h f r e s h l y p r e p a r e d d o u b l y distilled w a t e r . Sterilize t h e c o n t a i n e r s

to inhibit the g r o w t h o f m i c r o - o r g a n i s m s .
D i s s o l v e 4 4 . 6 1 g. N a P O 1 0 H O
4 2 7 2 in ca. 7 5 0 m l . w a t e r , a d d a b o u t 8 2 m l . 1 N H C 1 ,
check p H with the glass electrode, a n d m a k e u p to 1 0 0 0 ml. with water.
II. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 12 m M / 2 - N A D H ) :
D i s s o l v e 10.0 m g . N A D H - N a 2 in 1 m l . 1 % N a H C 0 3 solution.
III. 3 - H y d r o x y a c y l - C o A d e h y d r o g e n a s e , H O A D H (2 m g . p r o t e i n / m l . ) :
IV. P e r c h l o r i c a c i d (1 N ) :
D i l u t e 8.7 m l . 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h w a t e r .
V . P o t a s s i u m c a r b o n a t e (5 M ) :
D i s s o l v e 7 0 g. K C 0 2 3 in w a t e r a n d m a k e u p t o 100 m l .
Keep all solutions and suspensions in stoppered containers in a refrigerator at 0 - 4 °C. T h e H O A D H

suspension (III) is stable for at least 12 m o n t h s under these conditions. M a k e u p fresh N A D H solution (II)
weekly. The other solutions keep indefinitely.
* e. g. from D r . T h . Schuchardt, M u n i c h , G e r m a n y .
Procedure
Collection of sample: Take tissue samples from laboratory animals with "quick-freeze" t o n g s
(cf. " C e l l a n d T i s s u e D i s i n t e g r a t i o n " , p . 4 0 0 ) .
Deproteinization: W o r k at t h e h i g h e s t c o n c e n t r a t i o n p o s s i b l e b e c a u s e o f t h e l o w C o A c o n t e n t
of biological material. Deproteinization 1 + 3 with 1 N H C 1 0 4 solution (IV), neutralization
with 5 N K C 0 2 3 s o l u t i o n ( V ) . A v o i d o v e r - t i t r a t i o n o n n e u t r a l i z a t i o n , p r e f e r a b l y adjust t o
p H 6.0.
Stability of sample: C o A is m o s t s t a b l e in t h e p H r a n g e 4 . 5 - 6 . 0 . A c c o r d i n g t o results w i t h

this a s s a y C o A is s t a b l e f o r at least 2 4 h o u r s at + 4 ° C . C o A d e c o m p o s e s r a p i d l y in a l k a l i n e
s o l u t i o n ; s u c h c o n d i t i o n s s h o u l d b e a v o i d e d in t h e t r e a t m e n t o f t h e s a m p l e .
Preliminary treatment of sample
For determination of total CoA: D i s s o l v e C o A preparations in water to give a concentration

o f a b o u t 0.5 m g . / m l . , a n d d i l u t e C o A s o l u t i o n s a c c o r d i n g l y ; c o o l 10 m l . in a test t u b e t o 0 ° C ,
a d d 0.01 m l . o f t h i o g l y c o l l i c a c i d , a d j u s t t o p H 9 . 0 ( i n d i c a t o r p a p e r o r g l a s s e l e c t r o d e ) w i t h
a p p r o x . 0.1 m l . 2 N N a O H , a n d a l l o w t o s t a n d for 15 m i n . in a n i c e b a t h . A d d 0.01 m l . o f
d i k e t e n e a n d s h a k e v i g o r o u s l y f o r 3 m i n . at 0 ° C . T h e d i k e t e n e d r o p l e t s d i s s o l v e . C h e c k p H
e v e r y m i n u t e a n d k e e p at 7.3 ( i n d i c a t o r p a p e r o r g l a s s e l e c t r o d e ) .
For determination of CoA-SH: D i s s o l v e C o A preparations in distilled water t o give a c o n

c e n t r a t i o n o f a b o u t 0 . 5 m g . / m l . , a n d d i l u t e C o A s o l u t i o n s a c c o r d i n g l y ; c o o l 10 m l . t o 0 ° C
a n d adjust p H t o 8 . 0 ( i n d i c a t o r p a p e r o r g l a s s e l e c t r o d e ) w i t h 2 N N a O H . A d d 0.01 m l . o f
d i k e t e n e a n d s h a k e v i g o r o u s l y f o r 3 m i n . at 0 ° C , k e e p i n g t h e p H at 7.3 ( i n d i c a t o r p a p e r o r g l a s s
electrode).
T h e v o l u m e o f t h e m i x t u r e c a n b e c a l c u l a t e d b y a d d i t i o n o f all t h e v o l u m e s a d d e d . F o r s m a l l
quantities o f sample, the v o l u m e o f the solution m a y be decreased to about 3.0 m l .
Coenzyme A 1971
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 2 . 2 6 m l . ; r o o m t e m p e r
In t h e c a s e o f s a m p l e s o l u t i o n s w i t h l o w c o n c e n t r a t i o n s , u p t o 1 m l . m a y b e u s e d ( w i t h c o r r e s
p o n d i n g l y l e s s buffer s o l u t i o n ) .
Buffer s o l u t i o n (I) 2.00 ml. 88 m M

Sample, pretreated 0.20 ml. u p t o 0.11 m M
N A D H solution (II) 0.05 ml. 0.27 m M
M i x , read extinction E . x
H O A D H suspension (III) 0.01 m l . 9/*g./ml. = 4 5 0 m U / m l .
M i x ; after r e a c t i o n s t o p s ( 2 t o 3 m i n . ) r e a d e x t i n c t i o n
E . E -E
2 l 2 = AE.
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f t h e e n z y m e b y a d d i t i o n o f a further
0.01 m l . o f H O A D H s u s p e n s i o n (III), a n d s u b t r a c t t h e result f r o m A E .
Calculations
T h e reaction proceeds stoichiometrically u n d e r the above conditions. F o r m u l a (2) on p . 312 is therefore

valid. T h e result is obtained in /rniole C o A / m l . of sample after p r e t r e a t m e n t . W i t h 0.20 ml. sample in the
assay mixture, the following relationships are valid for the C o A c o n c e n t r a t i o n in the s a m p l e :
c = A E x 1.852 A E x 1.817 JEx3.32 [/imole/ml.]

c = A E x 1.422 AEx 1.394 AEx 2.55 [mg./ml.]
However, these values m u s t be multiplied by the factor
p ^ s a m p l e solution after pretreatment
^ s a m p l e solution before pretreatment
A further factor m u s t be applied in the case of deproteinized tissue e x t r a c t s :
tissue weight P , .
A/
v 5 x
Too HC1
° 4
^ p v
after neutralisation ^ ^ tissue weight
x y j y j
P = percentage of the v o l u m e of fluid (v/v) in the tissue,

D = specific gravity of the tissue, which can be taken as unity in most cases for the sake of simplicity.
A s t a n d a r d deviation of s = 0 . 8 /ig./cuvette a n d a coefficient of variation of 1.5% were found with p u r e

substance (54 /tg./cuvette).
N o r m a l V a l u e s , see p . 1 9 6 7 .
Interference in assay technique: T h e presence of other dehydrogenases in the H O A D H preparation

results in false values. Incomplete solution of the diketene droplets d u e to t o o short or not intensive
enough shaking results in areas of the assay mixture not being at the correct p H . Failure to maintain
the correct p H during the p r e t r e a t m e n t causes incomplete reaction as well as incorrect p H in the assay
mixture.
S p ec ific ity of M e t h o d
Decker 2,
reports the following t u r n o v e r n u m b e r s (mole x m i n " 1
per 100000 g protein) for an enzyme
obtained from pig heart for the acetoacetyl derivatives: C o A 20900, d e p h o s p h o - C o A 13200, pantetheine
11700, N-(acetyl-/?-alanyl)cysteamine 4630, N-acetylcysteamine 3900. Similar t u r n o v e r n u m b e r s were
found for a n u m b e r of synthetic m o d e l substances (2'-deoxybisnorpantetheine derivatives). T h u s some
fragments of C o A also react in the d e t e r m i n a t i o n with H O A D H . This can generally be recognised by a
"creep r e a c t i o n " after the reaction with C o A has occurred. E x t r a p o l a t i o n is not generally possible,
particularly when a mixture of the fragments is present.
P o s s i b i l i t i e s for Increased S e n s i t i v i t y
The sensitivity can be increased by a factor of a b o u t one h u n d r e d if the d e t e r m i n a t i o n is carried out

fluorimetrically . 9
R e f e r e n c e s , see p . 1 9 8 1 .
Determination of CoA-SH with Phosphotransacetylase

End-Point Method
T h e determination of C o A with phosphotransacetylase, P T A ( A c e t y l - C o A : o r t h o p h o s p h a t e acetyl-

transferase, E C 2.3.1.8) by the end-point m e t h o d (see p. 103) was developed by E. R. Stadtman . 13
PTA
catalyses the reversible transfer of acetyl g r o u p s between p h o s p h a t e a n d C o A 1 4 1 5
.
Principle
(7) CoA-SH + C H C O - O P 0 H
3 3 2 ^ = CoA-S-COCH 3 + H P0
3 4
Acetyl-CoA absorbs m o r e strongly than C o A at 233 n m . As was shown by W. Seubert

1 6 11
with the model
substance S-acetyl-N-succinylcysteamine, the extinction coefficient at 233 n m is e 233 = 4.44 x 10 6
cm. /
2
mole. T h e increase in extinction at 233 n m is measured. Only C o A - S H reacts, since no reducing reagent
is a d d e d ; acetyl-CoA does n o t react.
Coenzyme A 1973
The equilibrium lies on the r i g h t at p H 8.0 and 28 ° C :

7
yj_ [Ac-CoA] [phosphate]

[acetylphosphate] [CoA]
T h e reaction can be m a d e practically quantitative by the use of an excess of acetylphosphate. T h e assay

mixture should be < 0 . 1 m M in H P O j -
a n d acetyl-CoA a n d < 10 m M in N a . T h e enzyme requires K
+ +
or NH4 ions for a c t i v a t i o n . T h e alkali c o n c e n t r a t i o n d u e to the a m m o n i u m sulphate content of the enzyme

18
suspension and the K +

content of the acetylphosphate lie in the range of m a x i m u m activation.
Equipment
S p e c t r o p h o t o m e t e r c a p a b l e o f a c c u r a t e m e a s u r e m e n t s at 2 3 3 n m .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 4. P h o s p h o t r a n s a c e t y l a s e , P T A
2. Acetylphosphate from CI. kluyveri, crystalline suspension in
potassium-lithium salt, C H 0 P K L i .
2 3 4 Com 3.2 M a m m o n i u m sulphate solution; ^ 1 0 0 0
mercial p r e p a r a t i o n s , see p . 524. U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n s , see
3. H y d r o c h l o r i c a c i d , A . R., 1 N p. 507.
Purity of Reagents
T h e P T A should contain less t h a n 0.01 % deacylase, acetyl-CoA thiolases, a n d p h o s p h a t a s e s . The activity

with d e p h o s p h o - C o A as the substrate should not exceed 1% of the activity with C o A .
M a k e u p all s o l u t i o n s w i t h f r e s h l y p r e p a r e d d o u b l y d i s t i l l e d w a t e r . Sterilize t h e c o n t a i n e r s t o
inhibit the g r o w t h o f m i c r o - o r g a n i s m s .
I. Tris buffer (0.1 M ; p H 7 . 6 ) :
Dissolve 12.1 g. t r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e in ca. 5 0 0 m l . w a t e r , adjust to
p H 7.6 ( g l a s s e l e c t r o d e ) w i t h a b o u t 7 0 m l . 1 N h y d r o c h l o r i c a c i d , a n d m a k e u p t o 1 0 0 0 m l .
with water.
II. A c e t y l p h o s p h a t e (0.1 M ) :
D i s s o l v e 2 0 . 3 m g . a c e t y l p h o s p h a t e K L i salt, in 1 m l . w a t e r .
III. P h o s p h o t r a n s a c e t y l a s e , P T A (0.1 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n a s r e q u i r e d w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n ( p H ^ 8 . 0 )
t o 1 m g . p r o t e i n / m l . F r o m this d i l u t i o n , m a k e u p t h e d a y ' s r e q u i r e m e n t b y further
dilution 1 + 9 .
K e e p all solutions and suspensions in stoppered containers in a refrigerator at 0 - 4 °C. K e e p the acetyl
p h o s p h a t e solution (II) cool d u r i n g the analysis, a n d p r e p a r e freshly every 2 days. T h e 1 mg./ml. enzyme
suspension is stable for u p to a b o u t 6 m o n t h s at 0 - 4 °C.
Procedure
Collection of sample: T h e d e t e r m i n a t i o n is s u i t a b l e f o r t h o s e t i s s u e e x t r a c t s w i t h fairly h i g h

C o A c o n t e n t s a n d in t h e a b s e n c e o f i n t e r f e r i n g f a c t o r s ( e . g . f o r y e a s t e x t r a c t s ; s e e p . 1 9 6 7 ) .
T h e m o s t s u i t a b l e C o A c o n t e n t f o r s o l u t i o n s is a r o u n d 0 . 5 m g . / m l . W i t h l o w e r c o n c e n t r a t i o n s ,
the q u a n t i t y o f s a m p l e m a y b e i n c r e a s e d , a n d c o r r e s p o n d i n g l y l e s s b u f f e r is t h e n u s e d .
Stability of sample: C o A is m o s t s t a b l e i n t h e p H r a n g e 4 . 5 - 6 . 0 . I n t h i s r a n g e t h e c o n c e n t r a t i o n
o f C o A s o l u t i o n s ( d e t e r m i n e d b y t h i s m e t h o d ) d e c r e a s e s b y a b o u t 2 % in 2 4 hr. at 4 ° C a n d b y
a b o u t 6 % at r o o m temperature.
Assay System
W a v e l e n g t h : 2 3 3 n m ; l i g h t p a t h : 1 c m . ( q u a r t z c u v e t t e s ) ; final v o l u m e : 2 . 3 1 m l . ; r o o m t e m p e r
ature. M e a s u r e m e n t a g a i n s t a c u v e t t e c o n t a i n i n g tris buffer ( s o l u t i o n I ) ; in t h e c a s e o f s a m p l e s
with high extinctions, u s e 0.2 m l . in the reference cuvette.

Acetylphosphate solution (H) 0.10 ml. 4.3 m M
Sample 0.20 ml. up to 0.09 m M
M i x a n d read extinction E t
P T A suspension (III) 0.01 m l . 0.43 / i g . / m l . = 4 3 0 m U / m l .
Mix. F o l l o w increase in extinction until the reaction

s t o p s , t h e n r e a d E . E — Ej = A E.
2 2
D e t e r m i n e t h e i n c r e a s e i n e x t i n c t i o n d u e t o t h e a d d i t i o n o f t h e e n z y m e b y a d d i t i o n o f a further
0.01 m l . o f P T A s u s p e n s i o n ( I I I ) , a n d s u b t r a c t t h e result f r o m A E.
Calculations
T h e reaction proceeds stoichiometrically u n d e r t h e a b o v e conditions. F o r m u l a (2, 3) o n p . 312 is there

fore valid, the value t o be used as t h e extinction coefficient being e 3 3 = 4.44 c m / ^ m o l e . T h e C o A con
2
2
centration of the sample is
c = J E x 2 . 6 0 [/miole/ml.]
c = z t E x 2 . 0 0 [mg./ml.]
Where biological material h a s been analysed, it is necessary t o multiply by a factor (eqn. (6), p . 1971)
that takes the dilutions into a c c o u n t .
W i t h p u r e substance (160 /zg./cuvette) a s t a n d a r d deviation of s = 1.6 //g./cuvette was found. T h e coefficient

of variation is 1 % .
Coenzyme A 1975
N o r m a l Values, see p. 1967.
Interference in assay technique: Interference often results from an excessively high extinction at 233 n m ,
particularly in the case of biological samples.
Na +
concentrations a b o v e 10 m M a n d large quantities of L i +
cause interference , but the L i
7 +
c o n t e n t of
the acetylphosphate a d d e d has n o appreciable effect. T h e inhibitor effect can be c o m p e n s a t e d for by
increased K +
or NH4 concentrations .
18
Higher c o n c e n t r a t i o n s of acetyl-CoA or p h o s p h a t e interfere by displacement of the e q u i l i b r i u m , with 19
the result that the reaction n o longer proceeds to completion. W i t h c o n c e n t r a t i o n s of one of these c o m p o u n d s
in excess of 3 m M in the assay mixture (initial c o n c e n t r a t i o n of the o t h e r c o m p o u n d a s s u m e d to be 0), the
error exceeds 1%. If b o t h c o m p o u n d s are present, the e r r o r can be calculated from the law of mass action.
It can be decreased by increasing the acetylphosphate concentration.
In alkaline conditions glutathione accepts acetyl g r o u p s from acetyl-CoA by n o n - e n z y m a t i c t r a n s f e r . 19
In the assay described here ( p H 7.6) this reaction is n o t very r a p i d . A c c o r d i n g t o

20 1 9
the rate of the transfer
is o p t i m u m at p H 8 . 1 - 9 . 0 , a n d at p H < 7.0 is practically zero. Even when glutathione is a d d e d to the
assay mixture in the form of i m p u r e C o A solutions or tissue extracts, the e r r o r d u e to this n o n - e n z y m a t i c
reaction can be disregarded because of the short reaction times.
P T A from CI. kluyveri is specific for C o A - S H . In the absence of reducing substances, P T A does n o t react
with oxidized C o A , d e a m i n o - C o A (N. O. Kaplan, cited i n ) , or acyl-CoA. A c c o r d i n g t o , m o r e o v e r ,
2 0 7 2 1
it does not react with d e p h o s p h o - C o A . W i t h relatively large quantities (10 ug./determination) of crystalline
e n z y m e , however, we have observed a very distinct reaction with d e p h o s p h o - C o A , as h a d already been
22
described for less highly purified e n z y m e . If small quantities of enzyme are used as indicated, the inter
23
ference is insignificant; in any case it can be eliminated by extrapolation. T h e activity of the P T A t o w a r d s

d e p h o s p h o - C o A is p r o b a b l y due to a slight non-specificity of the enzyme, a n d n o t to a c o n t a m i n a n t ; the
relative activity with d e p h o s p h o - C o A does n o t decrease with progressive purification of the enzyme.
The enzyme from CI. kluyveri does n o t react with 4 ' - p h o s p h o p a n t e t h e i n e . Phosphotransacetylases from
20
other micro-organisms can also react with p a n t h e t h e i n e . 24
References, see p. 1981.
Catalytic Assay*
In the presence of arsenate, C o A can be determined with p h o s p h o t r a n s a c e t y l a s e by a kinetic m e t h o d . 1 4
T h e kinetic d e t e r m i n a t i o n described b e l o w is simpler to carry out a n d can be followed continuously. C o A

9
is converted into acetyl-CoA with p h o s p h o t r a n s a c e t y l a s e as described a b o v e . T h e acetyl-CoA is converted

into citrate with oxaloacetate (citrate synthase, C S ; Citrate oxaloacetate-lyase, E C 4.1.3.7), with re
generation of C o A , which re-enters the cycle. T h e rate of t r a n s f o r m a t i o n of C o A is indicated by the N A D -
dependent formation of oxaloacetate (malate dehydrogenase, M D H ; L - M a l a t e : N A D H oxidoreductase,
E C 1.1.1.37).
* In collaboration with H . Mollering.

Principle
(8) Acetylphosphate+CoA-SH , P h o s
P °-
h
, Acetyl-S-CoA + P h o s p h a t e
transacetvlase r
()9 Acetyl-S-Co A + Oxaloacetate + H 0 2 ^ — • Citrate + C o A - S H

synthase
(10) Malate + N A D +
, m a l a t e
» Oxaloacetate + N A D H + H +
dehydrogenase
See also scheme on p . 132.

The rate of formation of N A D H , as measured by the rate of the increase in extinction at 340 (334,365) n m ,
is p r o p o r t i o n a l to the q u a n t i t y of C o A a n d acetyl-CoA (assuming c o n s t a n t conditions). T h e measure
ments are carried out in the presence of dithiothreitol, which increases the reaction rate a n d also causes
CoA-S-S-CoA to be included in the d e t e r m i n a t i o n .
To differentiate the two forms of C o A , the sample can be pretreated with N - e t h y l m a l e i m i d e 25
in a second
assay; excess is removed by subsequent addition of dithiothreitol.
The activity of the indicator enzyme M D H m u s t not be limiting. Increasing quantities of enzyme lead to a
m a x i m u m in the reaction rate, while excessively large quantities of M D H result in a decrease again. T h e
o p t i m u m a m o u n t of M D H is 9.8 U / m l . assay mixture is o p t i m u m at 1.3 U CS/ml. (limiting).
The same result is obtained regardless of whether P T A or CS is chosen as limiting, but the less pure enzyme
should be a d d e d in limiting a m o u n t s . T h e q u a n t i t y of the other enzymes should n o t be t o o high, since the
reaction rate otherwise decreases. F o r 0 . 0 8 - 1 . 3 n m o l e C o A in the d e t e r m i n a t i o n , we established the
o p t i m u m conditions as 9.8 U M D H / m l . , 7 U P T A / m l . a n d 1.3 CS/ml. of assay mixture. There is a limit
to the possibility of increasing the sensitivity of the d e t e r m i n a t i o n by a general increase in the quantities
of enzyme. T h e quantities of N-ethylmaleimide a n d dithiothreitol a d d e d a n d the reaction times used are
adequate.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r c a p a b l e o f a c c u r a t e m e a s u r e m e n t s at 3 3 4 , 3 4 0 ,
o r 3 6 5 n m , p r e f e r a b l y w i t h a r e c o r d i n g d e v i c e . C o n s t a n t - t e m p e r a t u r e cell h o l d e r . L a b o r a t o r y
centrifuge.
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 7. P h o s p h o t r a n s a c e t y l a s e , P T A
2. L-( — ) - M a l i c a c i d , p u r e from CI. kluyveri, crystalline suspension in
3. P o t a s s i u m h y d r o x i d e s o l u t i o n , A . R., 1 N 3.2 M a m m o n i u m sulphate solution; ^ 1000 U /
4. Acetylphosphate mg. (25 °C). C o m m e r c i a l p r e p a r a t i o n s , see p. 507.
potassium-lithium salt, C H 0 P K L i .
2 3 4 Com 8. C i t r a t e s y n t h a s e , C S
mercial p r e p a r a t i o n s , see p. 524. from pig heart, crystalline suspension in 3.2 M
5. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , NAD ammonium sulphate solution; ^110 U/mg.
free acid. C o m m e r c i a l p r e p a r a t i o n s , see p. 545. (25 °C). C o m m e r c i a l p r e p a r a t i o n s , see p. 443.
6. M a l a t e d e h y d r o g e n a s e , MDH 9. P e r c h l o r i c a c i d , A . R., 7 0 % ( w / w ) , s p . gr.
from pig h e a r t ; suspension in 3.2 M a m m o n i u m 1.67
sulphate s o l u t i o n ; ^ 1100 U / m g . (25 °C). C o m 10. P o t a s s i u m c a r b o n a t e , A . R .
mercial p r e p a r a t i o n s , see p . 485.
Coenzyme A 1977
11. C o e n z y m e A , C o A 12. D i t h i o t h r e i t o l
free acid; d e t e r m i n e the content in the assay Cleland's reagent, pure
with P T A as described on p . 1972. C o m m e r c i a l 13. N-Ethylmaleimide
p r e p a r a t i o n s , see p . 528.
Purity of Reagents
Interfering c o n t a m i n a n t s of the enzymes are mainly N A D H oxidase, L D H , a n d oxaloacetate decarboxy

lase. T h e enzyme used should contain not m o r e t h a n 0.01 % of each of these. T h e C o A p r e p a r a t i o n used
as the s t a n d a r d should be as p u r e as possible ( ^ 8 0 % C o A ) a n d should be substantially free from de-
p h o s p h o - C o A , since this also reacts in the kinetic determination, t h o u g h very slowly (see below).
M a k e u p all s o l u t i o n s w i t h freshly p r e p a r e d d o u b l y d i s t i l l e d w a t e r . Sterilize the c o n t a i n e r s t o

i n h i b i t the g r o w t h o f m i c r o o r g a n i s m s .
I. T r i e t h a n o l a m i n e / m a l a t e ( 0 . 2 M t r i e t h a n o l a m i n e ; 15 m M m a l a t e ) :
D i s s o l v e 3.71 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e a n d 201 m g . m a l i c a c i d in a p p r o x . 5 0 m l .
o f w a t e r , a d j u s t t o p H 8.0 w i t h c a . 16 m l . 1 N p o t a s s i u m h y d r o x i d e s o l u t i o n , a n d m a k e
u p t o 100 m l . w i t h w a t e r .
II. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e / a c e t y l p h o s p h a t e (ca. 15 m M N A D ; 5 0 m M a c e t y l
phosphate) :
D i s s o l v e 120 m g . N A D a n d 100 m g . C H 0 P K L i in 1 0 . 0 m l . w a t e r .
2 3 4
III. M a l a t e d e h y d r o g e n a s e , M D H ( 1 4 0 0 U / m l . * ) :
IV. Phosphotransacetylase, P T A (1000 U / m l . * ) :
V. Citrate synthase, C S (140 U / m l . * ) :
V I . S t a n d a r d C o A s o l u t i o n ( 1 0 pg. C o A / m l . ) :
D i s s o l v e 5 m g . C o A ( e x a c t c o n t e n t a n a l y s e d b y the e n d - p o i n t m e t h o d ) in 2 5 . 0 m l .
w a t e r , a n d d i l u t e 5 m l . this s o l u t i o n t o 100 m l . w i t h w a t e r .
V I I . P e r c h l o r i c a c i d (1 N ) :
D i l u t e 8.7 m l . 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h w a t e r .
V I I I . P o t a s s i u m c a r b o n a t e (5 M ) :
D i s s o l v e 7 0 g. K C 0 2 3 in w a t e r a n d m a k e u p t o 100 m l .
IX. Dithiothreitol (20 m M ) :
D i s s o l v e 12.3 m l . d i t h i o t h r e i t o l in 4 m l . w a t e r .
X. N-Ethylmaleimide (20 m M ) :
D i s s o l v e 10 m g . o f N - e t h y l m a l e i m i d e in 4 m l . w a t e r .
* It is necessary to w o r k in U / m l (instead of mg/ml), since the activity ratio of the enzymes is decisive for
the success of the d e t e r m i n a t i o n .
K e e p all solutions a n d suspensions in a refrigerator at 0 t o 4 °C. P r e p a r e fresh solutions I, II, I X , a n d X

daily. U s e solution V I within a few h o u r s . T h e M D H (III) a n d C S (V) enzyme suspensions are stable for
12 m o n t h s a n d t h e P T A suspension (IV) for 6 m o n t h s ; the other solutions keep indefinitely.
Procedure
Collection of sample: Take tissue samples from laboratory animals with "quick-freeze" t o n g s
(cf. " C e l l a n d T i s s u e D i s i n t e g r a t i o n " , p . 4 0 0 ) ; adjust o t h e r s o l u t i o n s a s far a s p o s s i b l e t o
a b o u t 10 pg. C o A / m l .
Deproteinization: Deproteinize the frozen samples 1 + 3 with 1 N H C 1 0 4 solution (VII), a n d

neutralize the extract with 5 N K C0
2 3 (solution VIII); avoid over-titration, preferably
adjusting to p H 6.0.
Stability of sample: C o A is m o s t s t a b l e i n t h e p H r a n g e b e t w e e n 4 . 5 a n d 6. In t h i s r a n g e , t h e
c o n c e n t r a t i o n o f C o A s o l u t i o n s ( m e a s u r e d b y t h i s m e t h o d ) d e c r e a s e s b y a b o u t 2 % in 2 4 hr. a t
4 ° C a n d b y a b o u t 6 % at r o o m t e m p e r a t u r e . W i t h m o r e d i l u t e s o l u t i o n s o x i d a t i o n o c c u r s
rapidly.
Coenzyme A 1979
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 3 0 m l . ; t e m p e r a t u r e :
25 °C ( t h e r m o s t a t ) . R e a d a g a i n s t air.
M a k e up a blank with water instead o f s a m p l e and a C o A standard with 0.05 ml. standard
C o A s o l u t i o n ( V I ) i n s t e a d o f s a m p l e for e a c h series o f d e t e r m i n a t i o n s . Treat b o t h o f t h e s e
m i x t u r e in a c c o r d a n c e w i t h t h e m e t h o d for C o A - S H + a c e t y l - C o A + C o A - S - S - C o A . The
s t a n d a r d c o n t a i n s 0.5 pg. C o A ( 0 . 6 5 3 n m o l e ) , a n d t h e C o A c o n c e n t r a t i o n in the d e t e r m i n a t i o n
is 0 . 3 0 2 pM.
CoA-SH + Acetyl-CoA
acetyl-CoA + CoA- Concentration
-f CoA- S-S-CoA in a s s a y m i x t u r e
S-S-CoA
Sample 0.05 ml. 0.05 ml. u p t o 6 0 pM CoA

0.05 ml. 0.46 m M
N-Ethylmaleimide solution (X) N-ethylmaleimide
0.30 ml. 0.20 ml.
Water
Only for the determination of acetyl-CoA + CoA-S-S-CoA: Mix
well a n d i n c u b a t e for 10 m i n .
Triethanolamine/
malate solution (I) 1.50 m l . 1.50 m l . 140 m M
triethanolamine;
10.5 m M m a l a t e
Dithiothreitol solution (IX) 0.05 ml. 0.10 ml. 0.46 (0.93) m M
dithiothreitol
M i x a n d i n c u b a t e for 15 m i n .
NAD/acetylphosphate
solution (II) 0.20 ml. 0.20 ml. 1.4 m M N A D ;
4.6 m M acetylphosphate
M D H suspension (III) 0.015 ml. 0.015 ml. 9.8 U / m l .
P T A suspension (IV) 0.015 ml. 0.015 ml. 7 U/ml.
M i x , a n d wait until reaction s t o p s (ca. 5 min.).
CS suspension (V) 0.02 ml. 0.02 ml. 1.3 U / m l .
M i x , a n d start m e a s u r e m e n t o f t h e i n c r e a s e in e x t i n c t i o n w i t h
t i m e after a p p r o x . 1 m i n . , p r e f e r a b l y w i t h a p e n r e c o r d e r . A l t e r
natively, read extinction E 2 after e x a c t l y a further 15 m i n . E — 2
- Ej = A E. Subtract A E o f the blank from A E o f the standard

a n d o f t h e s a m p l e . C o n v e r t t o J E / m i n . (A E / m i n . ) S a m p l e and
(A E / m i n . ) S t are u s e d in t h e c a l c u l a t i o n .
Calculations
T h e reaction proceeds linearly for 15 min. u p to extinction changes of 0.570 at 340 n m or 0.300 at 365 n m :
the reaction rate A E/min is p r o p o r t i o n a l to the quantity of C o A . T h e C o A c o n c e n t r a t i o n of the sample is
therefore given by
i
c= x (^ / We [nmole/ml]
n Ky E m i n
C s t
(A E / m i n )L.
f /I F./min St
c S t = concentration of the s t a n d a r d C o A solution in nmole/ml.

Where biological material has been analysed, it is necessary to multiply by a factor (eqn. (6), p . 1971) that
takes the dilutions into account.
A s t a n d a r d deviation of s—0.0097 fig. C o A/cuvette was found with pure substance (0.539 pg. CoA/cuvette).
T h e coefficient of variation is 1.8%.
N o r m a l V a l u e s , see p. 1 9 6 7 .
Interference in assay technique: Insufficient purity of the enzymes results in false values, which are usually
too low. If the enzymes used are of the stated purity (see p . 1977), additions of the following c o m p o u n d s
at the a m o u n t s shown (in a m o l a r relationship to a m o u n t of C o A present) d o no interfere: pyruvate
(10-fold), lactate (50-fold), oxaloacetate (5-fold) a n d citrate (10-fold).
With t o o high a m o u n t s of C o A the A E/15 min. at 340 n m is > 0.570/15 min. and the curves are non-linear
(decreasing rate of reaction). T h e assay must then be repeated with smaller a m o u n t s of C o A . T h e u p p e r
limit for the reaction rate c o r r e s p o n d s to ca. 1.3 x 1 0 ~ mole/cuvette or 10 fig. C o A / c u v e t t e ; a
9
fifteenth
of this a m o u n t can still be measured accurately.
In the end-point m e t h o d by this p r i n c i p l e ' 26 27
it is necessary to apply a correction because of the difference
between the increase in N A D H a n d the C o A converted (see p . 1 1 2 - 1 1 7 a n d 1527). This correction is
not necessary with the kinetic assay, because it is standardized with a C o A s t a n d a r d preparation a n d in
addition there is only a very small conversion.
The smaller a m o u n t s of sample used in the kinetic m e t h o d in c o m p a r i s o n to the end-point m e t h o d means
that most of the possible sources of error are eliminated. In particular the interference from p h o s p h a t e
or acetyl-CoA does not play any role in the kinetic m e t h o d .
In addition to C o A - S H , oxidized C o A (CoA-S-S-CoA) and acetyl-CoA also react, the reaction rates
being equal. The separate determination of C o A - S H on the one h a n d a n d C o A - S - S - C o A and acetyl-CoA
on the other is possible by the m e t h o d described, but not the differentiation of C o A - S - S - C o A and acetyl-
C o A , which is less i m p o r t a n t in any case, since the c o n c e n t r a t i o n of C o A - S - S - C o A is usually low.
Specificity in relation to other derivatives of C o A is almost complete. D e p h o s p h o - C o A reacts at less t h a n
1 % of the rate of C o A , a n d 4 ' - p h o s p h o p a n t e t h e i n e does not react at all. D e a m i n o - C o A (N. O. Kaplan,
cited i n ) also does not react.
7
Simplification
If one dispenses with the determination of C o A - S - S - C o A and the separate determination of acetyl-CoA
and C o A - S H , the addition of dithiothreitol can be omitted a n d the m e a s u r e m e n t s m a d e directly (see ) 9
with a consequent saving in time.

Coenzyme A 1981
References
1 F. Lynen, E. Reichert & L. Rueff, Liebigs A n n . 578, 1 [1951].

2 F. Lynen, Federat. P r o c . 12, 683 [1953].
3 K. Decker, D i e aktivierte Essigsaure, F . E n k e , Stuttgart 1959.
4 N. O. Kaplan & F. Lipmann, J. biol. C h e m . 174, 37 [1948].
5 G. D. Novelli in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c Press, N e w York
1957, Vol. I l l , p . 916.
6 F. Lipmann & L. C Tattle, J. biol. C h e m . 159, 21 [1945].
7 E. R. Stadtman in S. P. Colowick & N. O. Kaplan: M e t h o d s in E n z y m o l o g y . A c a d e m i c Press, N e w York
1955, Vol. I, p . 596.
8 R. W. von Korff, J. biol. C h e m . 200, 401 [1953].
9 G. Michal, Fresenius' Ztschr. analyt. C h e m . 243, 649 [1968].
10 K. Decker & F. Lynen, 3rd. C o n g r . Intern. Biochim., C o m m u n . 36, Brussels 1955.
11 F. Lynen & O. Wieland in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c Press,
N e w York 1955, Vol. I, p . 566.
12 H. R. Mahler in C. Long: Biochemists H a n d b o o k . Spon, L o n d o n 1961, p. 332.
1
13 E. R. Stadtman, unpublished c o m m u n i c a t i o n .
14 E. R. Stadtman, G. D. Novelli & F. Lipman, J. biol. C h e m . 191, 365 [1951].
15 H. Chantrenne & F. Lipmann, J. biol. C h e m . 187, 757 [1950].
16 E.R. Stadtman, J. cell. c o m p . Physiol. 41, 89 [1953].
17 W. Seubert, personal c o m m u n i c a t i o n .
18 E. R. Stadtman, J. biol. C h e m . 196, 527 [1952].
20 G. Michal, unpublished c o m m u n i c a t i o n .
21 T. P. Wang, L. Shuster & N. O. Kaplan, J. Amer. chem. Soc. 74, 3204 [1952].
22 H. U. Bergmeyer, H. Klotzsch & G. Lang, Angew. C h e m . 72, 807 [1961].
23 T. P. Wang in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c Press, New York
1955, Vol. II, p . 649.
24 G. M. Brown, F e d e r a t . Proc. 16, 159 [1957].
25 / . B. Allred&D. G. Guy, A n a l . Biochem. 29, 293 [1969].
26 W. Bucket & H. Eggerer, Biochem. Z. 343, 29 [1965].
27 H. U. Bergmeyer & H. Mollering, Biochem. Z. 344, 167 [1966].
Fluorimetric Assay
Peter Bryan Garland
With small a m o u n t s of sample or with a low C o A content in the sample it is necessary to use fluorimetric
assay m e t h o d s to obtain the required sensitivity. If accurate s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s are
required a m o u n t s of C o A above 10 n m o l e are necessary because the sensitivity of commercial spectro
p h o t o m e t e r s is limited by the b a c k g r o u n d noise of ca. ± 0.002 extinction units. Higher sensitivity can be
obtained only with the m o r e expensive d o u b l e - b e a m instruments. Given a suitable enzymatic reaction
fluorimetry provides a simpler, cheaper m e t h o d .
Application of Method: In biochemistry for studies of mechanisms involved in metabolic control, for
enzyme kinetics and for enzyme mechanisms.
Principle
(1) C o A S H + 2-Oxoglutarate + N A D + o x o g l u t a r a t e
> Succinyl-CoA + C 0 2 + NADH 4- H +
dehydrogenase
The enzyme catalyses the complete conversion of C o A . T h e reaction is irreversible; 1 mole N A D is

1
reduced per mole C o A . T h e N A D H is measured s p e c t r o p h o t o m e t r i c a l l y or, as here,

1
fluorimetrically . 2
* 2-Oxoglutarate: lipoate oxidoreductase (decarboxylating a n d acceptor-succinylating), E C 1.2.4.2.

Optimum Conditions of Measurements
T h e Michaelis constant ( K ) of the enzyme for C o A at < 0 . 1 ^ M is r e m a r k a b l y l o w . T h e reaction curve

M
2
is apparently zero order (linear) until all the C o A is succinylated.

As the enzyme reacts at V m a x practically t h r o u g h o u t the d e t e r m i n a t i o n , low concentrations of C o A
can be rapidly determined. This m e a n s t h a t the time-dependent deviations of the zero p o i n t of a highly
sensitive fluorimeter are decreased t o a m i n i m u m .
With fluorimetric measurements the inhibition by the p r o d u c t s is n o p r o b l e m . T h e enzyme is inhibited
by N A D H and succinyl-CoA, if the ratio [ N A D ] : [ N A D H ] falls below 50; in this assay the concentration
of the products is < 1 fiM. In the case of s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s p r o d u c t inhibition results
in non-linear reaction curves; this can be overcome by a higher N A D c o n c e n t r a t i o n or by use of the
3-acetyl analogue instead of N A D . Succinyl-CoA is also relatively labile at neutral p H ; non-enzymatic
hydrolysis results in the liberation of C o A S H , which enters the reaction again. T h e hydrolysis increases
with increasing p H ; therefore the C o A assays should be carried out at p H 7 . 0 - 7 . 2 (not higher).
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r for d e t e r m i n a t i o n o f N A D H ; fluorimeter
with recorder and zero offset; b e n c h centrifuge; e q u i p m e n t for e n z y m e isolation.
Reagents
1. P o t a s s i u m d i h y d r o g e n a r s e n a t e 7. C o e n z y m e A , C o A
2. P o t a s s i u m h y d r o x i d e , A . R . free acid, commercial p r e p a r a t i o n , see p . 528.
3. P o t a s s i u m hydroxide, A . R., 2 N 8. 2-Mercaptoethanol
4. 2-Oxoglutaric acid 9. L - C y s t e i n e h y d r o c h l o r i d e • H 0 2
Commercial p r e p a r a t i o n , see p . 548. 10. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA

5. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , disodium salt, E D T A - N a H -2 H 0 2 2 2
NAD 11. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris

free acid, commercial p r e p a r a t i o n , see p. 545. 1 2 . P e r c h l o r i c a c i d , s p . gr. 1.67, c a . 7 0 % ( w / w )
6. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o 13. Oxoglutarate d e h y d r o g e n a s e , OxoGDH
tide, N A D H from pig heart according t o ; 4
1-3 U/mg.
d i s o d i u m salt, N A D H - N a ; commercial p r e p (25 °C). F o r isolation, see Appendix p. 1986.

2
Purity of Reagents
The enzyme should be free from N A D H oxidase and glutamate dehydrogenase.
U s e only doubly, glass-distilled water.

I. A r s e n a t e buffer (0.1 M ; p H 7 . 2 ) :
D i s s o l v e 9 g. K H A s 02 4 in 4 5 0 m l . d i s t i l l e d w a t e r , a d d s o l i d K O H w i t h stirring until t h e
p H is 7.2
II. A r s e n a t e buffer ( 0 . 5 M ; p H c a . 1 2 . 3 ) :
D i s s o l v e 4 5 g. K H A s 0 2 4 a n d 2 8 g. K O H i n d i s t i l l e d w a t e r a n d m a k e u p t o 5 0 0 m l .
III. T r i s ( l M ) :
D i s s o l v e 12.1 g. tris in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
Coenzyme A 1983
I V . 2 - O x o g l u t a r a t e (0.1 M ) :
D i s s o l v e 7 3 m g . 2 - o x o g l u t a r i c a c i d a n d 121 m g . tris in 5 m l . d i s t i l l e d w a t e r .
V. Nicotinamide-adenine dinucleotide (10 m M ) :
D i s s o l v e 7 0 m g . N A D in 8 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 6 - 7 w i t h tris s o l u t i o n (III)
a n d d i l u t e t o 10 m l . w i t h d i s t i l l e d w a t e r .
V I . R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 0.2 m M / ? - N A D H ) :
Dissolve 1 mg. N A D H - N a 2 in 5.0 m l . a r s e n a t e buffer (I). A n a l y s e this s t a n d a r d s o l u t i o n
for t h e fluorimeter s p e c t r o p h o t o m e t r i c a l l y a c c o r d i n g t o p. 2 0 5 2 .
V I I . C o e n z y m e A (ca. 0.5 m M ) :
D i s s o l v e 1 m g . C o A in 1 m l . i c e - c o l d d i s t i l l e d w a t e r .
VIII. Cysteine (50 m M ) :
D i s s o l v e 1 7 . 6 m g . c y s t e i n e h y d r o c h l o r i d e in 2 m l . distilled w a t e r .
IX. Perchloric acid (0.6 N ) :
X . Perchloric acid (1.8 N ) :
Dilute 15.6 ml. 70% perchloric acid to 100 ml. with distilled water.
X I . O x o g l u t a r a t e d e h y d r o g e n a s e , O x o G D H (2 m g . p r o t e i n / m l . ) :
D i l u t e t h e e n z y m e a c c o r d i n g l y w i t h 10 m M p h o s p h a t e buffer ( p H 7.2) a n d s t o r e 0.5 m l .
p o r t i o n s at - 2 0 ° C .
XII. 2-Mercaptoethanol (0.15 M ) :
D i l u t e 0 . 1 2 m l . m e r c a p t o e t h a n o l w i t h 8.8 m l . d i s t i l l e d w a t e r .
Store solutions I, II, III, IX a n d X at 0 - 4 ° C ; they are stable indefinitely. P r e p a r e the oxoglutarate solution
(IV) a n d N A D solution (V) every 4 - 6 weeks a n d store at - 20 to - 30 ° C ; p r e p a r e the C o A solution (VII)
freshly each day a n d store at — 20 to — 30 °C. P r e p a r e the N A D H solution (VI) a n d the cysteine solution
(VIII) freshly each day. T h e O x o G D H solution (XI) loses ca. 5 0 % of its activity per m o n t h at - 2 0 °C.
A p r e p a r a t i o n should be sufficient for at least 6 m o n t h s . Repeated freezing a n d t h a w i n g results in losses
of activity, therefore the p r e p a r a t i o n is frozen in 0.5 ml. portions.
Procedure
C o l l e c t t i s s u e s w i t h f r e e z e - c l a m p s (see p. 4 0 0 ) . C o o l m i c r o - o r g a n i s m s , cell or e n z y m e f r a c t i o n s
b y rapid a d d i t i o n t o i c e - c o l d p e r c h l o r i c a c i d .
Deproteinization:
P o w d e r f r o z e n t i s s u e a n d e x t r a c t w i t h 5.0 m l . i c e - c o l d 0.6 N p e r c h l o r i c a c i d ( s o l u t i o n I X ) p e r
g. t i s s u e . R a p i d l y m i x cell s u s p e n s i o n s o r cell f r a c t i o n s w i t h 2 v o l . i c e - c o l d 1.8 N p e r c h l o r i c
a c i d ( s o l u t i o n X ) . G e n e r a l l y t h e d e p r o t e i n i z e d e x t r a c t s h o u l d b e 0.6 N w i t h r e s p e c t t o p e r c h l o r i c
a c i d a n d n o t l e s s t h a n 0 . 2 pM C o A S H for fluorimetry. C e n t r i f u g e f o r 5 m i n . at 2 0 0 0 g, p o u r off
t h e s u p e r n a t a n t fluid a n d n e u t r a l i z e . T h e a c i d s o l u b l e p r e c i p i t a t e c o n t a i n s l o n g - c h a i n a c y l - C o A
a n d c a n b e a n a l y s e d a c c o r d i n g t o p . 2 0 1 5 . M i x 3 m l . p r o t e i n - f r e e s u p e r n a t a n t fluid a n d 0.5 m l .
a r s e n a t e s o l u t i o n ( I I ) ; s l o w l y a d d w i t h stirring 2 N K O H until t h e p H is 6 . 5 - 7 . 0 . A l l o w t o s t a n d
for 30 m i n . at 0 ° C , a n d r e m o v e t h e K C 1 0 4 b y c e n t r i f u g a t i o n for 2 m i n . at 2 0 0 0 g.
C o A S H is s l o w l y o x i d i z e d t o t h e d i s u l p h i d e ; t h e s a m p l e s s h o u l d b e a n a l y s e d t h e s a m e d a y .
Assay System
Spectrophotometric measurements: W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m for t h e d e t e c t i o n o f

N A D H ; light p a t h : 1 c m . ; final v o l u m e : 2 . 0 0 5 m l . ; r o o m t e m p e r a t u r e ; read a g a i n s t c u v e t t e s
c o n t a i n i n g distilled w a t e r i n s t e a d o f O x o G D H s o l u t i o n ( X I ) .
Fluorimetric measurements: Light source, m e d i u m pressure mercury lamp, xenon lamp,

100 w a t t t u n g s t e n - q u a r t z - i o d i n e l a m p . P r i m a r y filter: 365 n m o r 3 3 4 n m for t h e m e r c u r y l a m p ;
w i d e b a n d interference filter 3 3 0 - 3 5 0 n m for t h e x e n o n a n d t u n g s t e n l a m p s . S e c o n d a r y filter:
Wratten 2 E w i t h c u t off b e l o w 4 2 0 n m . C u v e t t e s : 1 c m . f o u r - s i d e d c u v e t t e s , e x c i t a t i o n a n d e m i s
s i o n t h r o u g h the front face. F i n a l v o l u m e : 2.0 m l . , r o o m t e m p e r a t u r e . R e a d a g a i n s t air.
Reagent mixture ( p H 7.2):
A r s e n a t e buffer ( s o l u t i o n I) 10 ml.
N A D solution (V) 0.2 m l .
2-Oxoglutarate solution (IV) 0.4 ml.
EDTA 7.5 m g .
P r e p a r e freshly e a c h d a y , j u s t b e f o r e u s e w a r m t o 3 0 ° C .
Reagent mixture 1.060 m l . 50 m M arsenate, 2 m M O x o G

0.1 m M N A D , 1 m M EDTA
Cysteine solution (VIII) 0.040 ml.
Sample + water 0.900 ml.
M i x , adjust r e c o r d e r t o z e r o a n d r e c o r d b a s e line for

30 sec.
O x o G D H solution (XI) 0.005 ml. 5 pglmX.
M i x a n d after 1 m i n . t h e r e a c t i o n is c o m p l e t e . N o t e t h e
final v a l u e .
O x o G D H solution (XI) 0.005 ml.
T h i s a d d i t i o n o f e n z y m e s e r v e s t o m e a s u r e the fluor
e s c e n c e d u e t o the e n z y m e *
N A D H solution (VI) 0.005 ml. 5 pM NADH
T h i s a d d i t i o n o f N A D H is t o s t a n d a r d i z e the fluori
meter.
* By addition of phosphotransacetylase at this point the acetyl-CoA content of the sample can be
determined, see p. 1972.
Coenzyme A 1985
Calculations
Three fluorescence differences are m e a s u r e d :
A ¥ after 1st addition of O x o G D H

l
A F after 2nd addition of O x o G D H

2
A F after addition of N A D H , equivalent to C pM

3 NADH
In the assay
c = A F l
~ A F l
x 2 C [nmole/2 ml.]
^F 3
To convert to n m o l e C o A in the sample the dilutions in the preliminary t r e a t m e n t of the sample a n d the
a m o u n t of sample taken for the assay must be taken into account.
T h e b a c k g r o u n d noise of the instrument limits the sensitivity a n d precision of the m e t h o d : it c o r r e s p o n d s

in this assay to 50 p m o l e C o A in the fluorimeter cuvette.
N o r m a l Values
" N o r m a l " values are only an indication, because the acylation of C o A in tissues a n d m i t o c h o n d r i a is
considerably d e p e n d e n t on their metabolic state. T h e following values have been found in rat o r g a n s :
Heart 1
30-50 nmole/g. fresh wt.
Liver 1
8 0 - 1 5 0 nmole/g. fresh wt.
Epididymal adipose tissue 5
4 nmole/g. fresh wt.
Liver m i t o c h o n d r i a 2
0 . 2 - 1 . 5 n m o l e / m g . protein
In the in vitro incubation of tissues in which C o A is t o be determined, n o substance should be a d d e d

which absorbs at the excitation wavelength (e. g. 2,4-dinitrophenol), which fluoresces (e. g. F M N ) or which
acts as an electron acceptor (e. g. phenazine m e t h o s u l p h a t e ) . If such c o m p o u n d s are used they must be
removed before the d e t e r m i n a t i o n of C o A .
All solutions m u s t be particularly free from dust.
Recycling of succinyl-CoA is a small error even in the worst case because the rate of the enzyme-catalysed
reaction in c o m p a r i s o n t o t h a t of the recycling is extremely rapid. T h e final c o n c e n t r a t i o n of succinyl-CoA
in the cuvette determines t h e r a t e of recycling; the r a t e is therefore a m o r e i m p o r t a n t source of e r r o r
in the s p e c t r o p h o t o m e t r i c m e t h o d . In a n y case the a m o u n t of O x o G D H can be increased so that the
determination is complete within a few seconds a n d then recycling c a n be ignored.
N A D H can be reoxidized u n d e r the following c o n d i t i o n s : a m m o n i a in the sample together with g l u t a m a t e
dehydrogenase as a c o n t a m i n a n t of the O x o G D H ; p y r u v a t e in the sample together with lactate d e h y d r o
genase as a c o n t a m i n a n t of O x o G D H ; oxaloacetate in the sample together with m a l a t e dehydrogenase
as a c o n t a m i n a n t of O x o G D H . If the sample contains these c o m p o u n d s , the O x o G D H must be freed
from the c o n t a m i n a t i n g enzymes of lower molecular weight by centrifugation for 1 hr. at 120000 g.
O x o G D H is a multi-enzyme complex a n d sediments, whereas o t h e r enzymes remain in the s u p e r n a t a n t
fluid.
The K M value of O x o G D H for d e p h o s p h o - C o A is 6 0 - 7 0 / / M ; in this fluorimetric assay d e p h o s p h o - C o A

is not determined. However, it can in principle be determined with O x o G D H spectrophotometrically.
Succinyl-CoA in tissue extracts is deacylated d u r i n g the t r e a t m e n t of the sample a n d a p p e a r s as C o A .
unless all the t r e a t m e n t steps are carried t h r o u g h in a few minutes. O t h e r thiolesters, e.g. acetyl-CoA
and C o A disulphide d o n o t interfere, provided they d o not u n d e r g o conversion to C o A S H (e. g. by high p H
or by S H reagents).
Further A p p l i c a t i o n s
Coenzyme A a n d certain derivatives are p r o d u c t s of n u m e r o u s enzyme reactions. Assay m e t h o d s for these

p r o d u c t s are the basis for the d e t e r m i n a t i o n of the c o r r e s p o n d i n g enzyme activities.
Examples :
(a) P a l m i t a t e : C o A ligase, ( E C 6.2.1.3)
Palmitate + A T P + C o A S H . , M g 2
^ P a l m i t o y l - C o A + A M P + PPi
(b) P a l m i t o y l - C o A c a r n i t i n e O-palmitoyltransferase, ( E C 2.3.1.21)
Palmitoyl-CoA + ( L ) - C a r n i t i n e , C o A S H + Palmitoyl-(L)-carnitine
(c) Acetyl-CoA c a r n i t i n e 0 - a c e t y t r a n s f e r a s e , (EC 2.3.1.7)
Acetyl-CoA + (L)-Carnitine , Acetyl-(L)-Carnitine + CoASH
(d) Acyl-CoA + E n z y m e - S H t Acyl-S-Enzyme + CoASH
(e) Transferases, general, e. g. p a l m i t o y l - C o A deacylase, where R = H
Palmitoyl-CoA + R — O H • Palmitoyl-O—R + C o A S H
Several of these possible applications have already been d e s c r i b e d 6 - 8

. If C o A is liberated the reaction can
be followed directly by coupling with the fluorimetric d e t e r m i n a t i o n with O x o G D H . T h e low K M value
of O x o G D H for C o A S H m e a n s t h a t the system is suitable as an indicator reaction, a n d that there is
scarcely any induction period. T h e high sensitivity of the fluorimetric assay permits the determination
according to equations (b) a n d (e) of p a l m i t o y l - C o A concentrations below 3 uM. A further advantage of
the fluorimetric technique is the fact t h a t by use of a fluorimeter with front-face cuvettes for excitation a n d
emission, even turbid suspensions of subcellular organelles can be m e a s u r e d directly (e. g. reactions (b)
and (c)).
Appendix
Isolation of 2-Oxoglutarate Dehydrogenase 4
Cut u p 4 fresh pig hearts (ca. 600 g) into cubes ( 1 - 2 cm.), homogenize with 3 - 4 1 . 30 m M potassium p h o s
p h a t e buffer ( p H 7.4; 1 m M E D T A ) for 30 sec. at 0 °C in a mixer. Adjust the p H to 7.4 with 10 N K O H
a n d homogenize for a further 30 sec. M a i n t a i n the p H at 7.4. Centrifuge for 15 min. at 1000 g; filter t h r o u g h
gauze. Adjust the filtrate to p H 5.4 with 10% (v/v) acetic acid a n d centrifuge for 20 min. at 10000 g. Discard
the s u p e r n a t a n t fluid, suspend the precipitate ( m i t o c h o n d r i a ) in 1.51. water a n d centrifuge again for 20 min.
at 10000 g. Suspend the precipitate in 500 ml. distilled water, adjust p H to 7.2 with 10 N K O H a n d freeze
at - 2 0 ° to - 30 °C. After 1 0 - 1 2 hr. t h a w the frozen m i t o c h o n d r i a . R e p e a t the freezing a n d thawing twice
a n d then centrifuge for 30 min. at 18000 g. T h e s u p e r n a t a n t fluid contains the enzyme.
Coenzyme A 1987
F u r t h e r purification: Slowly a d d with stirring 38.5 g. a m m o n i u m acetate/100 ml. s u p e r n a t a n t fluid;

maintain the p H at 7.4. Allow t o stand for 30 min. at 0 °C, centrifuge for 30 min. at 18000 g a n d discard
the precipitate; a d d slowly t o each 100 ml. s u p e r n a t a n t fluid 45 g. a m m o n i u m acetate a n d adjust the p H
to 7.4 with 10 N K O H . Allow to stand for 30 min. at 0 °C a n d centrifuge for 30 min. at 18000 g. Dissolve
the slightly yellow precipitate in 3 to 6 ml. p o t a s s i u m p h o s p h a t e buffer ( p H 7.4; 1 m M E D T A ) .
R e m o v e the a m m o n i u m acetate by dialysis against 2 changes of 1000 ml. buffer. T h e salt-free enzyme is
slowly inactivated by repeated freezing a n d thawing. It is therefore preferable to freeze the solution in
0.25 ml. portions and store t h e m at — 20 °C. T h e loss of activity is ca. 50% per m o n t h .
References
1 P. B. Garland, Biochem. J. 92, 10C [1964].

2 P. B. Garland, D. Shepherd & D. W. Yates, Biochem. J. 97, 587 [1965].
3 V. Massey, Biochim. Biophys. A c t a 38, 447 [I960].
4 D. R. Sanadi, J. W. Littlefield & R. M. Bock, J. biol. C h e m . 197, 851 [1952].
5 R. M. Denton & M. L. Halperin, Biochem. J. 110, 27 [1968].
6 P. B. Garland & D. W. Yates in J. M. Tager, S. Papa, E. Quagliariello & E. C. Slater: " M i t o c h o n d r i a l
Structure a n d C o m p a r t m e n t a t i o n " , p . 385, Adriatica Editrice, Bari, 1967.
7 B. Middleton, A b s t r a c t s 4th Meeting F E B S , Universitetsforlaget Oslo, 1967, p . 135.
8 J. R. Williamson & B. E. Corkey in S. P . Colowick & N. O. Kaplan: M e t h o d s in Enzymology A c a d e m i c
Press, N e w York, Vol. 13, p . 434.
Acetyl-Coenzyme A
UV-Spectrophotometric Assay
Karl D e c k e r
Acetyl-CoA is the c o m m o n intermediate in the oxidation of c a r b o h y d r a t e s , fatty acids a n d certain a m i n o

acids, a n d it is the point of entry for terminal oxidation in the citric acid cycle. Acetyl-CoA is also the
precursor for the biosynthesis of fatty acids, acetoacetate and steroids. Consequently it is found in all
living cells. It has not been detected in extracellular fluids (blood). Assay m e t h o d s for acetyl-CoA are
based on the enzymatic acetylation of a r o m a t i c amines, on the arsenolysis of acetyl-CoA catalysed by
phosphotransacetylase, P T A ( A c e t y l - C o A : o r t h o p h o s p h a t e acetyltransferase, E C 2.3.1.8) or on the
synthesis of citrate from oxaloacetate in the presence of citrate synthase, CS (Citrate oxaloacetate-lyase,
EC 4.1.3.7). T h e latter m e t h o d is n o w the one of choice because of its specificity, simplicity and the com
mercial availability of the necessary p u r e enzymes. By the systematic studies of Bucket a n d Eggerer1
and also Pearson 2

the m e t h o d has been greatly improved b o t h experimentally a n d theoretically.
Principle
(1) Acetyl-CoA + O x a l o a c e t a t e " + H 0 2

2 Citrate 2 -
+ CoASH
(2) L-Malate 2 -
+ NAD +
_^!PiL O x a l o a c e t a t e 2 -
+ NADH + H +
(3) L-Malate 2 -
+ Acetyl-CoA + N A D +
+ H 0 ^ 2 Citrate 2 -
+ NADH + H +
+ CoASH
Malate dehydrogenase, M D H (L-malate: N A D oxidoreductase, E C 1.1.1.37) is the indicator enzyme

and the M D H reaction is a " p r e c e d i n g indicator r e a c t i o n " (see p . 112).
The formation of N A D H , as measured by the change of extinction at 340 (334, 365) nm, is the basis of the
assay, but it is not directly p r o p o r t i o n a l to the a m o u n t of acetyl-CoA.
E q u a t i o n s (1) a n d (2) are p H - d e p e n d e n t .
Kl = [Citrate] [ C o A S H ] = 1 2 x ^ 2 5 t
[Oxaloacetate] [Acetyl-CoA] [ H 0 ] 2
v , _ [Oxaloacetate] [ N A D H ] _^ ^ x 1 Q _ 5 ^ ^ 2 $ O Q 3
[L-Malate] [ N A D ]
KJ = , ^ [pirate] [CoASH] [NADH] = 2 5
[L-Malate] [Acetyl-CoA] [ N A D ] [ H 0 ] 2
A l t h o u g h increase of p H favours the formation of citrate it is not advisable to exceed p H 8.1, because
b o t h the hydrolysis of acetyl-CoA a n d the influence on the indicator reaction affect the accuracy of the
determination. Since citrate synthase is not saturated with oxaloacetate u n d e r the conditions of the
assay a n d therefore is n o t fully active, relatively high concentrations of this enzyme (ca. 0.1 to 0.2 U )
are required in order to complete the reaction within a reasonable time.
Acetyl-CoA 1989
Equipment
Spectrophotometer or spectrum-line photometer s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at

3 4 0 , 3 3 4 o r 365 n m ; b e n c h c e n t r i f u g e ; e q u i p m e n t for f r e e z e - c l a m p i n g s e e p. 4 0 0 .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 8. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ,
KH P0 2 4 NAD
2. P o t a s s i u m h y d r o x i d e , A . R., c a . 8 N free acid, commercial preparation, see p. 545.
3. P o t a s s i u m h y d r o g e n c a r b o n a t e , 9. M a l a t e d e h y d r o g e n a s e , MDH
K H C 0 , A . R.
3 from pig heart; suspension in 3.2 M ammonium
4 . P e r c h l o r i c a c i d , A . R . , s p . gr. 1.67; sulphate solution; ^ 7 2 0 U/mg. (25 °C); com-
ca. 7 0 % ( w / w ) merial preparation, see p. 485.
5. M a l i c a c i d , s u i t a b l e for b i o c h e m i c a l u s e 10. C i t r a t e s y n t h a s e , C S
6. H y d r o c h l o r i c a c i d , A . R., 1 N from pig heart; crystalline suspension in 3.2 M
7. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris ammonium sulphate solution; ^ 70 U/mg.
(25 °C); commercial preparation, see p. 443.
Purity of Reagents
Malate dehydrogenase must be free from citrate synthase, N A D H oxidase and fumarase. The malate
must not contain oxaloacetate (see below).
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y distilled w a t e r .

I. P o t a s s i u m d i h y d r o g e n p h o s p h a t e ( 0 . 2 M ) :
D i s s o l v e 2 . 7 2 2 g. K H P 0 2 4 in distilled w a t e r a n d m a k e u p t o 100 m l .
II. P o t a s s i u m h y d r o g e n c a r b o n a t e (ca. 1 M ) :
D i s s o l v e 10 g. K H C 0 3 in distilled w a t e r a n d m a k e u p t o 100 m l .
III. P e r c h l o r i c a c i d (ca. 4 M ) :
D i l u t e 35 m l . 7 0 % H C 1 0 4 t o 100 m l . w i t h distilled w a t e r .
I V . P e r c h l o r i c a c i d ( c a . 0.5 M ) :
D i l u t e 10 m l . 4 N H C 1 0 4 w i t h 7 0 m l . distilled w a t e r .
V . P o t a s s i u m D L - m a l a t e s o l u t i o n (0.1 M ) :
D i s s o l v e 1.34 g. DL-malic a c i d in c a . 5 0 m l . distilled w a t e r , s l o w l y a d d 2 0 m l . 1 M KHCO3
s o l u t i o n (III) w i t h stirring ( f o a m s ) a n d d i l u t e t o 100 m l . T h i s s o l u t i o n u s u a l l y c o n t a i n s
traces o f o x a l o a c e t a t e ; b e f o r e u s e h e a t for 10 m i n . at 100 ° C t o r e m o v e t h e s e traces.
1
T h e h e a t i n g c a n b e r e p e a t e d several t i m e s .
V I . Tris buffer (0.5 M ; p H 8 . 1 ) :
D i s s o l v e 6.05 g. tris in 25 m l . 1 N H C 1 a n d d i l u t e t o 100 m l . w i t h distilled w a t e r .
V I I . N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 10 m M / ? - N A D ) :
D i s s o l v e 7 . 4 m g . N A D in a b o u t 0.5 m l . d i s t i l l e d w a t e r , n e u t r a l i z e w i t h a f e w d r o p s
K H C O 3 s o l u t i o n (II) a n d d i l u t e t o 1 m l .
VIII. Malate dehydrogenase, M D H (0.5 m g . / m l . ) :

D i l u t e s t o c k s u s p e n s i o n a c c o r d i n g l y w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n a n d m i x .
I X . Citrate s y n t h a s e , C S ( 3 0 U / m l . ) :
D i l u t e t h e s t o c k s u s p e n s i o n a c c o r d i n g l y w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n a n d
mix.
T h e solutions are stable for m o n t h s at 4 °C. T h e malate solution should be stored sterile. Store buffer
and alkaline solutions in well-stoppered polyethylene bottles.
Procedure
Deproteinization:
If p o s s i b l e o b t a i n t i s s u e s a m p l e s b y t h e f r e e z e - s t o p m e t h o d (see p . 4 0 0 ) , m i x w i t h t w o v o l u m e s
c o l d 0.5 N p e r c h l o r i c a c i d ( s o l u t i o n I V ) in a c e n t r i f u g e t u b e a n d h o m o g e n i z e as rapidly as
p o s s i b l e (see p . 4 0 0 ) . A d d 1/10 v o l u m e o f 4 M p e r c h l o r i c a c i d ( s o l u t i o n III) t o s o l u b l e s a m p l e s
a n d m i x t h o r o u g h l y . A f t e r a l l o w i n g t o s t a n d for 5 m i n . in the c o l d n e u t r a l i z e m o s t o f t h e
a c i d b y d r o p w i s e a d d i t i o n ( w i t h s h a k i n g ) o f 8 N K O H a n d t h e n c a r e f u l l y adjust t o p H 6 . 3 - 6 . 7
with K H C 0 3 s o l u t i o n (II). T h i s p r o c e d u r e a v o i d s e x p o s u r e o f the s a m p l e t o t o o strong
alkali a n d t h e r e f o r e m i n i m i z e s t h e h y d r o l y s i s o f a c e t y l - C o A . It is sufficient t o c h e c k t h e
p H w i t h i n d i c a t o r p a p e r u s i n g a t h i n g l a s s r o d t o o b t a i n a s a m p l e . If t h e p H e x c e e d s p H 7
i m m e d i a t e l y a d d several d r o p s o f p h o s p h a t e s o l u t i o n (I).
C e n t r i f u g e off p r e c i p i t a t e d p r o t e i n a n d p o t a s s i u m p e r c h l o r a t e at 3 0 0 0 g f o r 5 m i n . a n d p o u r
off s u p e r n a t a n t fluid a s q u a n t i t a t i v e l y a s p o s s i b l e i n t o a m e a s u r i n g c y l i n d e r . W a s h t h e preci
p i t a t e w i t h a little c o l d w a t e r , c e n t r i f u g e a n d c o m b i n e t h e w a s h i n g w i t h t h e s u p e r n a t a n t .
U s e a p o r t i o n o f this s o l u t i o n w i t h o u t further t r e a t m e n t for t h e d e t e r m i n a t i o n .
If the a c e t y l - C o A c o n t e n t is v e r y l o w it is p o s s i b l e t o c o n c e n t r a t e t h e e x t r a c t ; for t h e m e t h o d ,
see .5
A q u e o u s s o l u t i o n s o f a c e t y l - C o A at b e t w e e n p H 3 a n d 6 c a n b e s t o r e d d e e p - f r o z e n for w e e k s .
In a l k a l i n e s o l u t i o n h y d r o l y s i s o c c u r s v e r y r a p i d l y , a n d i n s t r o n g a c i d t h e a c t i v i t y d e c r e a s e s
gradually . 4
Acetyl-CoA 1991
Assay System

a t u r e ; r e a d a g a i n s t air.
Sample + water 0.80 ml. u p t o ca. 0.2 /imole

acetyl-CoA/ml.
Tris buffer (VI) 0.80 ml. 2 0 0 m M tris
DL-Malate solution (V) 0.10 ml. 5 m M L-malate
N A D solution (VII) 0.30 ml. 1.5 m M / ? - N A D
M i x , f o l l o w t h e initial e x t i n c t i o n u n t i l c o n s t a n t a n d
read E . 0
M D H suspension (VIII) 0.005 ml. 1.25 jug./ml. = 9 0 0 m U / m l .
M i x , f o l l o w t h e e x t i n c t i o n until c o n s t a n t a n d r e a d
E,. E j — E = 0 AE,.
CS suspension (IX) 0.005 ml. 1.07 pg./ml = 75 m U / m l .
M i x , follow extinction until constant and read E . 2
E - E =
2 xAE . 2
Calculations
Since in this m e t h o d of estimation the indicator reaction is a preceding one, there is n o direct stoichio-
metry between the a m o u n t of acetyl-CoA reacting a n d the m e a s u r e d increase in N A D H ' . 1 2
T h e deviation is particularly serious if the reaction is started with CS (after a t t a i n m e n t of the M D H

equilibrium).
With a final v o l u m e in the cuvette of 2.01 ml. the /miole acetyl-CoA in the p o r t i o n of sample t a k e n for
the assay are o b t a i n e d by t h e following e q u a t i o n . 1
M»nole = ^2X2.01 / AE, \

e \ A E 1 + A E 2 J
W h e r e E [cm. //miole] has a value of 6.22 at 340 n m , 6.1 at 334 n m and 3.4 at 365 n m .
2
Eight d e t e r m i n a t i o n s o n a n acetyl-CoA solution gave a value of 182 + 6 n m o l e ( + S.D.). This gives

1
a coefficient of variation of 3 . 3 % .
N o r m a l Values
R a t liver contains 15 to 20 n m o l e acetyl-CoA/g. fresh w t . ' ' . T h e c o n t e n t is very d e p e n d e n t o n the

6 7 8
nutritional state of the a n i m a l a n d o n h o r m o n a l factors.

C o n t a m i n a t i o n of the reagents, especially the malate, with oxaloacetate leads to low values. If the sample
contains appreciable a m o u n t s of N A D H (this is not the case with acid-treated samples) t o o high values
are obtained. Pyruvate or lactate also interfere in the assay in a similar way. C o A derivatives of long-chain
fatty acids inhibit C S , b u t u n d e r the a b o v e conditions interference from a q u e o u s extracts or samples
does n o t occur. If the activity of C S in the assay mixture is t o o low a n d m o r e t h a n 30 min. is required to
complete the reaction, s p o n t a n e o u s hydrolysis of acetyl-CoA a n d decarboxylation of oxaloacetate m u s t
be taken into account.
Specificity
CS is specific with regard to the acid c o m p o n e n t ; o t h e r acyl-CoA derivatives d o n o t react. C o A S H c a n n o t

be replaced by pantetheine p h o s p h a t e , pantetheine of N-acetylcysteamine. A c e t y l - d e p h o s p h o - C o A has
a low affinity for CS a n d reacts in the assay. Occurrence of a c e t y l - d e p h o s p h o - C o A in vivo has not been
reported.
O t h e r M e t h o d s for D e t e r m i n a t i o n o f A c e t y l - C o A
Acetyl-CoA can be determined with C S alone according to e q u a t i o n (1); in this case the decrease of ex
tinction of the thiol ester at 232 n m 1 , 4 , 9
o r the f o r m a t i o n of free S H g r o u p s (e.g. with n i t r o p r u s s i d e )
10
is measured. Neither m e t h o d is suitable for m e a s u r e m e n t s on tissue extracts or samples which contain

m u c h U V absorbing material or m e r c a p t a n s . In the s p e c t r o p h o t o m e t r i c m e t h o d particular care should
be taken with regard to the m a i n t e n a n c e of an exact p H , because the extinction coefficients of acetyl-Co A
a n d oxaloacetate are p H - d e p e n d e n t . 1
T h e assay of acetyl-CoA by enzymatic acetylation of a r o m a t i c amines has a low substrate specifity

( n u m e r o u s other acyl thiolesters react). A r y l a m i n e acetylase is p r e p a r e d from pigeon liver acetone p o w d e r ; 1 1
n o commercial p r e p a r a t i o n s are available. In a d d i t i o n acetyl-CoA can be determined by arsenolysis

with p h o s p h o t r a n s a c e t y l a s e from Clostridium kluyveri; the assay is based o n the m e a s u r e m e n t of the
decrease in extinction at 232 n m . This m e t h o d is the most specific. Its m a i n disadvantage lies in the
9
difficulty of carrying o u t m e a s u r e m e n t s on samples which have a strong U V a b s o r p t i o n .

A modification of this m e t h o d has been described by Garland et a l . , in which the C o A liberated by the
1 2
arsenolysis is determined specifically by the use of 2-oxoglutarate d e h y d r o g e n a s e (2-oxoglutarate: lipoate-

oxidoreductase (acceptor acylating), E C 1.2.4.2). This m e t h o d combines the specificity of p h o s p h o t r a n s
acetylase with the m e a s u r e m e n t of N A D H formation at 340 n m . 2-Oxoglutarate dehydrogenase is pre
pared according to Sanadi et a l . , a n d m u s t be highly purified.
1 3
P r e p a r a t i o n s of this enzyme are n o t available commercially.

T h e sensitivity of all the assay m e t h o d s linked to the pyridine nucleotides can be increased by fluorimetry
(see p . 1993). A radiochemical m e t h o d for the d e t e r m i n a t i o n of acetyl-CoA with CS in the presence of
[ C l - o x a l o a c e t a t e has been described by Prinz et a l .
14 1 4
(see p . 1994).
U n d e r suitable conditions non-enzymatic m e t h o d s can also be used. Examples of these m e t h o d s are
determination as the a c e t o h y d r o x a m a t e , the delayed nitroprusside reaction a n d U V s p e c t r o s c o p y . 4
References
1 W. Bucket & H. Eggerer, Biochem. Z . 343, 29 [1965].

2 D. J. Pearson, Biochem. J. 95, 23C [1965].
3 K. Burton & T. H. Wilson, Biochem. J. 54, 86 [1953].
4 K. Decker, Die aktivierte Essigsaure. F e r d . E n k e , Stuttgart 1959.
5 O. Wieland, G. Loffler, L. Weiss & I. Neufeldt, Biochem. Z. 333, 10 [I960].
Acetyl-CoA 1993
6 W. M. Bortz & F. Lynen, Biochem. Z. 339, 77 [1963].

7 O. Wieland& L. Weiss, Biochem. biophys. Res. C o m m . 10, 333 [1963].
8 P. K. Tubbs & P. B. Garland, Biochem. J. 93, 550 [1964].
9 E. R. Stadtman, J. Cell. C o m p . Physiol. 41, 89 [1953].
10 R. Grunert & P. Phillips, Arch. Biochem. Biophysics 30, 217 [1951].
11 H. Tabor, A. H. Mehler & E. R. Stadtman, J. biol. C h e m . 204, 127 [1953].
13 D. R. Sanadi, J. W. Littlefield & R. M. Bock, J. biol. C h e m . 197, 851 [1952].
14 W. Prinz, W. Schoner, U. Haag & W. Seubert, Biochem. Z. 346, 206 [1966].
Fluorimetric Assay
Peter Bryan Garland
Only a small p r o t i o n of the total C o A content of tissues is in the form of a c e t y l - C o A ; s p e c t r o p h o t o m e t r i c

assays therefore require relatively large a m o u n t s of sample. T h e two fluorimetric m e t h o d s described here
are modifications of the s p e c t r o p h o t o m e t r i c m e t h o d for the determination of acetyl-CoA (see p . 1988)
a n d the fluorimetric m e t h o d for C o A S H (see p . 1981) using oxoglutarate dehydrogenase. Only deviations
from the cited m e t h o d s are therefore given below.
Determination with Citrate Synthase and Malate Dehydrogenase

Principle
See e q u a t i o n s ( 1 ) a n d ( 2 ) o n p . 1988.
In the s p e c t r o p h o t o m e t r i c m e t h o d the M D H reaction yields a b o u t 30 pM N A D H , which is a b o u t fifty-
fold t o o high for a highly sensitive fluorimeter. Even with efficient null-point c o m p e n s a t i o n the ratio of
b a c k g r o u n d 'noise' to deflection is t o o high. T h e initial concentration of L-malate a n d / o r N A D must
therefore be reduced so t h a t only ca. 1 /zM N A D H is present at equilibrium.
Equipment
F l u o r i m e t e r w i t h r e c o r d e r , a p p r o p r i a t e l a m p s a n d filters (see p. 1 9 8 2 ) .
R e a g e n t s and P r e p a r a t i o n o f S o l u t i o n s
A s for t h e s p e c t r o p h o t o m e t r i c m e t h o d , s e e p . 1 9 8 9 .
Procedure
Standardize the fluorimeter by the addition o f a k n o w n a m o u n t o f a c e t y l - C o A to the experi

m e n t a l a s s a y m i x t u r e after t h e e n d o f t h e r e a c t i o n . T h e a c e t y l - C o A s o l u t i o n m u s t b e p r e v i o u s l y
s t a n d a r d i z e d s p e c t r o p h o t o m e t r i c a l l y . P r o c e e d a s for t h e s p e c t r o p h o t o m e t r i c m e t h o d (see
p. 1991) b u t p i p e t t e o n l y 0.01 m l . D L - m a l a t e s o l u t i o n ( V ) a n d o n l y 0 . 0 0 5 m l . N A D s o l u t i o n
( V I I ) ; m a k e u p t h e v o l u m e w i t h d i s t i l l e d w a t e r . T h e final c o n c e n t r a t i o n s i n t h e a s s a y a r e
t h e n 0.5 m M L - m a l a t e a n d 2 5 pM 0-NAD.
Sensitivity
This depends on the fluorimeter. A b a c k g r o u n d noise which is equivalent to 0.02 /zM acetyl-CoA in the
cuvette can be overcome w i t h o u t difficulty.
Determination with Oxoglutarate Dehydrogenase and Phosphotransacetylase

Principle
(1) Acetyl-CoA + O H " P T A

> Acetate" + CoASH
V J J
arsenate
T h e arsenolysis of acetyl-CoA is irreversible; the C o A S H formed can be determined with oxoglutarate

dehydrogenase according to the description on p . 1981. O n successive addition of oxoglutarate d e h y d r o
genase and P T A , C o A S H can be determined first in a cuvette followed by acetyl-CoA. Unlike the assay
which requires citrate synthase a n d m a l a t e dehydrogenase the molecular ratio of N A D :acetyl-CoA here
is 1 : 1. T h e fluorimeter is standardized with N A D H , as for the determination of C o A (p. 1984).
Sensitivity
0.02 uM Acetyl-CoA in the fluorimeter cuvette.
N o r m a l Values
D e p e n d i n g on the metabolic state, rat liver m i t o c h o n d r i a 1 2

contain 0.02 to 1.3 n m o l e acetyl-CoA/mg.
protein. R a t epididymal fat p a d s contain 1 nmole/g. fresh wt.
3
A c e t y l - d e p h o s p h o - C o A is n o t e s t i m a t e d .
2
References

2 G. Michal & H. U. Bergmeyer in " M e t h o d s of E n z y m a t i c Analysis", A c a d e m i c Press, N e w Y o r k ,
1963, p . 512.
Radiochemical Assay
Wilhelm Schoner and Werner Seubert
T h e spectrophotometric estimation of acetyl-CoA is n o t sufficiently sensitive for determinations on small

samples of tissue (needle biopsies a n d kidney cortex slices, etc.) a n d consequently a radioactive assay
has been developed. T h e sensitivity of the assay is determined by the specific radioactivity of the [ 4 - C ] - 14
a s p a r t a t e used.
Acetyl-CoA 1995
Acetyl-CoA a n d [ 4 - C ] - o x a l o a c e t a t e are converted into [ l - C ] - c i t r a t e with citrate synthase, CS (Citrate

14 14
oxaloacetate-lyase, E C 4.1.3.7). T h e [ 4 - C ] - o x a l o a c e t a t e is formed from [ 4 - C ] - a s p a r t a t e by t r a n s

14 14
a m i n a t i o n in the presence of 2-oxoglutarate a n d glutamate-oxaloacetate t r a n s a m i n a s e , G O T ( L - A s p a r t a t e :

2-oxoglutarate aminotransferase, E C 2.6.1.1).
Application of Method: I n biochemistry a n d clinical chemistry.
Principle
(1) [ 4 - C ] - A s p a r t a t e + 2-Oxoglutarate
14
[ 4 - C ] - O x a l o a c e t a t e 4- L - G l u t a m a t e
14
(2) [ 4 - C ] - O x a l o a c e t a t e + Acetyl-CoA + H 0 ^
14
2 [l- C]-Citrate +
14
CoASH
T h e radioactivity i n c o r p o r a t e d into citrate is m e a s u r e d after decarboxylation of the residual [ 4 - C ] - 14
oxaloacetate a n d separation of the [ 4 - C ] - a s p a r t a t e by high voltage electrophoresis. W i t h limiting

14
a m o u n t s of acetyl-CoA the radioactivity of the citrate is p r o p o r t i o n a l to the c o n c e n t r a t i o n of acetyl-CoA.
Tissue extracts c o n t a i n sufficiently high a m o u n t s of L-aspartate so t h a t the specific radioactivity of the

a d d e d a s p a r t a t e is diluted leading t o errors in the results. To exclude this source of error, a n excess of
[ 4 - C ] - o x a l o a c e t a t e is formed from [ 4 - C ] - a s p a r t a t e by t r a n s a m i n a t i o n before
14 14
the addition of the
tissue extract. T h e reaction of radioactive oxaloacetate with acetyl-CoA proceeds so rapidly t h a t there
is n o decrease in the specific radioactivity of the preformed [ 4 - C ] - o x a l o a c e t a t e by exchange (equation 1)
14
with cold a s p a r t a t e present in the s a m p l e . 1
Equipment
3 3 4 o r 365 n m ; h i g h v o l t a g e e l e c t r o p h o r e s i s a p p a r a t u s ; s c i n t i l l a t i o n c o u n t e r ; b e n c h c e n t r i f u g e ;
30 ° C w a t e r b a t h ; ice b a t h .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 9. 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-
2. P o t a s s i u m c a r b o n a t e , K C 0 , A . R.
2 3 benzene, dimethyl-POPOP (scintillation
3. P o t a s s i u m h y d r o x i d e , K O H , A . R . , grade)
4 N, 1 N 10. T o l u e n e , A . R.
4. H y d r o c h l o r i c a c i d , H C 1 , A . R . , 2 N 11. 2-Oxoglutarate
5. A c e t i c a c i d , A . R . , 2 N commercial p r e p a r a t i o n , see p . 548.
6. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , s p . 12. A c e t y l - c o e n z y m e A
gr. 1.67 lithium salt; c o m m e r c i a l p r e p a r a t i o n , see p . 524.
7. T r i s o d i u m c i t r a t e • 2 H 0 2 13. M e t h y l red
8. 2 , 5 - D i p h e n y l o x a z o l e , PPO (scintillation
grade)
14. D L - [ 4 - C ] - A s p a r t i c a c i d
1 4
16. C i t r a t e s y n t h a s e ( " c o n d e n s i n g e n z y m e " ) ,
specific radioactivity 1.9 to 3.4 m C / m M o l e * CS
15. G l u t a m a t e - o x a l o a c e t a t e t r a n s a m i n a s e , from pig heart, suspension in 3.2 M a m m o n i u m
GOT sulphate solution; ^ 7 0 U / m g . (25 °C). C o m
from pig heart, suspension in 3.2 M ammonium mercial p r e p a r a t i o n , see p . 443.
sulphate solution; ^ 180 U/mg. (25 °C). Com
mercial preparation, see p. 462.
Purity of Reagents
G O T a n d C S m u s t be free from deacylases a n d t h e 2-oxoglutarate should contain n o aspartate.
I. Tris buffer ( 0 . 1 M ; p H 7 . 5 ) :
D i s s o l v e 1.21 g. tris in w a t e r , a d d 4 . 0 6 m l . 2 N H C 1 a n d m a k e u p t o 1 0 0 m l . w i t h w a t e r .
II. 2 - O x o g l u t a r a t e (0.1 M ) :
D i s s o l v e 1 4 6 m g . 2 - o x o g l u t a r i c a c i d in a little w a t e r a n d a d j u s t t o p H 7 w i t h 1 N K O H
a n d m a k e u p t o 10 m l . w i t h w a t e r .
III. D L - [ 4 - C ] - A s p a r t i c a c i d (specific r a d i o a c t i v i t y 1.9 t o 3 . 4 m C / m M o l e ; 6 0 - 8 0 m M ) :
1 4
D i s s o l v e 0 . 0 5 m C D L - [ 4 - C ] - a s p a r t i c a c i d in a little w a t e r a n d a d j u s t t o p H 6 - 7 w i t h
1 4
4 N KOH.
I V . G O T / C S - m i x e d s u s p e n s i o n ( e a c h 10 m g . p r o t e i n / m l . ) :
M i x 0.1 m l . o f G O T a n d C S s t o c k s u s p e n s i o n s .
V . P o t a s s i u m c a r b o n a t e (5 M ) :
D i s s o l v e 6 9 g. K C 0 2 3 in w a t e r a n d m a k e u p t o 1 0 0 m l .
V I . S o d i u m citrate (0.1 M ) :
D i s s o l v e 2 9 4 m g . s o d i u m citrate in 10 m l . w a t e r .
VII. Methyl red indicator ( 0 . 0 5 % ) :
D i s s o l v e 5 0 m g . m e t h y l r e d in 1 0 0 m l . tris buffer ( s o l u t i o n I ) . F i l t e r t h e s o l u t i o n .
VIII. Perchloric acid (0.71 M ) :
Dilute 3 ml. 7 0 % perchloric acid with water to 70 ml.
IX. Scintillation fluid:
D i s s o l v e 4 g. P P O a n d 1 0 0 m g . d i m e t h y l - P O P O P in t o l u e n e a n d d i l u t e t o 1 0 0 0 m l .
X. A c e t y l - C o A stock solution ( 4 - 5 m M ) :
D i s s o l v e 1.0 m g . o f s y n t h e t i c a l l y p r e p a r e d a c e t y l - C o A ' 2 3
in 1.1 m l . w a t e r .
W h e n d e t e r m i n i n g t h e a c e t y l - C o A c o n t e n t b y t h e m e t h o d s o f Pearson*, Bucket a n d
Eggerer 5
o r Bergmeyer a n d Mollering 6
observe the necessary precautions (see also
"Spectrophotometric Determination o f A c e t y l - C o A " , p. 1988.
XI. A c e t y l - C o A standard solution ( 4 - 5 pM)\
D i l u t e 0.1 m l . a c e t y l - C o A s t o c k s o l u t i o n ( X ) w i t h 9 . 9 m l . i c e - c o l d distilled w a t e r .
U s e t h e s o l u t i o n immediately for the radioactive assay. Only use solutions w h o s e con
centration has just been determined.
* C o m m e r c i a l p r e p a r a t i o n from N e w E n g l a n d N u c l e a r C o r p o r a t i o n , Boston, M a s s . , U S A .
Acetyl-CoA 1997
Store solutions II, III and VI at - 1 5 °C, and all other solutions at 0 to 4 °C. Solutions II and VI are stable
for at least a year under these conditions. Check the specific radioactivity of the [4- C]-aspartate every 14
month. Apart from solution IV, all the other solutions are stable indefinitely.
Procedure
Collection:
O b t a i n tissue s a m p l e s b y f r e e z e - c l a m p i n g (refer t o " C e l l a n d T i s s u e D i s i n t e g r a t i o n " , p. 4 0 0 ) .
Deproteinization:
C a r r y o u t all o p e r a t i o n s at 0 ° C . A d d 1.5 m l . i c e - c o l d p e r c h l o r i c a c i d s o l u t i o n ( V I I I ) t o e a c h
g r a m o f t i s s u e a n d h o m o g e n i z e w i t h a n U l t r a t u r r a x in a p l a s t i c c e n t r i f u g e t u b e for c a . 3 0 - 4 5 sec.
C e n t r i f u g e t h e h o m o g e n a t e for 10 m i n . at 12 0 0 0 g. P o u r off t h e s u p e r n a t a n t fluid a n d e x t r a c t
t h e s e d i m e n t w i t h 1 m l . p e r c h l o r i c a c i d s o l u t i o n ( V I I I ) p e r g. t i s s u e . C o m b i n e t h e s u p e r n a t a n t
fluids. W i t h v i g o r o u s stirring, a n d w h i l e g a s s i n g w i t h N 2 adjust to a b o u t p H 2 by the addition
of 0.046 ml. K C 0 2 3 s o l u t i o n ( V ) p e r 2 m l . e x t r a c t . T h e n adjust t o p H 5 - 6 b y t h e s l o w a d d i t i o n
o f 1 N K O H ( c h e c k t h e p H after t h e a d d i t i o n o f e a c h d r o p o f K O H b y r e m o v i n g a s m a l l
a m o u n t o f extract with a capillary pipette and testing with indicator paper). M e a s u r e the v o l u m e
o f t h e e x t r a c t , c e n t r i f u g e off t h e p r e c i p i t a t e o f p o t a s s i u m p e r c h l o r a t e a n d u s e t h e s u p e r n a t a n t
fluid for t h e d e t e r m i n a t i o n o f a c e t y l - C o A .
A c e t y l - C o A is s t a b l e in t h e a c i d e x t r a c t o r at p H 4 - 6 for at least 2 h r . a n d t h e d e t e r m i n a t i o n
1
s h o u l d b e carried o u t w i t h i n this t i m e .
Preparation of Blank
A d d 0 . 0 2 5 m l . 4 N K O H t o 1 m l . e x t r a c t a n d i n c u b a t e for 3 0 m i n . at 30 ° C t o h y d r o l y s e t h e
a c e t y l - C o A . A d j u s t t o p H 5 - 6 w i t h a little c o n e . H C 1 0 4 a n d c e n t r i f u g e off t h e p r e c i p i t a t e
of potassium perchlorate.
Assay System
D e t e r m i n e t h e a c e t y l - C o A c o n t e n t o f t h r e e different a m o u n t s o f s a m p l e ( 0 . 0 7 ; 0 . 1 ; 0 . 1 3 m l . )
t o a v o i d errors d u e t o t h e i n c o m p l e t e s e p a r a t i o n o f t h e r a d i o a c t i v e c o m p o u n d s .
Treat t h e b l a n k c o n t a i n i n g t h e h y d r o l y s e d s a m p l e a s for t h e test. P r e p a r e s t a n d a r d s c o n t a i n i n g
0 . 0 2 , 0 . 0 4 a n d 0 . 0 6 m l . a c e t y l - C o A s t a n d a r d s o l u t i o n ( X I ) e q u i v a l e n t t o 1, 2 a n d 3 n m o l e .
P i p e t t e s u c c e s s i v e l y i n t o a test t u b e (1 c m . x 7.5 c m . ) : A m o u n t per assay mixture
Pipette o n the b o t t o m :
DL-[4- C]-Aspartate
1 4
(III) 0.001 ml. 6 0 - 8 0 nmole
P i p e t t e o n t h e e d g e o f t h e test t u b e :
2-Oxoglutarate (II) 0.001 ml. 100 n m o l e
G O T / C S mixture (IV) 0.002 ml.
W a s h s o l u t i o n s II a n d I V i n t o t h e test 2 0 pg. e a c h = 3.6 U G O T , 1.4 U C S
t u b e w i t h tris buffer (I) 0.05 ml.
5000 nmole
C o v e r t h e t u b e w i t h P a r a f i l m a n d i n c u b a t e for 5 m i n .
at 3 0 ° C .
S a m p l e (extract) 0.07 to u p to ca. 5 n m o l e

0.13 ml. acetyl-CoA
M i x a n d i n c u b a t e for a further 5 m i n . at 3 0 ° C . S t o p
t h e r e a c t i o n b y p l a c i n g t h e t u b e in a n ice-salt m i x t u r e
at - 2 °C.
Citrate solution (VI) 0.02 ml. 2000 nmole
M i x ; h e a t for 1 m i n . at 1 0 0 ° C w i t h s h a k i n g a n d c o o l
t o 0 ° C in a n ice b a t h .
Electrophoretic Separation of Radioactive Citrate from Aspartate
Treat t h e b l a n k as for t h e test. A p p l y t h e w h o l e r e a c t i o n m i x t u r e o n t o a n e l e c t r o p h o r e s i s

p a p e r ( 3 0 x 35 c m . , S c h l e i c h e r a n d S c h u l l , 2 0 4 3 a) in 0 . 0 2 t o 0 . 0 3 m l . p o r t i o n s a s a h a l f circle
o f 1.5 x 1 c m . at a d i s t a n c e 15 c m . f r o m t h e e n d o f t h e p a p e r a n d d r y in a s t r e a m o f air (fan).
S p r a y t h e p a p e r w i t h 2 N a c e t i c a c i d a n d s e p a r a t e t h e e x c e s s a s p a r t a t e f r o m the citrate b y h i g h
v o l t a g e e l e c t r o p h o r e s i s at 2 7 0 0 V a n d at — 5 ° C in t h e s a m e m e d i u m (3 hr.). D r y the e l e c t r o
p h o r e s i s p a p e r a n d s t e a m t o r e m o v e t h e last t r a c e s o f a c e t i c a c i d . W h e n t h e p a p e r is dry l o c a t e
t h e citrate s p o t b y careful s p r a y i n g w i t h m e t h y l red i n d i c a t o r s o l u t i o n ( V I I ) . Citric a c i d a p p e a r s
a s a red s p o t o n a y e l l o w b a c k g r o u n d , w h i c h h a s m i g r a t e d ca. 6 - 8 c m . t o w a r d s t h e a n o d e .
C u t t h e citrate s p o t o u t as a s q u a r e ( 4 x 4 c m . ) , treat w i t h c o n e . H C 1 f u m e s a n d d e t e r m i n e
t h e r a d i o a c t i v i t y in P P O / P O P O P s o l u t i o n ( I X ) .
T h e t r e a t m e n t w i t h H C 1 f u m e s is c o n t i n u e d u n t i l t h e a n i o n o f t h e i n d i c a t o r ( y e l l o w f o r m )
is c o n v e r t e d t o t h e h y d r o c h l o r i d e (red f o r m ) . In t h i s f o r m t h e i n d i c a t o r is n o t l e a c h e d f r o m
t h e p a p e r b y t h e s c i n t i l l a t i o n fluid a n d c o n s e q u e n t l y i n t e r f e r e n c e d u e t o s t r o n g q u e n c h i n g
is a v o i d e d .
Acetyl-CoA 1999
Calculations
Correct all sample values for the b l a n k , which h a s been o b t a i n e d by incubation of 0.1 ml. of hydrolysed
sample a n d subsequent separation of the reaction mixture u n d e r identical conditions to the non-hydrolysed
samples. O b t a i n the results by c o m p a r i s o n with k n o w n a m o u n t s of acetyl-CoA ( s t a n d a r d solution X I ) .
^ . . V x counts/min. , , . , „ , A
Content = - [nmole Acetyl-CoA/g. wet wt.]
spec. act. of a s p a r t a t e x g. x v
V = total v o l u m e of extract
v = v o l u m e of sample t a k e n for assay
g. = wt. of tissue in sample
W i t h a m e a n value of 12.3 n m o l e acetyl-CoA/ml. tissue extract the s t a n d a r d deviation was 0.475 n m o l e .

T h e coefficient of variation is 3.8%.
Normal Values
T h e acetyl-CoA content of a tissue d e p e n d s on its metabolic state. R a t liver was found to contain 16 to
104 nmole and rat kidney 16 to 44 n m o l e acetyl-CoA/g. wet w t . " . 9 1 3
Effects of drugs and other therapeutic measures . N o n e k n o w n .
Interference in the assay. If the separation of [ 4 - C ] - a s p a r t a t e a n d [ l - C ] - c i t r a t e is n o t complete, the

14 14
linear relationship between a m o u n t of sample a d d e d a n d the radioactivity in the citrate spot will n o t
hold. In this case the assay m u s t be repeated. Insufficient steaming of the p a p e r after electrophoresis
prevents the location of the citrate spots because of the acetic acid still present in the p a p e r . T h e acetic
acid can be driven off by steaming the p a p e r again. Failure to completely convert the indicator to the h y d r o
chloride form results in t o o low a c o u n t . This is seen by failure to o b t a i n a linear relationship between the
radioactivity in citrate a n d the a m o u n t of sample t a k e n . In this case the d e t e r m i n a t i o n m u s t be repeated.
T h e radiochemical assay h a s the same specificity as the s p e c t r o p h o t o m e t r i c assay; only acetyl-CoA

reacts.
References
1 W. Prinz, W. Schoner, U. Haag & W. Seubert, Biochem. Z. 346, 206 [1966].

2 E. J. Simon & D. Shemin, J. A m e r . C h e m . Soc. 75, 2520 [1953].
3 S. Ochoa in S. P. Colowick &N.O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c Press, Inc., N e w Y o r k
1955, Vol. 1, p . 688.
4 D. J. Pearson, Biochem. J. 95, 23 C [1965].
6 H. U. Bergmeyer & H. Mollering, Biochem. Z. 344, 167 [1966].
7 H. Biihring & J. Kuhnau, Klin. W s c h r . 38, 694 [I960].
8 O. Wieland, G. Loffler, L. Weiss & /. Neufeldt, Biochem. Z . 333, 10 [I960].
9 O. Wieland& L. Weiss, Biochem. Biophys. Res. C o m m u n . 10, 333 [1963].

10 W. M. Bortz & F. Lynen, Biochem. Z. 339,11 [1963].
12 W. Seubert & W. Prinz, Z. Klin. C h e m . 3, 209 [1965].
13 W. Tarnowski & M. Seemann, Z. physiol. C h e m . 348, 829 [1967].
Acetoacetyl-Coenzyme A
Karl Decker
Acetoaeetyl-CoA occurs as an intermediate in the /^-oxidation of fatty a c i d s a n d in the synthesis of 1
3-hydroxy-3-methylglutaryl-CoA 2
in cell-free systems. Acetoacetyl-CoA is n o t detectable in extracellular
fluids (blood).
Acetoacetyl-CoA is reduced by N A D H in the presence of 3-hydroxyacyl-CoA dehydrogenase, H O A D H
( L - 3 - H y d r o x y a c y l - C o A : N A D oxidoreductase, E C 1.1.1.35) to L - ( + ) - 3 - h y d r o x y b u t y r y l - C o A .
Application of Method: In the enzymology of fatty acid, acetoacetate a n d isoprenoid metabolism.
Principle
OH
(1) CH —C—CH —CO~SCoA + NADH + H

3 2
+
HOADH C H — C H — C H — C O ~ S C o A + N A D
t 3 2
+
T h e oxidation of N A D H , as measured by the decrease in extinction at 340 (334, 365) n m , is p r o p o r t i o n a l

to the a m o u n t of acetoacetyl-CoA.
A t neutral p H the equilibrium of reaction (1) favours the reduction of acetoacetyl-CoA. T h e equilibrium
constant
_ [Acetoacetyl-CoA] [ N A D H ] [H + ] 10"" 10
M at 25 ° C 3
[L-(+)-3-Hydroxybutyryl-CoA] [NAD ] +
T h e o p t i m u m p H for the d e t e r m i n a t i o n lies between p H 6.5 a n d 7.5. T h e o p t i m u m range of c o n c e n t r a t i o n s

of acetoacetyl-CoA in the assay system described here is 0 . 0 1 5 - 0 . 2 ^mole/cuvette.
Equipment
(334 or 365) n m .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 3. P o t a s s i u m h y d r o g e n c a r b o n a t e , KHC0 , 3
KH P0 2 4 A . R.
2. D i s o d i u m h y d r o g e n p h o s p h a t e , 4. P o t a s s i u m h y d r o x i d e , A . R . , c a . 8 N
Na HP0
2 4 • H 0
2 5. E t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d , EDTA
disodium salt, E D T A - N a H 2 2 • 2H 0 2
6. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo 7. 3 - H y d r o x y a c y l - C o A d e h y d r o g e n a s e ,
tide, N A D H HOADH
s o d i u m salt, N A D H - N a ; commercial p r e p a r a
2 crystalline from pig heart according to Stern*;
tion, see p . 545. ^ 9 U / m g . (25 °C); highly purified from sheep
liver, free from thiolase ( A c y l - C o A : acetyl-
CoA-C-acyltransferase, E C 2.3.1.16) according
to Lynen a n d Wieland ; 3
^ 2.5 U / m g . (25 °C) in
assay with N-acetyl-s-acetoacetylcysteamine.
Crystalline commercial preparation,see p . 474.
Purity of Reagents
3-Hydroxyacyl-CoA dehydrogenase must be free from /Mcetoacyl thiolase.
P r e p a r e all s o l u t i o n s w i t h m e t a l - f r e e , distilled w a t e r .
I. P h o s p h a t e buffer ( 0 . 2 M ; p H 6 . 8 ; 5 m M E D T A ) :
D i s s o l v e 1.361 g. K H P 0 , 1.78 g. N a H P 0 - 2 H 0 a n d 186 m g .
2 4 2 4 2 EDTA-Na H -2H 0 2 2 2
in distilled w a t e r a n d m a k e u p t o 100 m l .
II. P o t a s s i u m h y d r o g e n c a r b o n a t e (ca. 1 M ) :
III. R e d u c e d n i c o t i n a m i d e - a d e n i n e - d i n u c l e o t i d e (ca. 10 m M / ? - N A D H ) :
D i s s o l v e 9.3 m g . N A D H - N a 2 in 1 m l . d i s t i l l e d w a t e r .
I V . 3 - H y d r o x y a c y l - C o A d e h y d r o g e n a s e , H O A D H (ca. 3 m g . p r o t e i n / m l . ) :
If n e c e s s a r y , d i l u t e t h e s t o c k s u s p e n s i o n w i t h p h o s p h a t e buffer ( s o l u t i o n I).
Solutions of 3-hydroxyacyl-CoA dehydrogenase are stable for long periods at 0 °C. F r e q u e n t freezing
and thawing leads to a decrease in activity. T h e N A D H solution should be freshly prepared each week
and always be stored at 0 °C. To avoid bacterial c o n t a m i n a t i o n store the other solutions in a refrigerator.
Polyethylene bottles should be used for the buffer a n d alkaline solutions.
Procedure
S e e C h a p t e r " A c e t y l - C o A " , p . 1990. T h e n e c e s s a r y r e a g e n t s a n d s o l u t i o n s are d e s c r i b e d

there. C o n c e n t r a t e s o l u t i o n s b y f r e e z e - d r y i n g rather t h a n b y p h e n o l e x t r a c t i o n .
S o l u t i o n s o f a c e t o a c e t y l - C o A s h o u l d b e b e t w e e n p H 4 a n d 6 ; t h e y are s t a b l e for s e v e r a l m o n t h s
in t h e f r o z e n state. A c e t o a c e t y l - C o A is h y d r o l y s e d b y alkali j u s t a s r a p i d l y a s a c e t y l - C o A
a n d , like t h e latter, it is s e n s i t i v e t o t h e p r o l o n g e d a c t i o n o f s t r o n g a c i d . T h e p r e s e n c e o f h i g h
c o n c e n t r a t i o n s o f m e r c a p t a n s , e s p e c i a l l y c y s t e i n e , s h o u l d b e a v o i d e d a t p H v a l u e s greater
t h a n 6.5 ( t h i o l e x c h a n g e ) .
Acetoacetyl-CoA 2003
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 1.855 m l . ; r o o m

temperature; read against a control cuvette containing water instead o f sample.
Pipette successively into C o n c e n t r a t i o n in a s s a y

Test Control
cuvettes: mixture
P h o s p h a t e buffer (I) 0.50 ml. 0.50 ml. 54 m M phosphate

1.35 m M E D T A
S a m p l e + distilled water 1.30 m l . 1.30 m l . H 2 0 u p t o 100 juM
N A D H solution (III) 0.05 ml. 0.01 m l . 0.27 m M N A D H
M i x , f o l l o w extinction until constant a n d read E.l
H O A D H solution (IV) 0.005 ml. 0.005 ml. 8.1 pg./ml = 73 m U / m l .
M i x ; after c a . 3 - 4 m i n . r e a d e x t i n c t i o n E 2 which should be

constant. —E 2 = A E is u s e d f o r t h e c a l c u l a t i o n s .
T h e N A D H c o n c e n t r a t i o n is s o a r r a n g e d t h a t t h e e x t i n c t i o n a t 3 6 5 n m lies o n t h e m o s t a c c u r a t e
r a n g e o f t h e s p e c t r o p h o t o m e t e r . If m e a s u r e m e n t s a r e m a d e at 3 4 0 o r 3 3 4 n m , p i p e t t e o n l y
0 . 0 3 m l . N A D H s o l u t i o n i n t o t h e test c u v e t t e . T h e a d d i t i o n o f N A D H t o t h e c o n t r o l c u v e t t e
ensures that even w h e n E 2 is v e r y l o w t h e r e is still e x c e s s N A D H p r e s e n t in t h e test c u v e t t e .
It is r e c o m m e n d e d t h a t t h e a m o u n t o f a c e t o a c e t y l - C o A b e s o a r r a n g e d t h a t t h e e x t i n c t i o n
difference E x —E 2 does n o t exceed 0.5.
Calculations
The reaction proceeds quantitatively a n d stoichiometrically u n d e r the a b o v e conditions a n d therefore

the calculation formula (2) o n p . 312 applies.
With the above conditions the a m o u n t of acetoacetyl-CoA in the sample is given b y :
/zmole= 0.304 x AE x ^- 0.298 x AE x ^ 0.546 x zl E x —

v 2 v 2 v 2
where
Vj = total v o l u m e of acetoacetyl-CoA solution in ml.
v = volume of acetoacetyl-CoA solution taken for t h e assay in ml.
2
T h e reaction of 10 pmole C o A S H with diketene gave an acetoacetyl-CoA solution containing a m e a n

of 10.13 + 0.06 jumole ( s t a n d a r d deviation of 8 d e t e r m i n a t i o n s ) ; this gives a coefficient of variation of 0.6%.
Normal Values
T h e c o n c e n t r a t i o n of acetoacetyl-CoA in tissues is very l o w ; n o values are k n o w n .

Interference in the assay: T h e purest p r e p a r a t i o n s of 3-hydroxyacyl-CoA dehydrogenase are practically

free from thiolase. If this is not the case then acetyl-CoA will react according to the overall equilibrium
of the two r e a c t i o n s and lead to t o o high values. Similarly, acetoacetyl-CoA c a n n o t be determined in the
5
presence of free C o A , pantetheine, pantetheine p h o s p h a t e or N-acetyl-cysteamine with a n enzyme prepa

ration containing thiolase otherwise t o o low results will be obtained.
Specificity
3-Hydroxyacyl-CoA dehydrogenase does n o t show very m a r k e d specificity with regard to either the
m e r c a p t a n or 3-ketoacyl g r o u p . T h e thiol ester of pantetheine p h o s p h a t e ( K M = 0.22 m M ) , pantetheine
( K = 0.09 m M ) or N-acetylcysteamine ( K = 9 m M ) , as well as the C o A derivative of acetoacetic acid
M M
(K M = 0.04 m M ) are reduced by N A D H . T h e K 5

M values are for the enzyme from sheep liver; 3-hydroxy-
acyl-CoA dehydrogenase from pig liver shows only slight differences with respect to the K M values, but
there are m o r e i m p o r t a n t differences in the t u r n o v e r n u m b e r s .
It is c o m m o n l y agreed t h a t only one 3-hydroxyacyl-CoA dehydrogenase exists; t h a t is to say the enzyme
reacts with the 3-hydroxy- o r 3-ketoacyl-CoA derivatives of chain length u p to a b o u t C . A differentiation 2 0
of chain length is therefore n o t possible with this enzyme. O t h e r carbonyl c o m p o u n d s , for example 2-keto
acid derivatives, aldehydes, ketones a n d 3-keto acids n o t b o u n d to C o A d o n o t react. T h e reversible
reduction of acetoacetate to D-( — )-3-hydroxybutyrate is catalysed by a n o t h e r e n z y m e (p. 1836). 6
O t h e r M e t h o d s for t h e D e t e r m i n a t i o n o f A c e t o a c e t y l - C o A
Acetoacetyl-CoA is split by /?-ketoacyl-CoA thiolase according to the e q u a t i o n :
(2) Acetoacetyl-CoA + C o A S H ^ 2 Acetyl-CoA
M e a s u r e m e n t of the p H - d e p e n d e n t U V - a b s o r p t i o n of acetoacetyl-CoA at 300 n m , which can be increased

by M g 2 +
, is the basis of this m e t h o d ' . A n o t h e r m e t h o d for measuring acetoacetyl-CoA is to couple
5 7
reaction (2) with arylamine acetylase (Acetyl-CoA: arylamine-7V-acetyltransferase, E C 2.3.1.5) or with

8
citrate s y n t h a s e '9 10
(Citrate-oxaloacetate-lyase, E C 4.1.3.7) (p. 1988). A c e t o a c e t y l - C o A can be estimated
relatively quickly a n d simply by a non-enzymatic m e t h o d d e p e n d e n t on its U V - a b s o r p t i o n . T h e h y d r o -
5
xamic acid a n d the nitroprusside reactions are not applicable to 3-ketoacyl-CoA derivatives.
References
1 F. Lynen, F e d . P r o c . 72, 683 [1953].

2 H. Rudney, J. biol. C h e m . 227, 363 [1957].
3 F. Lynen & O. Wieland in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. Academic Press,
N e w Y o r k 1955, vol. I, p . 566.
4 J. R. Stern, Biochim. biophys. A c t a 26, 448 [1957].
5 K. Decker: Die aktivierte Essigsaure. F e r d . E n k e , Stuttgart 1959.
6 A. L. Lehninger & G. D. Greville, Biochim. biophys. Acta 12, 188 [1953].
7 / . R. Stern, J. biol. C h e m . 221, 33 [1956].
8 F. Lynen, K Decker, O. Wieland & D. Reinwein in G. Popjak & E. LeBreton: Biochemical Problems
of Lipids, Butterworths, L o n d o n 1956.
9 Z). S. Goldman, J. biol. C h e m . 208, 345 [1954].
10 J. R. Stern, M. J. Coon & A. Del Campillo, J. biol. C h e m . 221, 1 [1956].
Acrylyl-Coenzyme A
P. R o y V a g e l o s
Acrylyl-CoA is a m i n a t e d with N H C 1 a n d acrylyl-CoA aminase (/?-Alanyl-CoA a m m o n i a lyase, E C

4
4.3.1.6), with a c o r r e s p o n d i n g decrease in extinction at 263 n m . Acrylyl-CoA can be quantitatively

determined by m e a n s of this change in extinction . 1
Principle
/P ^ Acrylyl-CoA- + ^ °
(1) CH =CH-C
2 N + NH 3 + H +
H N-CH -CH -C
3 2 2
S-COA aminase S"CoA
The equilibrium c o n s t a n t of this reaction is 58 x 1 0

2 1 3
(mole/I.) - 2
at p H 7.5 and 25 °C. With excess
a m m o n i u m ions a mole /?-alanyl-CoA is formed per m o l e acrylyl-CoA. T h e d i s a p p e a r a n c e of acrylyl-
C o A is measured spectrophotometrically at 263 n m .
T h e conditions given below are o p t i m u m .
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for p r e c i s e m e a s u r e m e n t s at 2 6 3 n m .
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e precipitation, a m m o n i u m sulphate precipita

2. S o d i u m h y d r o x i d e , 2 N tion, dialysis, a d s o r p t i o n o n calcium p h o s p h a t e
3. A m m o n i u m c h l o r i d e gel, second a m m o n i u m s u l p h a t e precipitation,
4. A c r y l y l - C o A a m i n a s e dialysis). It is stable for at least one m o n t h at
purified 75-fold a c c o r d i n g t o from extracts of
1
- 20 °C.
Clostridium propionicum (protamine sulphate
D i s s o l v e 1 8 . 6 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in c a . 5 0 m l . d i s t i l l e d w a t e r , a d j u s t p H
t o 7.5 w i t h 2 N N a O H a n d d i l u t e t o 100 m l . w i t h d i s t i l l e d w a t e r .
II. A m m o n i u m c h l o r i d e ( 1 . 0 M ) :
D i s s o l v e 5.3 g. N H C 1 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
4
III. A c r y l y l - C o A a m i n a s e (ca. 75 m U / m l . ) :
Dilute the preparation obtained according t o 1
with distilled water. Store the solution
at - 2 0 ° C .
Procedure
Sample
T h e m e t h o d w a s d e s i g n e d for t h e d e t e r m i n a t i o n o f a c r y l y l - C o A a m i n a s e . It s h o u l d
1
be
a p p l i c a b l e t o a n y t y p e o f m a t e r i a l in w h i c h a c r y l y l - C o A is t h e o n l y c o m p o u n d h a v i n g a s t r o n g
a b s o r p t i o n at 2 6 3 n m .
Assay System
W a v e l e n g t h : 2 6 3 n m ; 1 m l . q u a r t z c u v e t t e s , light p a t h : 1 c m . ; final v o l u m e : 1 m l . ; r o o m
temperature. R e a d against reference cuvette containing distilled water instead o f sample.
Triplicate d e t e r m i n a t i o n s .
Concentration in
assay mixture
S a m p l e + distilled water 0.80 ml. 20-100 pM

Buffer (I) 0.05 ml. 50 m M
N H C 1 solution
4 (II) 0.10 ml. 0.1 M
M i x and read extinction E . x
Enzyme solution (III) 0.05 ml. 3.75 m U / m l .
M i x , r e a d e x t i n c t i o n s e v e r y 3 0 sec. until t h e r e is n o
further d e c r e a s e . M u l t i p l y t h e final e x t i n c t i o n s E 2 by
1.05 ( d i l u t i o n f a c t o r ) , s u b t r a c t t h e m e a n f r o m t h e m e a n
o f t h e initial e x t i n c t i o n s . AE = E t — 1.05 E 2 is u s e d
T h e a m o u n t o f e n z y m e u s e d s h o u l d b e sufficient t o b r i n g t h e r e a c t i o n t o c o m p l e t i o n in 1 - 3
m i n . ; m o r e e n z y m e d e c r e a s e s t h e specificity.
Calculations
The decrease in extinction at 263 n m is strictly p r o p o r t i o n a l to the a m o u n t of acrylyl-CoA between 0.01

and 0.13 /miole/ml. The acrylyl-CoA content of the assay mixture is calculated from the extinction co
efficient of 6.7 c m . / / m i o l e for the thioester b o n d a c r y l y l - C o A . H e n c e
2 3
AE
c = [/zmole/ml.]
Specificity and S o u r c e s o f Error
C r u d e extracts of Clostridium propionicum, which have been grown o n jft-alanine instead of a-alanine,
m a y have acrylyl-CoA aminase activity (up to 1.36 U / m g . ) . Since so little protein is required such crude
1
extracts can be used directly in this m e t h o d without risk of interference from side reactions.
Acrylyl-CoA aminase reacts with the following c o m p o u n d s (in descending o r d e r of reactivity): acrylyl-
C o A , c r o t o n y l - C o A , acrylyl-pantetheine, c r o t o n y l - p a n t e t h e i n e . It reacts slowly with acrylyl-N-acetyl-
1
Acrylyl-CoA 2007
t h i o e t h a n o l a m i n e , b u t n o t at all with acrylyl-S-thiopropionic acid a n d crotonyl-N-acetylthioethanol-

amine. T h e rates of reaction differ e n o r m o u s l y : e. g. acrylyl-CoA a m i n a s e reacts with c r o t o n y l - C o A at
only 5 % of the rate with acrylyl-CoA. M e a s u r e m e n t s of kinetics of the reaction give an indication which
c o m p o u n d in the experimental material reacts. If m o r e enzyme is used in the assay m i x t u r e t h a n is stated
above, the d e t e r m i n a t i o n will be less specific a n d will also include other thiolesters reacting with acrylyl-
C o A aminase.
References
1 P. R. Vagelos, J. M. Earl & E. R. Stadtman, J. biol. C h e m . 234, 490 [1959].

2 P. R. Vagelos & /. M. Earl, J. biol. C h e m . 234, 2272 [1959].
3 F. Lynen, Fed. P r o c . 12, 683 [1953].
Benzoyl Coenzyme A and other Coenzyme A Derivatives
Gerhard Michal and Hans Ulrich Bergmeyer
The assay m e t h o d described here is applicable not only to benzoyl-CoA, but also to all C o A thioesters.
Unlike those described in other chapters it is not limited to a special type of thioester.
The thioester is hydrolysed at alkaline p H , and the C o A liberated is then determined by one of the usual
enzymatic assay m e t h o d s (see C h a p t e r " C o e n z y m e A " ) . The most suitable m e t h o d for the determination
of C o A is the m e t h o d with 3-hydroxyacyl-CoA dehydrogenase, H O A D H (L-3-Hydroxyacyl-CoA: N A D
oxidoreductase, EC 1.1.1.35) after conversion of the C o A to acetoacetyl-CoA with diketene.
Principle
O O
(1) / Vc-S-CoA + 2 OH" —• / Vc-O" + CoA-S" + H 0
2
CH =C-0
(2) * • + CoA-SH —* CH -CO-CH -CO-S-CoA
3 2
(3) CH3-CO-CH2-CO-S-C0A + NADH + H + H Q A D H

> C H 3 - C H O H - C H 2 - C O - S - C 0 A + NAD+
The decrease of N A D H , as measured by the change in extinction at 340 (334, 365) n m , is p r o p o r t i o n a l

to the a m o u n t of acyl-CoA present.
The half-time of the hydrolysis of the thioesters of saturated fatty acids of chain length C to C ( p K = 2 8 a
= 4.7 — 4.9) in 0.1 N N a O H is only 1 - 2 min. at 30 °C. T h e crotonyl derivative is somewhat m o r e stable.
Generally the thioesters with low p K values are hydrolysed m o r e rapidly in alkali t h a n those with high
a
values . The hydrolysis time stated here is sufficient for all the i m p o r t a n t thioesters. Stauff a n d Jaenicke
1 2
give m a n y p K values.
a
C o A - S H is easily oxidized to C o A - S - S - C o A in alkaline m e d i u m . Therefore to obtain quantitative con

version it is necessary to introduce a reduction step before the enzymatic reaction. This is included in the
H O A D H m e t h o d (see p . 1968). If an acyl-CoA derivative is formed during the course of the reaction, e.g.
in the P T A m e t h o d for C o A (see p . 1972) preliminary treatment with a reducing agent is not possible
because of the transfer of the acyl g r o u p to the S H reducing a g e n t s . 3
Equipment
3 3 4 or 365 n m .
R e a g e n t s , see p . 1 9 6 9 .
Benzyl-CoA a n d other C o e n z y m e A Derivatives 2009
S e e p . 1 9 6 9 ; in a d d i t i o n :
V I . S o d i u m h y d r o x i d e (0.1 N ) :
D i l u t e 5.0 m l . s o l u t i o n III t o 100 m l . w i t h d i s t i l l e d w a t e r .
Procedure
T h e m e t h o d is m a i n l y s u i t a b l e for purified p r e p a r a t i o n s . B e n z o y l - C o A is p a r t i c u l a r l y s t a b l e
in t h e p H r a n g e 4 . 5 - 6 . T h e r e is n o m e a s u r a b l e d e c r e a s e w i t h t h e H O A D H a s s a y in 2 d a y s at
0 °C. T h e other acyl derivatives have varying stabilities.
Assay System
A d j u s t s o l u t i o n s t o 0.1 N N a O H b y a d d i t i o n o f N a O H ( s o l u t i o n III) a n d a b o u t t o 0 . 7 m M
a c y l - C o A t h i o e s t e r ( w i t h m e d i u m c h a i n - l e n g t h a c y l g r o u p s a b o u t 0.7 m g . / m l . ) . In t h e c a s e o f
s o l i d s w e i g h o u t ca. 7 m g . a n d d i s s o l v e in 10 m l . 0.1 N N a O H ( s o l u t i o n V I ) . A l l o w t o s t a n d at
r o o m t e m p e r a t u r e (ca. 2 2 °C) for 2 0 m i n . a n d t h e n a d j u s t t o p H 9 . 0 w i t h 1 N H C 1 ( s o l u t i o n I)
a n d n o t e t h e c h a n g e in v o l u m e .
T h e n a l l o w t h e s a m p l e t o react w i t h t h i o g l y c o l l i c a c i d a n d d i k e t e n e a c c o r d i n g t o t h e p r o c e d u r e
o n p. 1970, and analyse by the H O A D H m e t h o d .
Calculations
The relationships expressed on a m o l a r basis are given on p . 1971. To calculate the c o n c e n t r a t i o n in m g . / m l .

the molecular weight of the thioester (for b e n z o y l - C o A : 870.6) must be used. T h e dilution factor should
also include the dilutions on addition of N a O H a n d HC1.
W i t h pure substance (77 /ig./cuvette) a s t a n d a r d deviation of 1.9 /ig./cuvette was o b t a i n e d ; coefficient

of variation is 2 . 5 % .
As described for the H O A D H assay (p. 1972).
References
1 F. Lynen & Th. Wieland, cited in E. R. Stadtman in S. P. Colowick & N. O. Kaplan: M e t h o d s in E n
zymology. Academic Press, N e w Y o r k 1957, vol. I l l , p . 934.
2 J. Stauff & R. Jaenicke in H. M. Rauen: Biochemisches Taschenbuch. Springer-Verlag, B e r l i n - G o t t i n -
g e n - H e i d e l b e r g - N e w Y o r k 1964, part 2, p. 7 1 .
Butyryl-CoA and CoA Derivatives of Higher
Saturated Fatty Acids
Colorimetric Determination
Werner Seubert
T h e usual m e t h o d s for the d e t e r m i n a t i o n of the C o A derivatives of s a t u r a t e d fatty acids depend on the

characteristic a b s o r p t i o n of the thioester b o n d at 2 3 3 n m o r o n the reaction of the thioester with hydroxy 1-
amine. In the first m e t h o d the a b s o r p t i o n of thioester b o n d is in the s a m e area as t h a t of the adenosine
moiety a n d to exclude the c o n t r i b u t i o n of the a d e n o s i n e moiety the m e a s u r e m e n t s are m a d e against a
hydrolysed sample (difference spectrum). In the second m e t h o d the c o n c e n t r a t i o n of the C o A derivatives
is obtained from the a b s o r p t i o n of the ferric complex of the resulting h y d r o x a m i c acid. T h e hydrolysis of
long-chain acyl C o A derivatives, even in a nitrogen a t m o s p h e r e , results in the formation of products which
a b s o r b in t h e s a m e r a n g e as the thioester. D e t e r m i n a t i o n of c o n c e n t r a t i o n by difference spectrum is there
fore often n o t valid. B o t h m e t h o d s are also non-specific a n d insensitive.
T h e principle of using redox dyes as h y d r o g e n acceptors for the d e t e r m i n a t i o n of the activity of d e h y d r o
genases was developed by Thunberg . This m e t h o d is also applicable t o the determination of C o A derivatives
1
of the higher saturated fatty acids by m e a n s of acyl-CoA dehydrogenase ( A c y l - C o A : (acceptor) oxidore

ductase, E C 1 . 3 . 9 9 . 3 ) 2
a n d the "electron-transferring flavoprotein" ( E T F ) * . T h e acyl-CoA d e h y d r o
3
genase is well suited for the d e t e r m i n a t i o n of long-chain C o A derivatives because it is specific for the C o A
esters of the C 4 to C 1 6 fatty acids.
E T F can transfer h y d r o g e n t o synthetic h y d r o g e n acceptors, such as p o t a s s i u m ferricyanide o r dichloro-
p h e n o l i n d o p h e n o l , as well a s t o c y t o c h r o m e c. D i c h l o r o p h e n o l i n d o p h e n o l is used in this m e t h o d because
of its high extinction coefficient.
Principle
(1) R — C H — C H — C O — S C o A + A c y l - C o A d e h y d r o g e n a s e - F A D -*
2 2
- • R — C H = C H — C O — S C o A + Acyl-CoA d e h y d r o g e n a s e - F A D H 2
* '
(2) Acyl-CoA dehydrogenase-FADH + E T F - F A D - * A c y l - C o A dehydrogenase-FAD + E T F - F A D H
2 2
, I
(3) E T F - F A D H + Dichlorophenolindophenol—»ETF-FAD + Leucodichlorophenolindophenol

2
T h e decolorization of d i c h l o r o p h e n o l i n d o p h e n o l as measured by the c h a n g e of extinction at 5 7 8 n m is

p r o p o r t i o n a l t o the a m o u n t of the acyl-CoA derivative present.
T h e r e d o x potentials for the systems b u t y r y l - C o A / c r o t o n y l - C o A ( R = C H ) a n d dichlorophenolindo-

3
p h e n o l / l e u c o d i c h l o r o p h e n o l i n d o p h e n o l are Eq = -0.015 4
a n d Eq = + 0 . 2 2 respectively. The reaction
goes virtually t o completion a n d with a n excess of d i c h l o r o p h e n o l i n d o p h e n o l acyl-CoA is quantitatively
* N o system n u m b e r h a s been assigned by the E n z y m e C o m m i s s i o n .

Butyryl-CoA and C o A Derivatives of Higher Saturated Fatty Acids 2011
converted to dehydroacyl-CoA (Fig. 1). The amount of dye decolorized is proportional to the added
acyl-CoA derivative.
0.300
0.200
in
UJ
0.100
Fig. 1. Standardization of the method with C o A
derivatives of different chain length
Capronyl-CoA ( C ) + 6
Caprylyl-CoA ( C ) 8•
Caprinyl-CoA ( C ) O 1 0 0.01 0.02
Palmityl-CoA ( C ) •
1 6 julMoI Adenine-
Equipment
S p e c t r o p h o t o m e t e r o r p h o t o m e t e r for a c c u r a t e m e a s u r e m e n t s at a r o u n d 6 0 0 n m ; bench
centrifuge.
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 6. " E l e c t r o n - t r a n s f e r r i n g flavoprotein",
KH P0 2 4 ETF
2. D i s o d i u m h y d r o g e n p h o s p h a t e , N a H P 0 2 4
from pig liver mitochondria, ^0.11 U/mg.
3. 2,6-Dichlorophenolindophenol, (30 °C)*. Isolation,see p. 2014.
sodium salt 7. " A c y l - C o A d e h y d r o g e n a s e "
4. I o d i n e , s u b l i m e d from pig liver mitochondria , ^ 0 . 1 8 2
U/mg.
5. P o t a s s i u m i o d i d e (30 °C)*. Isolation,see p. 2014.
P r e p a r e all s o l u t i o n s w i t h freshly p r e p a r e d , d o u b l y d i s t i l l e d w a t e r .
I. P h o s p h a t e buffer ( 0 . 0 6 7 M ; p H 7 . 0 ) :
D i s s o l v e 7 . 1 2 g. N a H P 0
2 4 • 2 H 0 a n d 3 . 5 4 g. K H P 0
2 2 4 in distilled w a t e r a n d m a k e
up to 1000 ml.
* 1 U is the amount of enzyme which, under the conditions of the assay (0.5 ml. final volume, 0.05 /miole 2
butyryl-CoA, 30 °C), decolorizes 1 /miole 2,6-dichlorophenolindophenol per min.

II. 2 , 6 - D i c h l o r o p h e n o l i n d o p h e n o l ( 0 . 3 m g . / m l . ; p H 7 . 0 ) :
D i s s o l v e 3.0 m g . N a salt o f t h e d y e w i t h buffer ( s o l u t i o n I) a n d m a k e u p t o 10 m l .
III. I o d i n e - p o t a s s i u m i o d i d e ( 0 . 0 1 N ) :
D i s s o l v e 1.8 g. K I in a little d i s t i l l e d w a t e r in a 1 0 0 0 m l . v o l u m e t r i c flask, a d d 1.27 g.
i o d i n e , s t o p p e r flask a n d s h a k e u n t i l all t h e i o d i n e is d i s s o l v e d . If n e c e s s a r y , a d d s o m e
m o r e K I c r y s t a l s . D i l u t e w i t h d i s t i l l e d w a t e r t o 1 0 0 0 m l . S t o r e t h e s o l u t i o n in a b r o w n
bottle.
IV. "Electron-transferring flavoprotein", E T F (12 mg. protein/ml.):
D i l u t e the e n z y m e suspension (30 to 40 m g . protein/ml.) prepared according to p. 2014.
w i t h p h o s p h a t e buffer ( s o l u t i o n I) t o g i v e 12 m g . p r o t e i n / m l .
V. " A c y l - C o A dehydrogenase", (10 mg. protein/ml.):
D i l u t e t h e e n z y m e s u s p e n s i o n ( 3 0 t o 4 0 m g . p r o t e i n / m l . ) w i t h p h o s p h a t e buffer ( s o l u t i o n I).
P r e p a r e the d i c h l o r o p h e n o l i n d o p h e n o l solution freshly each week. T h e enzyme p r e p a r a t i o n s are stable

for a b o u t 6 m o n t h s at —15 °C.
Procedure
T h e conditions described for collection, t r e a t m e n t a n d stability of the sample u n d e r " R a d i o c h e m i c a l

Assay for D e t e r m i n a t i o n of A c e t y l - C o A " (see p. 1994) also apply here. It should be n o t e d however t h a t
u n d e r these conditions only the C o A derivatives of the C to C 4 1 2 fatty acids go into solution. T h e higher
h o m o l o g u e s remain in the residue because of their insolubility in acid. T h e present m e t h o d is therefore
most suitable for the d e t e r m i n a t i o n of synthetically p r e p a r e d acyl-CoA derivatives.
C o A derivatives of saturated fatty acids are stable virtually indefinitely when stored as dry powders in a
desiccator at 0 °C. The stability of a q u e o u s solutions of these c o m p o u n d s is related to the stability of
the thioester b o n d . The thioester b o n d of short-chain S-acyl-N-acetylcysteamine derivatives is hydrolysed
in a few m i n u t e s by 0.1 N K O H . T h e stability increases with increase in chain length of the fatty acid.
8
T h e thioester b o n d is very stable between p H 3 a n d 4. Acetyl-CoA can be heated at between p H 3.5 a n d

5 for 15 min. at 100 °C w i t h o u t appreciable d e c o m p o s i t i o n . A q u e o u s solutions of C o A derivatives should
5
therefore be stored at slightly acid p H a n d at low t e m p e r a t u r e s (0 to —15 °C).

Butyryl-CoA a n d C o A Derivatives of H i g h e r S a t u r a t e d F a t t y Acids 2013
Assay System
W a v e l e n g t h : H g 578 n m ; light p a t h : 1 c m . ; final v o l u m e : 1.07 m l . ; r o o m t e m p e r a t u r e ; r e a d

a g a i n s t a reference c u v e t t e c o n t a i n i n g buffer s o l u t i o n I i n s t e a d o f s a m p l e .
Sample ( b u f f e r e d w i t h s o l u t i o n I) 1.00 m l . u p t o 2 5 fiM acyl-CoA

Dichlorophenolindophenol(solutionll) 0.05 ml. 75 fiM dichlorophenolindophenol
M i x a n d f o l l o w t h e e x t i n c t i o n c h a n g e s in b o t h c u v e t
tes until c o n s t a n t ( a b o u t 5 min.). Set the extinction o f
the reference cuvette at E = 0.6. R e a d the extinction
E A o f t h e test c u v e t t e a g a i n s t t h e r e f e r e n c e c u v e t t e .
M i x into each cuvette.
E T F suspension (IV) 0.01 m l . 1 2 0 /zg./ml.

Acyl-CoA dehydrogenase (V) 0.01 m l . 100 pg./ml.
Continually adjust the extinction o f the reference

c u v e t t e t o E = 0.6. F o l l o w t h e d e c r e a s e in e x t i n c t i o n
in t h e test c u v e t t e . R e a d E 2 w h e n a c o n s t a n t v a l u e is
o b t a i n e d (ca. 5 - 1 0 m i n . ) .
Ej — E 2 = A E is u s e d for t h e c a l c u l a t i o n s .
Calculations
T h e reaction proceeds stoichiometrically u n d e r the described conditions. T h e calculation formula ( 2 )

on p . 312 applies. T h e extinction coefficient of d i c h l o r o p h e n o l i n d o p h e n o l is 15.6 c m . / / m i o l e * at 600 n m
2
a n d 14.3 c m . / / m i o l e at 578 n m a n d therefore:

2
c= AE x 0.0748 AE x 0.0685 [/miole/ml.]
Dilution of the sample d u r i n g p r e t r e a t m e n t m u s t be allowed for separately.
Replicate determination of a c a p r y l - C o A solution gave a m e a n value of 4.76 /imole/ml. with a deviation

of 0.2. Coefficient of variation is 4 . 2 % .
* T h e published values for the extinction coefficient of d i c h l o r o p h e n o l i n d o p h e n o l at 600 n m vary

between 16.0 a n d 19.0 c m . / / i m o l e ' . T h e extinction coefficient given here was o b t a i n e d by use of
2 2 6 - 8
p u r e C o A derivatives of butyric, caproic, caprylic, capric a n d palmitic a c i d s ' . T h e purity was j u d g e d

8 9
by means of the S-acyl o r a d e n i n e c o n t e n t a n d n o c o n t a m i n a n t s a b s o r b i n g at 260 n m c o u l d be detected

chromatographically in these p r e p a r a t i o n s . T h e ratios of the c o n t e n t s of S-acyl: a d e n i n e : p h o s p h a t e
were between 1 : 1 : 2.99 a n d 1 : 1 : 3 . 1 6 . T h e r e was a linear relationship between the a d e n i n e c o n t e n t
8
of the C o A derivatives a n d the A E values o b t a i n e d by the enzymatic m e t h o d of d e t e r m i n a t i o n , a n d

this was independent of the chain length of the fatty acid (Fig. 1).
If the samples c o n t a i n large a m o u n t s of reducing agents, these m u s t be oxidized with iodine before the
d e t e r m i n a t i o n . To avoid a n excess of iodine, first determine the a m o u n t of iodine required for a p o r t i o n
of t h e sample a n d then titrate the sample (apart from a small fraction) t o a definite yellow colour. R e d u c e
the excess iodine by addition of the small a m o u n t of u n t r e a t e d sample. Small a m o u n t s of reducing c o m
p o u n d s still present a r e oxidized before t h e d e t e r m i n a t i o n with a n excess of d i c h l o r o p h e n o l i n d o p h e n o l .
C o A samples used for the p r e p a r a t i o n of acyl-CoA derivatives by t r e a t m e n t with the corresponding
acid anhydrides m u s t be checked for their glutathione content. A n y glutathione present will react with
the acid a n h y d r i d e t o give the S-acyl glutathione derivative a n d subsequently this will be hydrolysed to
glutathione by deacylases present in the enzyme p r e p a r a t i o n s . This leads t o high values for the acyl-CoA
in the assay because the glutathione will decolorize the d i c h l o r o p h e n o l i n d o p h e n o l .
A c y l - C o A d e h y d r o g e n a s e is specific for the C o A derivatives of the C 4 to C 1 8 fatty acids. G l u t a t h i o n e

derivatives d o n o t react.
Appendix
Acyl-CoA Dehydrogenase and "Electron-transferring Flavoprotein" (ETF)
T h e activity of b o t h enzymes is m e a s u r e d by the s a m e m e t h o d as described for the determination of the

acyl-CoA derivatives. T h e m e a s u r e of the activity is the rate of decolorization of the dye. Since a combinat
ion of b o t h enzymes is required for the t r a n s p o r t of the substrate hydrogen t o the acceptor only the activity
of t h e enzyme which limits t h e overall reaction c a n be m e a s u r e d . To follow the purification of the acyl-
C o A d e h y d r o g e n a s e by assays of its activity an excess of E T F m u s t be added. T h e converse applies in
the purification of E T F . It is therefore necessary t o purify o n e of the enzymes " b l i n d " .
"Electron-transferring flavoprotein" (ETF) .3

Extract 100 g. acetone p o w d e r of pig liver m i t o c h o n d r i a
with tris-acetate buffer ( p H 7.5) a n d precipitate t h e inactive protein in the extract with Z n lactate. By
addition of solid a m m o n i u m sulphate to the s u p e r n a t a n t o b t a i n four fractions at 35, 50, 65 and 8 5 %
s a t u r a t i o n . C o m b i n e the fractions between 50 a n d 6 5 % a n d 65 a n d 8 5 % saturation, dialyse a n d then
precipitate inactive protein by addition of Z n lactate. F r a c t i o n a t e the filtrate with alcohol at - 1 5 °C
( 1 0 , 1 6 a n d 4 0 % alcohol). E T F occurs in the third fraction ( 1 6 - 4 0 % ) . After dialysis of the protein solution
fractionate with solid a m m o n i u m sulphate. F r a c t i o n a t e the 6 5 - 9 0 % fraction at p H 8.1 with a m m o n i u m
sulphate. A p r e p a r a t i o n suitable for the acyl-CoA assay is obtained between 7 0 - 7 5 % saturation.
Acyl-CoA dehydrogenase*. Extract 300 g. acetone p o w d e r of pig liver m i t o c h o n d r i a with p h o s p h a t e

buffer, centrifuge a n d fractionate the extract with solid a m m o n i u m sulphate. Dialyse the precipitate
obtained between 55 a n d 6 5 % s a t u r a t i o n a n d then heat for 2 min. at 57 °C. Centrifuge off denatured
protein a n d precipitate inactive protein by addition of Z n lactate t o the s u p e r n a t a n t fluid. F r a c t i o n a t e
with alcohol a n d then fractionate the precipitate o b t a i n e d between 18 a n d 2 3 % alcohol with a m m o n i u m
s u l p h a t e at p H 8.1.
References
1 C. Oppenheimer & L. Pinaussen, Die M e t h o d i k d e r F e r m e n t e , G e o r g T h i e m e , Leipzig 1929, vol. I l l p . 8.

2 F. L. Crane, S. MU, J. G. Hauge, D. E. Green & H. Beinert, J. biol. C h e m . 218, 701 [1956].
3 F. L. Crane & H. Beinert, J. biol. C h e m . 218, 111 [1956].
Butyryl-CoA a n d C o A Derivatives of Higher S a t u r a t e d F a t t y Acids 2015
4 / . G. Hauge, J. A m e r . c h e m . Soc. 78, 5 2 6 6 [1956].

5 E. Stadtman, J. biol. C h e m . 196, 535 [1952].
6 R. E. Basford & F. M. Huennekens, J. A m e r . c h e m . Soc. 77, 3871 [1955].
7 D. A. Green, S. MU & H. R. Mahler, J. biol. C h e m . 206,7 [1954].
8 W. Seubert, Dissertation Univers. M u n i c h [1955].
9 W. Seubert, Biochem. P r e p . 7, 80 [1959].
Fluorimetric Determination with Oxoglutarate Dehydrogenase

Peter Bryan Garland
Hydrolysis of acyl-CoA gives free C o A which can be determined s p e c t r o p h o t o m e t r i c a l l y ' 1 2

or fluori-
metrically . T h e m e t h o d is virtually identical with the fluorimetric d e t e r m i n a t i o n of C o A with o x o g l u t a r a t e
3
dehydrogenase (see p . 1981) a n d therefore only the preliminary t r e a t m e n t of the sample is described in
detail here.
Application of Method: In biochemistry in studies o n the regulation of intermediary m e t a b o l i s m a n d for

the determination of acyl-CoA synthetase activity.
Principle
C o A thioesters of fatty acids of chain length C 1 0 a n d a b o v e are insoluble in acid, while those of chain
length C 8 a n d below are s o l u b l e . If biological material such as liver is extracted with perchloric acid,
1
the long-chain fatty acyl C o A esters are precipitated with proteins. T r e a t m e n t of the acid-insoluble
precipitate with alkali a n d SH-reagents results in quantitative conversion of the acyl-CoA to C o A S H a n d
the corresponding free fatty acids. T h e C o A is acid-soluble a n d is determined by the usual m e t h o d s .
T h e p H for the hydrolysis is i m p o r t a n t ; t o o low a p H results in incomplete deacylation, while t o o high

a p H results in loss of C o A .
Reagents, Preparation of Solutions
a n d Stability of Solutions see p . 1 9 8 2 - 1 9 8 3 .
Procedure
D e p r o t e i n i z e s a m p l e s a s d e s c r i b e d for t h e d e t e r m i n a t i o n o f C o A o n p . 1 9 8 3 . R e s u s p e n d t h e
a c i d - i n s o l u b l e p r e c i p i t a t e in 5.0 m l . i c e - c o l d 0 . 6 M H C 1 0 4 ( s o l u t i o n X ) a n d c e n t r i f u g e for
5 m i n . at 2 0 0 0 g. D i s c a r d t h e s u p e r n a t a n t fluid. D r y t h e t u b e s a s w e l l a s p o s s i b l e b y d r a i n i n g
a n d w i p i n g i n s i d e w i t h filter p a p e r . R e s u s p e n d t h e p r e c i p i t a t e in 5.0 m l . d i s t i l l e d w a t e r , a d d
0.1 m l . 2 - m e r c a p t o e t h a n o l s o l u t i o n ( X I V ) a n d a d j u s t t o p H 1 2 . 5 - 1 3 . 0 w i t h c a . 0 . 2 t o 0 . 5 m l .
( n o t e e x a c t v o l u m e ) 2 N K O H . Test w i t h i n d i c a t o r p a p e r . T h e a c i d - i n s o l u b l e m a t e r i a l v i r t u a l l y
c o m p l e t e l y d i s s o l v e s t o f o r m a g e l a t i n o u s s o l u t i o n ; m i x t h o r o u g h l y . A l l o w t o s t a n d for 3 0 m i n .
at r o o m t e m p e r a t u r e , t h e n c o o l t o 0 ° C a n d a d d 0.3 m l . 7 0 % ( w / w ) H C 1 0 . C e n t r i f u g e for
4
5 m i n . at 3 0 0 0 g. d e c a n t t h e s u p e r n a t a n t fluid a n d n e u t r a l i z e for t h e C o A d e t e r m i n a t i o n a s
described o n p. 1983.
A c i d - i n s o l u b l e p r e c i p i t a t e s o f d e n a t u r e d p r o t e i n a n d l o n g - c h a i n a c y l - C o A c a n b e s t o r e d at
— 25 ° C for at l e a s t 4 8 hr. w i t h o u t l o s s o f a c y l - C o A . A l t h o u g h t h e n e u t r a l i z e d s a m p l e c o n t a i n s
2 - m e r c a p t o e t h a n o l t h e r e is a s l o w l o s s o f C o A at 0 ° C o r —25 ° C . T h e a l k a l i n e h y d r o l y s i s
a n d the analysis s h o u l d be carried o u t o n the s a m e day.
A s for the fluorimetric d e t e r m i n a t i o n of C o A .
N o r m a l Values
Heart 2
1 0 - 2 0 nmoles/g. fresh wt.
Liver ' 1 2
3 0 - 5 0 nmoles/g. fresh wt.
Epididymal fat p a d 4
2 nmoles/g. fresh wt.
Liver ( m i t o c h o n d r i a ) 3
0 . 2 - 1 . 0 nmoles/mg. protein
All acid-insoluble C o A derivatives are determined. In simple systems, e.g. the determination of the
activity of p a l m i t o y l - C o A synthetase in rat liver microsomes, the acid-soluble C o A can be taken to be
p a l m i t o y l - C o A . In m o r e complex systems it should be b o r n e in m i n d that proteins can bind C o A .
References

2 P. B. Garland, Biochem. J. 92, 10c [1964].
Crotonyl-Coenzyme A
Karl Decker
T h e ^-oxidation of even-numbered fatty acids results in the formation of c r o t o n y l - C o A from butyryl-

CoA 1 , 2
. T h e further m e t a b o l i s m of the u n s a t u r a t e d c o m p o u n d proceeds by way of L - ( + ) - 3 - h y d r o x y b u t y r y l -
C o A to a c e t o a c e t y l - C o A ' . 1 3
Application of Method: In enzyme studies on fat metabolism.
Principle
(1) CH -CH = CH-CO~SCoA + H 0

3 2 CH -CH(OH)-CH -CO~SCoA
3 2
(2) CH -CH(OH)-CH -CO~SCoA

3 2 + NAD +
(
H Q A P H
- CH - C O - C H
3 2 -CO~SCoA +
+ NADH + H +
T h e enzymes c r o t o n a s e (L-3-Hydroxyacyl-CoA hydro-lyase, E C 4.2.1.17) a n d 3-hydroxyacyl-CoA d e h y d r o

genase ( L - 3 - H y d r o x y a c y l - C o A : N A D oxidoreductase, E C 1.1.1.35) have been crystallized. The increase
in N A D H , as m e a s u r e d by the extinction change at 340 (334, 365) n m is p r o p o r t i o n a l to the a m o u n t of
crotonyl-CoA.
The equilibrium constant of the c r o t o n a s e reaction (1) is 0.0618 l . / m o l e 4

(for equilibrium constant of
reaction (2) a n d its dependence o n pH,see p. 2022). T h e o p t i m u m c o n c e n t r a t i o n range in the assay system
described here is 0 . 0 1 5 - 0 . 2 pmole crotonyl-CoA/cuvette.
T h e time t a k e n for the assay a n d the length of contact of c r o t o n y l - C o A with the alkaline assay m e d i u m
m u s t be reduced to a m i n i m u m because of the alkali lability of the acylmercaptans. Therefore the crotonyl-
C o A should be a d d e d immediately before the start of the reaction a n d highly active enzyme p r e p a r a t i o n s
should be used to minimize the reaction time. Since the conversion of c r o t o n y l - C o A is only quantitative
at p H > 9, it is necessary to check with indicator p a p e r on completion of the reaction t h a t the p H of the
assay mixture h a s n o t fallen below 9. If this is the case, then the sample is t o o strongly buffered. T h e a m o u n t
of 0.1 N K O H required to o b t a i n the correct p H is determined a n d the assay is repeated with the inclusion
of the requisite q u a n t i t y of 0.1 N K O H just before the distilled water.
Equipment
334 or 365 n m .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 4. P o t a s s i u m h y d r o x i d e , A . R . , c a . 8 N
KH P0 2 4 5. H y d r o c h l o r i c a c i d , A . R . , c o n e ,
2. D i s o d i u m h y d r o g e n p h o s p h a t e , (ca. 3 7 % w / w )
Na HP0 2 4 • 2H 02 6. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris
3. P o t a s s i u m h y d r o g e n c a r b o n a t e , 7. E t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d , E D T A
K H C 0 , A . R. 3 disodium salt, E D T A - N a H • 2 H 0 2 2 2
8. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , i. C r o t o n a s e
NAD crystalline from beef liver according to Stern et
free acid; commercial p r e p a r a t i o n , see p . 545. a l . , suspended in 0.02 M p h o s p h a t e
7
buffer
( p H 7.4) containing 3 m M E D T A ; ^ 2000
9. 3 - H y d r o x y a c y l - C o A d e h y d r o g e n a s e ,
U / m g . (25 ° C ) ; isolation, see A p p e n d i x on
HOADH
p.2021.
from sheep liver or pig h e a r t (see B. IV. 4.7);
6
^ 9 U / m g . (25 °C); crystalline commercial

Purity of Reagents
Enzymes prepared according to the cited publications are sufficiently p u r e for this assay. H O A D H must
be free from crotonase. Both enzymes must be free from enoyl-CoA isomerase a n d 3-hydroxyacyl-CoA-3-
epimerase in case the sample contains J - e n o y l - C o A o r isocrotonyl-CoA.
3
P r e p a r e all s o l u t i o n s w i t h fresh distilled w a t e r .

I. P o t a s s i u m h y d r o g e n c a r b o n a t e ( c a . 1 M ) :
II. Tris buffer (ca. 0.5 M ; p H 9 . 5 ; 5 m M E D T A ) :
D i s s o l v e 12.1 g. tris a n d 3 7 2 m g . E D T A - N a H 2 2 • 2 H 0 in 150 m l . distilled w a t e r , a d d
2
0.35 ml. c o n e . HC1 and dilute to 200 ml. with distilled water.
III. P h o s p h a t e buffer ( 2 0 m M ; p H 7 . 4 ; 3 m M E D T A ) :
Mix 4 ml. K H P 0 2 4 s o l u t i o n ( 2 . 7 2 2 g./lOO m l . ) + 16 m l . N a H P 0
2 4 s o l u t i o n ( 3 . 5 6 1 g./
100 m l . ) , a d d 2 2 3 m g . E D T A - N a H 2 2 • H 0 and dilute to 200 ml. with C 0 - f r e e (boiled)
2 2
distilled w a t e r .
I V . N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 10 m M / ? - N A D ) :
D i s s o l v e 7.4 m g . N A D in a b o u t 0.5 m l . d i s t i l l e d w a t e r , n e u t r a l i z e w i t h a f e w d r o p s K H C 0 3
s o l u t i o n a n d d i l u t e w i t h distilled w a t e r t o 1 m l .
V . 3 - H y d r o x y a c y l - C o A d e h y d r o g e n a s e , H O A D H (3 m g . p r o t e i n / m l . ) :
Dilute the stock suspension with 0 . 2 % E D T A solution.
V I . C r o t o n a s e (5 \ig. protein/ml.):
D i l u t e t h e c r o t o n a s e s u s p e n s i o n (5 m g . p r o t e i n / m l . ) a c c o r d i n g l y w i t h p h o s p h a t e buffer
( s o l u t i o n III).
C o n c e n t r a t e d or dilute solutions of the two enzymes can be stored at 0 °C or in the frozen state without
appreciable loss of activity. F r e q u e n t freezing a n d thawing of the solutions causes deterioration. Neutral,
a q u e o u s solutions of N A D are stable at 0 °C o r in the frozen state for several weeks. All other solutions
are stable indefinitely, provided t h a t bacterial c o n t a m i n a t i o n is avoided by storage in a refrigerator.
Use polyethylene bottles for buffer and alkaline solutions.
Crotonyl-CoA 2019
Procedure
See C h a p t e r o n " A c e t y l - C o A " p . 1 9 8 8 . T h e n e c e s s a r y r e a g e n t s a n d s o l u t i o n s are d e s c r i b e d t h e r e .
A q u e o u s s o l u t i o n s o f c r o t o n y l - C o A at p H 3 - 7 c a n b e s t o r e d f r o z e n f o r l o n g p e r i o d s . T h e
t h i o l esters o f t h e a, ^ - u n s a t u r a t e d a c i d s are m o r e s t a b l e t o alkali t h a n o t h e r acyl m e r c a p t a n s
(refer t o ) . 8
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 1.965 m l . ; r o o m

Tris buffer (II) 0.70 ml. 178 m M tris

1.78 m M E D T A
N A D solution (IV) 0.05 ml. ca. 0.25 m M 0 - N A D
H O A D H solution (V) 0.01 m l . 15.3 jug./ml. = 138 m U / m l .
Sample + water 1.20 m l . u p t o 1 0 0 pM c r o t o n y l - C o A
M i x , f o l l o w t h e e x t i n c t i o n until c o n s t a n t l o r at least
30 sec. a n d read E . x
Crotonase solution (VI) 0.005 ml. 0 . 0 1 2 7 pg./ml = 25 m U / m l .
M i x . W h e n t h e r e a c t i o n is c o m p l e t e ( c o n s t a n t e x
t i n c t i o n for 1 m i n . ) r e a d E . E — E i = A E is u s e d
2 2
Calculations
The reaction is stoichiometric u n d e r the described conditions. T h e calculation formula (2) on p . 312
applies in this case. T h e a m o u n t of c r o t o n y l - C o A in the sample is given b y :
/miole - 0.322 x AE x ^ 0.316 x J E x ^ 0.578 x ^ E x ^

v 2 v 2 v 2
\ = volume of whole sample in ml.

l
v = volume of sample taken for assay in ml.

2

Conversion of 20 /miole C o A S H t o c r o t o n y l - C o A with crotonic a n h y d r i d e yielded a m e a n of 13.70
/miole c r o t o n y l - C o A with a s t a n d a r d deviation of 0.22 /miole (7 d e t e r m i n a t i o n s ) ; this gives a coefficient
of variation of 1.6%.
N o r m a l Values
T h e steady state concentration of c r o t o n y l - C o A in living cells m u s t be very l o w ; Garland et a l . 9

found
n o detectable intermediates of /^-oxidation in rat liver m i t o c h o n d r i a oxidizing p a l m i t o y l - C o A .
Effects of drugs and other compounds: None known.
Interference in the assay: T h e presence of L-(-h)-3-hydroxybutyryl-CoA in the sample, which would also
lead to a reduction of N A D , is indicated by an increase in extinction before addition of crotonase. If
E t has reached a steady value before the start of the reaction then the accuracy of the c r o t o n y l - C o A
assay is n o t affected. T h e m o s t i m p o r t a n t of the interfering c o m p o u n d s which react with b o t h crotonase
a n d 3-hydroxyacyl-CoA dehydrogenase (refer to "Specificity") are the higher a,/?-(trans)-unsaturated
fatty acids. M e r c a p t a n s a d d to the double b o n d of a,/?-enoylthiol esters a n d the resulting /?-thioethers
d o not r e a c t . A n excess of S H - c o m p o u n d s is therefore to be avoided when w o r k i n g with c r o t o n y l - C o A .
8
Specificity
Pure crotonase catalyses the h y d r a t i o n of J - t r a n s a n d ,d -cis-, but n o t J - e n o y l - C o A . C r o t o n a s e shows

2 2 3 1 0
n o m a r k e d specificity with regard to the chain length of the a,/?-unsaturated acids. ^ - t r a n s - C r o t o n y l - C o A

2
gives L-(-f-)-3-hydroxybutyryl-CoA which can be oxidized to acetoacetyl-CoA by H O A D H , while the

J -cis-derivative gives D - ( - ) - 3 - h y d r o x y b u t y r y l - C o A
2 10
which does n o t react with H O A D H . A J -cis-zJ -
3 2
trans-enoyl-CoA isomerase a n d a 3-hydroxyacyl-CoA-3-epimerase have been detected in the m i t o c h o n d r i a

of several o r g a n s . 10
C o n t a m i n a t i o n of c r o t o n a s e o r H O A D H by these enzymes could affect the specificity of the c r o t o n y l - C o A

assay.
C r o t o n a s e has a high degree of specificity with regard to the thiol c o m p o n e n t . Neither the N-acetylcystea-
mine or the glutathione derivative of c r o t o n i c acid are hydrated. C r o t o n y l p a n t e t h e i n e reacts with the
crystalline enzyme extremely slowly; however, in the presence of C o A S H c r o t o n a s e can act on this substrate
in the same m a n n e r as a t h i o l t r a n s c r o t o n y l a s e .
4
O t h e r M e t h o d s for D e t e r m i n a t i o n o f C r o t o n y l - C o A
The thiolase system can be a d d e d to the assay mixture a n d acetyl-CoA d e t e r m i n e d as described on p . 1988.
Crotonyl thiol esters have a characteristic a b s o r p t i o n spectrum with two p e a k s at 225 a n d 263 n m ( e =
1.06 x 10 a n d 6.5 x 1 0 c m ^ / m o l e ) . T h e decrease in extinction between 260 a n d 270 n m * o n h y d r a t i o n
7 6 1
can be used for the d e t e r m i n a t i o n of c r o t o n y l - C o A , if the 3-hydroxybutyryl-CoA formed in the reaction

is completely removed from the reaction mixture. This is accomplished by use of 3-hydroxyacyl-CoA
dehydrogenase, alcohol dehydrogenase, acetaldehyde a n d a catalytic a m o u n t of N A D . A n o t h e r m e t h o d
for the determination of c r o t o n y l - C o A is its reduction to b u t y r y l - C o A with N A D P H a n d an enzyme from
liver m i c r o s o m e s ' . N o n e of these m e t h o d s has any a d v a n t a g e over the p r o c e d u r e described here.
11 12
* In most cases c o m p e n s a t i o n for the nucleotide extinction in this range m u s t be m a d e .

Crotonyl-CoA 2021
Appendix
Isolation of Crotonase
T h e starting material is deep-frozen beef liver. T h e stages a r e : 1. Extraction with K H C 0 3 and cysteine,
p H 8.2. - 2. H e a t i n g to 55 °C at p H 5.5. - 3. A c e t o n e precipitation at - 5 °C, solution of the precipitate
in p h o s p h a t e buffer, p H 7.4 (3 m M E D T A ) a n d dialysis. - 4. A m m o n i u m sulphate fractionation between
40 a n d 6 5 % s a t u r a t i o n ; solution of precipitate in 20 m M potassium p h o s p h a t e buffer, p H 7.4 (3 m M
E D T A and 1 m M g l u t a t h i o n e ) ; dialysis. - 5. Crystallization by addition of 0.1 v o l u m e of ethanol to the
dialysed solution. Recrystallize twice.
References
1 W. Seubert & F. Lynen, J. A m e r . chem. Soc. 75, 2787 [1953].

2 D. E. Green, S. MU, H. R. Mahler & R. M. Bock, J. biol. C h e m . 206, 1 [1954].
3 J. R. Stern & A. Del Campillo, J. A m e r . chem. Soc. 75, 2277 [1955].
4 J. R. Stern & A. Del Campillo, J. biol. C h e m . 218, 985 [1956].
5 F. Lynen & O. Wieland in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c Press,
N e w Y o r k 1955, vol. I, p . 566.
6 / . R. Stern, Biochim. biophys. A c t a 26, 448 [1957].
7 J. R. Stern, A. Del Campillo & I. Raw, J. biol. C h e m . 218, 971 [1956].
8 K Decker: Die aktivierte Essigsaure. F e r d . E n k e , Stuttgart 1959.
9 P . B. Garland, D. Shepherd & D. W. Yates, Biochem. J. 97, 587 [1965].
10 W. Stoffel & H. Caesar, Hoppe-Seyler's Z. physiol. C h e m . 341, 76 [19651.
11 R.G. Langdon, J. A m e r . chem. Soc. 77, 5190 [1955].
12 W. Seubert, G. Greull & F. Lynen, Angew. C h e m . 69, 359 [1957].
L-(+)-3-Hydroxybutyryl-Coenzyme A
Karl Decker
L - ( + ) - 3 - H y d r o x y b u t y r y l - C o A occurs as an intermediate in the /^-oxidation of fatty acids by cell-free

s y s t e m s . So far it has n o t been detected in intact cells o r extracellular fluids (blood).
1
L - ( + ) - 3 - H y d r o x y b u t y r y l - C o A is oxidized to acetoacetyl-CoA by nicotinamide-adenine dinucleotide in

a reaction catalysed by 3-hydroxyacyl-CoA dehydrogenase, H O A D H ( L - 3 - H y d r o x y b u t y r y l - C o A : N A D
oxidoreductase, E C 1.1.1.35). This reaction is the reverse of t h a t used for the d e t e r m i n a t i o n of acetoacetyl-
C o A (p. 2001).
Application of Method: In studies o n the enzymology of fatty acid metabolism.
Principle
(1) CH —CH—CH CO~SCoA + NAD

3 2
+
t
H O A P I
L CH —C—CH CO~SCoA + NADH + H
3 2
+
OH O
The a m o u n t of N A D H formed, as measured by the c h a n g e in extinction at 340 (334, 365) n m , is p r o p o r t i o
nal to the L-(-|-)-3-hydroxybutyryl-CoA present.
T h e equilibrium c o n s t a n t :
[Acetoacetyl-CoA] [ N A D H ] [H ] +
1.9 x 1 0 " 1 0
at 25 ° C 2
[L-(+)-3-Hydroxybutyryl-CoA] [ N A D ] +
As with all pyridine nucleotide-linked reactions the equilibrium is d e p e n d e n t on p H a n d by use of a m o r e

alkaline m e d i u m it is displaced m o r e in favour of the oxo-derivate. A b o v e p H 8.5 the equilibrium is further
displaced because of enol dissociation which allows the quantitative oxidation of 3-hydroxybutyryl-CoA.
Because C o A derivatives are sensitive to the alkaline conditions of the assay mixture, a n a m o u n t of en
zyme sufficient to complete the oxidation in 1 to 2 min. m u s t be used. F o r the same reason the 3-hydroxy-
butyryl-CoA should be exposed to the alkaline m e d i u m for the m i n i m u m length of time. Consequently,
the solution of the C o A derivative is a d d e d last to the assay mixture a n d the reaction is started as soon
as possible. A t the end of the reaction it is necessary to check with indicator p a p e r whether the p H has
fallen below 9. If this is the case, the solution of the C o A derivative is t o o strongly buffered. T h e a m o u n t
of 0.1 N K O H required to m a i n t a i n the correct p H in the reaction mixture is determined in a preliminary
test a n d then this a m o u n t of K O H is a d d e d before the enzyme. U n d e r the conditions of the assay system
described below suitable extinction changes are obtained with 0 . 0 1 5 - 0 . 2 /miole L - ( + ) - 3 - h y d r o x y b u t y r y l -
CoA
Equipment
334 or 365 n m .
L-(+)-3-Hydroxybutyryl-CoA 2023
Reagents
1. P o t a s s i u m h y d r o g e n c a r b o n a t e , KHC0 , 3 7. 3 - H y d r o x y a c y l - C o A dehydrogenase,
A . R. HOADH
2. P o t a s s i u m h y d r o x i d e , A . R . , c a . 8 N crystallized from pig h e a r t according to Stern*,
3. H y d r o c h l o r i c a c i d , c o n e , c a . 3 7 % ( w / w ) , ^9 U / m g . (25 ° C ) ; o r highly purified from
A. R. sheep liver according to Lynen a n d WielancP
4. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris ^ 2 . 5 U / m g . (25 °C) in the assay with N-acetyl-

s-acetoacetyl cysteamine. C o m m e r c i a l prepa
5. E t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d , E D T A
ration, see p . 474.
sodium salt, E D T A - N a H - 2 H 0 2 2 2
6. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , N A D
free acid, commercial p r e p a r a t i o n , see p . 545.
Purity of Reagents
T h e enzyme p r e p a r a t i o n s o b t a i n e d according to Stern 3

or Lynen a n d WielancP are sufficiently pure for
this determination.
P r e p a r e all s o l u t i o n s w i t h fresh d i s t i l l e d w a t e r .
I. P o t a s s i u m h y d r o g e n c a r b o n a t e ( c a . 1 M ) :
D i s s o l v e 10 g. K H C 0 3 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
II. Tris buffer ( c a . 0.5 M ; p H 9 . 5 ; 5 m M E D T A ) :
D i s s o l v e 12.1 g. tris a n d 3 7 2 m g . E D T A - N a H - 2 H 0 in 150 m l . d i s t i l l e d w a t e r , a d d
2 2 2
0.35 m l . c o n e . H C 1 a n d d i l u t e w i t h d i s t i l l e d w a t e r t o 2 0 0 m l .
III. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( c a . 10 m M /?-NAD):
D i s s o l v e 7 . 4 m g . N A D in a b o u t 0.5 m l . d i s t i l l e d w a t e r , n e u t r a l i z e w i t h a f e w d r o p s o f
KHC0 3 s o l u t i o n (I) a n d d i l u t e w i t h d i s t i l l e d w a t e r t o 1 m l .
I V . 3 - H y d r o x y a c y l - C o A d e h y d r o g e n a s e , H O A D H (3 m g . p r o t e i n / m l . ) :
Dilute the stock suspension accordingly with 0 . 2 % E D T A solution.
Solutions of 3-hydroxyacyl-CoA d ehydro genase are stable for long periods at 0 ° C ; frequent freezing a n d
t h a w i n g results in loss of activity. Solutions of N A D are stable for several weeks at 0 °C, or longer if
deep-frozen. T h e o t h e r solutions s h o u l d be stored in a refrigerator t o prevent bacterial g r o w t h a n d u n d e r
these conditions are stable indefinitely. Polyethylene bottles should be used for the buffer a n d alkaline
solutions.
Procedure
S e e C h a p t e r o n " A c e t y l - C o A " ( p . 1 9 8 8 ) . T h e n e c e s s a r y r e a g e n t s a n d s o l u t i o n s are d e s c r i b e d

there.
A q u e o u s s o l u t i o n s o f 3 - h y d r o x y a c y l - C o A at p H 3 - 6 . 5 c a n b e s t o r e d for a l o n g p e r i o d in t h e
f r o z e n state.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; r o o m t e m p e r a t u r e ; final v o l u m e :
1.96 m l . ; read a g a i n s t air.
Tris buffer (II) 0.70 ml. 179 m M t r i s ;

1.79 m M E D T A
N A D solution (III) 0.05 ml. ca. 0.25 m M £ - N A D
Sample + water 1.20 m l . u p t o 1 0 0 pM
L-(+)-3-HB-CoA
M i x , f o l l o w t h e initial e x t i n c t i o n for at least 3 0 sec.

until c o n s t a n t a n d r e a d E x
H O A D H solution (IV) 0.01 m l . 15 M g / m l . = 138mU/ml.
Mix, and w h e n the reaction has ceased (constant

e x t i n c t i o n for m o r e t h a n 1 m i n . ) r e a d E . E
2 2 — Ej =
= A E is u s e d for t h e c a l c u l a t i o n s .
Calculations
The reaction proceeds quantitatively a n d stoichiometrically u n d e r the described conditions. T h e calcu

lation formula (2) on p. 312 applies. T h e a m o u n t of L ( + ) - 3 - h y d r o x y a c y l - C o A in the sample is given b y :
wmole = 0.321 x z l E x - ^ - 0.315 x ^ E x ^ 0.576 xÊx-^-

v 2 v 2 v 2
v = volume of the whole sample in ml.

x
v = volume of sample taken for assay in ml.

2
T h e reaction of 10 ^ m o l e C o A S H with /?-butyrolactone gave a m e a n of 4.86 /zmole L-( + ) - 3 - H B - C O A

with a standard deviation of 0.06 /rniole; this gives a coefficient of variation of 1.2%.
N o r m a l Values
L-(-h)-3-Hydroxybutyryl-CoA has n o t been detected in tissues or b o d y fluids, see Garland et a l . . 4

L-(+)-3-Hydroxybutyryl-Co A 2025
Interference in assay: T h e higher h o m o l o g u e s of L - ( + ) - 3 - h y d r o x y b u t y r y l - C o A a n d several o t h e r L - ( - f ) - 3 -

hydroxyacyl-thiol esters can interfere (see "Specificity").
Specificity
3-Hydroxyacyl-CoA d e h y d r o g e n a s e is n o t very specific with regard to the m e r c a p t a n c o m p o n e n t . T h e

3-hydroxybutyryl derivatives of p a n t e t h e i n e p h o s p h a t e , p a n t e t h e i n e , N-acetylcysteamine a n d several
other m e r c a p t a n s (see ) are oxidized. T h e C o A derivatives of all the higher h o m o l o g u e s of L - ( + ) - 3 - h y d r o x y -
5
butyric acid u p t o a b o u t C 2 0 also react. T h e r e is, however, a high specificity with regard to the 3-hydroxy-
acylmercaptan g r o u p i n g R — C H ( O H ) — C H C O — S — R : neither 2-hydroxyacyl derivatives, n o r the free
2
acids or p r i m a r y or secondary alcohols are oxidized. T h e selectivity of the enzyme for stereoisomers is
just as r i g o r o u s : only the C o A derivatives of the L - ( + ) - 3 - h y d r o x y a c i d s 6
are oxidized a n d only this
isomer is formed on reduction of acetoacetyl-CoA. A l t h o u g h H O A D H from sheep liver is specific for
N A D , Stern 2,
found t h a t the enzyme from pig h e a r t can also use N A D P as h y d r o g e n acceptor (the relative
rates with N A D a n d N A D P are 10 : 1).
O t h e r M e t h o d s for t h e D e t e r m i n a t i o n o f L - ( + ) - 3 - H y d r o x y b u t y r y l - C o A
T h e d e t e r m i n a t i o n can be m a d e m o r e sensitive if 3-hydroxyacyl-CoA d e h y d r o g e n a s e , thiolase (Acetyl-CoA:

acetyl-CoA C-acetyltransferase, E C 2.3.1.9), m a l a t e d e h y d r o g e n a s e ( L - M a l a t e : N A D oxidoreductase,
E C 1.1.1.37) a n d citrate synthase (Citrate-oxaloacetate lyase, E C 4.1.3.7) are used in a coupled assay:
for each mole of L-( + )-3-hydroxybutyryl-CoA 3 moles of N A D are r e d u c e d :
(2) L-(+)-3-Hydroxybutyryl-CoA + 2 L-Malate " + 3 N A D 2 +

+ 2 H 0 -»
2
-+ 2 C i t r a t e " + C o A S H + 3 N A D H + 3 H
2 +
Usually the greater expenditure o n enzymes is h a r d l y justified by the a d v a n t a g e gained. T h e same is

true for the coupled assay in which p-nitroaniline is acetylated in the presence of H O A D H , thiolase,
arylamine acetylase (Acetyl-CoA :arylamine-AT-acetyltransferase, E C 2.3.1.5), N A D a n d catalytic a m o u n t s
of C o A (or p a n t e t h e i n e ) :
(3) L-(+)-3-Hydroxybutyryl-CoA + 2 p-Nitroaniline + NAD +
- » 2 p-Nitroacetanilide + C o A S H + N A D H + H +
In the absence of interfering substances, especially thioesters, n o n - e n z y m a t i c assay m e t h o d s can be used. 5
References
1 F. Lynen, Fed. P r o c . 12, 683 [1953].

2 F. Lynen & O. Wieland in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c Press,
N e w Y o r k , vol. I, p . 566.
3 J. R. Stern, Biochim. biophys. A c t a 26, 448 [1957].
5 K Decker: Die aktivierte Essigsaure. F e r d . E n k e , S t u t t g a r t 1959.
6 A. L. Lehninger & G. D. Greville, Biochim. b i o p h y s . A c t a 12, 188 [1953].
3-Hydroxy-3-methylglutaryl-Coenzyme A
Joachim Knappe
3-Hydroxy-3-methylglutaryl-coenzyme A ( H M G - C o A ) is cleaved by H M G - C o A lyase (3-Hydroxy-

3-methylglutaryl-CoA acetoacetate-lyase, E C 4.1.3.4) t o acetoacetate a n d a c e t y l - C o A . This reaction can 1
be used for the determination of H M G - C o A because the equilibrium strongly favours cleavage. T h e m o s t
convenient m e t h o d is to couple it with one of the s p e c t r o p h o t o m e t r i c m e t h o d s for the determination of
acetyl-CoA (see p . 1988) . 2
C o m b i n a t i o n with the acetyl-CoA a s s a y 3

involving m a l a t e d e h y d r o g e n a s e , M D H ( L - M a l a t e : N A D
oxidoreductase, E C 1.1.1.37) a n d citrate synthase, CS (Citrate oxaloacetate-lyase, E C 4.1.3.7) is employed
here.
Application of Method: In biochemistry. T h e sensitivity is n o t sufficient for direct determinations on

tissue extracts. A preliminary c o n c e n t r a t i o n of the sample, for example, with phenol, is necessary , b u t 4
refer to p . 2029.
Principle
(1) HMG-CoA H M G
~ C o A lyase
> Acetoacetate + Acetyl-CoA
(2) L-Malate + N A D +
Oxaloacetate + N A D H +
+ H +
(3) Oxaloacetate + Acetyl-CoA + H 0 2 Citrate + C o A
T h e increase in N A D H , measured by the c h a n g e in extinction at 340 (334, 365) n m , is used to calculate

the a m o u n t of H M G - C o A present.
T h e rate of the overall reaction is dictated by the comparatively low activity of H M G - C o A lyase. T h e
p H - o p t i m u m of the enzyme from ox liver is at p H 9 . 5 . However, it is r e c o m m e n d e d to carry out the
1 1
assay in the range between p H 7.8 a n d 8.5, in order avoid s p o n t a n e o u s hydrolysis of H M G - C o A .

It is necessary to a d d thiols (thioglycollic acid or glutathione) to protect o r reactivate the enzyme. T h e
enzyme requires m a g n e s i u m ions.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e f o r m e a s u r e m e n t s at 3 4 0 , 3 3 4 o r
365 n m ; b e n c h c e n t r i f u g e .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 6. D L - M a l i c a c i d
2. H y d r o c h l o r i c a c i d , A . R . , 0.5 N 7. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ,
3. M a g n e s i u m c h l o r i d e , M g C l - 6 H 0 , A . R .
2 NAD
4. Thioglycollic acid free acid; commercial p r e p a r a t i o n , see p . 545.
5. P o t a s s i u m h y d r o x i d e , A . R . , 1 N
3-Hydroxy-3-methylglutaryl-CoA 2027
8. M a l a t e d e h y d r o g e n a s e , MDH 10. H M G - C o A l y a s e
from pig heart, crystalline suspension in 3.2 M P r e p a r a t i o n s from ox liver containing ^ 0.1
a m m o n i u m s u l p h a t e ; ca. 700 U / m g . (25 °C). U / m g . (20 °C), which are free from NADH
C o m m e r c i a l p r e p a r a t i o n , see p . 485. oxidase, are suitable. For preparation, see
9. C i t r a t e s y n t h a s e , C S A p p e n d i x p . 2029.
from pig heart, crystalline suspension in 3.2 M
ammonium sulphate; ca. 70 U/mg. (25°).
C o m m e r c i a l p r e p a r a t i o n , see 443.
P r e p a r e all s o l u t i o n s w i t h freshly d i s t i l l e d w a t e r .
I. Tris buffer (ca. 0.5 M ; p H 8 . 0 ) :
D i s s o l v e 6 g. t r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e in 5 0 m l . d i s t i l l e d w a t e r a n d m a k e u p t o
100 m l . w i t h 1 N H C 1 ; c h e c k p H w i t h a g l a s s e l e c t r o d e .
D i s s o l v e 2 . 0 3 g. M g C l • 6 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
2 2
III. T h i o g l y c o l l a t e ( 0 . 3 5 M ) :
D i s s o l v e 0 . 2 4 m l . 8 0 % t h i o g l y c o l l i c a c i d in c a . 4 m l . d i s t i l l e d w a t e r , adjust t o c a . p H 6
w i t h 1 N K O H a n d m a k e u p w i t h d i s t i l l e d w a t e r t o 10 m l .
IV. D L - M a l a t e ( 0 . 1 M ) :
D i s s o l v e 1.34 g. D L - m a l i c a c i d in d i s t i l l e d w a t e r , n e u t r a l i z e w i t h 1 N K O H a n d m a k e u p
with distilled water to 100 ml.
V . N i c o t i n a m i d e - a d e n i n e - d i n u c l e o t i d e (ca. 2 0 m M / ? - N A D ) :
D i s s o l v e 30 m g . N A D in 1 m l . d i s t i l l e d w a t e r , a d j u s t t o p H c a . 6 w i t h 0.1 N K O H a n d
m a k e up with distilled water to 2 ml.
V I . M a l a t e d e h y d r o g e n a s e , M D H (5 m g . p r o t e i n / m l . ) :
Use suspension undiluted.
VII. Citrate synthase, C S (2 m g . protein/ml.):
Use suspension undiluted.
VIII. H M G - C o A lyase:
S o l u t i o n p r e p a r e d a c c o r d i n g t o p . 2 0 2 9 c o n t a i n i n g c a . 2 - 4 m g . p r o t e i n / m l . (in 10 m M
tris buffer, p H 7 . 5 ) .
P r e p a r e solution III freshly each d a y ; store I, II, IV, V, VI, V I I , stoppered, in a refrigerator (0° to 4 °C).
T h e dialysed, a m m o n i u m sulphate-free solution of H M G - C o A lyase is stable for several m o n t h s a t —18 ° C ;
repeated freezing a n d t h a w i n g results in loss of activity.
Procedure
If n e c e s s a r y , d e p r o t e i n i z e t h e s a m p l e s w i t h p e r c h l o r i c a c i d (final c o n c e n t r a t i o n c a . 3 %) in a n
ice b a t h . C a r e f u l l y a d j u s t t h e s u p e r n a t a n t fluid t o p H 5 w i t h 1 N K O H , a l l o w t o s t a n d 10 m i n .
in t h e ice b a t h a n d c e n t r i f u g e off t h e K C 1 0 . B r i n g t h e s a m p l e s t o r o o m t e m p e r a t u r e b e f o r e
4
assay.
Stability:
L i k e all C o A - t h i o l esters H M G - C o A is s e n s i t i v e t o o x i d i s i n g a g e n t s (e. g. ether p e r o x i d e s ,

H 0 ) a n d alkali (half-life t i m e for H M G - C o A h y d r o l y s i s at 15 ° C in 0.1 N K O H is 8 m i n . ) .
2 2
A t p H 4 t o 5 ( o p t i m u m ) H M G - C o A is s t a b l e for several d a y s a t 0 ° C , w h i l e at - 1 8 ° C it is
s t a b l e virtually indefinitely.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; l i g h t p a t h : 1 c m . ; final v o l u m e : 1.80 m l . ; r o o m t e m p e r
a t u r e ; read a g a i n s t air. If s t r o n g l y c o l o u r e d H M G - C o A l y a s e p r e p a r a t i o n s are u s e d , d e t e r m i n e
the i n c r e a s e in e x t i n c t i o n d u e t o a d d i t i o n o f l y a s e in a b l a n k c u v e t t e c o n t a i n i n g w a t e r i n s t e a d o f
sample. Subtract this a m o u n t from E . 2
Pipette successively into cuvettes C o n c e n t r a t i o n in a s s a y m i x t u r e
Sample, deproteinized, where necessary

m a k e up with water 1.00 m l . u p to ca. 0.15 m M HMG-CoA
Tris buffer (I) 0.40 ml. 0.11 M
MgCl 2 solution (II) 0.20 ml. 11 m M
Thioglycollate solution (III) 0.02 ml. 4 mM
Malate solution (IV) 0.05 ml. 3 mM
N A D solution (V) 0.05 ml. 0.55 m M
Mix. Read E . 0
M D H suspension (VI) 0.001 ml. c a . 3 / i g . / m l . = 2.1 U / m l .

CS suspension (VII) 0.002 ml. ca. 2 Mg/ml. = 140 m U / m l .
M i x . F o l l o w e x t i n c t i o n until c o n s t a n t ( f e w s e c . )
and read E . x
H M G - C o A lyase solution (VIII) 0.08 ml. ca. 20 m U / m l .
M i x . F o l l o w extinction until c o n s t a n t (10 - 3 0 min.).

Read E . 2
Calculations
It is necessary to include in the general formula a c o r r e c t i o n : 5
T h e concentration of H M G - C o A in the sample taken for assay is then calculated as follows
AE x 0.295/v AE x 0.289/v AE x 0.529/v [^mole/ml.]

v = volume of sample in assay.
Multiply by the molecular weight 911 to convert to ug.
3-Hydroxy-3-methylglutaryl-CoA 2029
According to o u r m e a s u r e m e n t s the s t a n d a r d deviation with a b o u t 0.1 /miole H M G - C o A is 4 % .
N o r m a l Values
T h e a m o u n t of H M G - C o A in rat liver is a b o u t 10 n m o l e per g. fresh w t . . 9
W h e n analysing biological samples the possibility of e r r o r m u s t be considered because of the impurity of the
H M G - C o A lyase p r e p a r a t i o n s described below. F o r example, the reaction of o t h e r metabolites in the sample
with c o n t a m i n a t i n g N A D - l i n k e d dehydrogenases. A suitable c o n t r o l which excludes this possibility is to
establish the r e q u i r e m e n t for citrate synthase (start reaction with C S instead of H M G - C o A lyase) o r t o
incubate the sample with K O H (final c o n c e n t r a t i o n 0.2 N ) for 30 min. at r o o m t e m p e r a t u r e (hydrolysis of
HMG-CoA).
Specificity
H M G - C o A lyase reacts only with the naturally occurring H M G - C o A isomer, whose configuration (S) is
given by the biochemical relation t o (R)-mevalonic a c i d . 6
Acetyl-CoA can be determined in the same assay system: the extinction is m e a s u r e d before a n d after
addition of CS (refer also to p . 1991). H M G - C o A is then m e a s u r e d by the addition of H M G - C o A lyase.
O t h e r M e t h o d s for D e t e r m i n a t i o n o f H M G - C o A
Ross a n d Wieland 10
have used the m o r e sensitive m e a s u r e m e n t of N A D H fluorescence instead of a b s o r p t i o n .
This allows tissue extracts to be analysed without preliminary c o n c e n t r a t i o n . T h e modified assay system
contains 20 m M t r i e t h a n o l a m i n e buffer ( p H 7.6), 5 m M M g C l , 5 m M glutathione, 2 m M malate, 0.4 m M
2
N A D and the enzymes. S t a n d a r d i z a t i o n with a k n o w n a m o u n t of acetyl-CoA (internal s t a n d a r d ) .

A n o t h e r s p e c t r o p h o t o m e t r i c assay for H M G - C o A involves coupling reaction (1) with the acetylation of
p-nitroaniline by acetyl-CoA catalysed by arylamine acetyltransferase ( A c e t y l - C o A : arylamine N-acetyl-
transferase, E C 2.3.1.5) (see p . 2004). F o r its application to the assay of H M G - C o A in liver extracts, see . 4
A r y l a m i n e acetyltransferase is significantly inhibited by C o A S H a n d long-chain acyl-CoA derivatives such

as palmityl-CoA ( ^ 10 ; u M ) a n d this can result in a considerable increase in the time required for the
7
assay.
Appendix
Isolation of H M G - C o A Lyase from Beef Liver * 8
T h e following steps are u s e d :

1. P r e p a r a t i o n of acetone-dried p o w d e r from ox liver.
2. Extraction of the dry p o w d e r with p h o s p h a t e buffer.
* The enzyme is designated as " H M G - C o A cleavage e n z y m e " a n d " E n z y m e A " in the cited work. W h e n
the activity is m e a s u r e d in the c o m b i n e d assay with arylamine acetyltransferase: 28 " e n z y m e u n i t s "
= 1 m U (20 °C). - P r e p a r a t i o n of a highly purified, yet less stable enzyme has been described r e c e n t l y . 11
3. H e a t i n g to 50 °C (20 min.).
4. F r a c t i o n a t i o n with acetone at - 8 °C.
5. Precipitation with zinc acetate solution; extraction with p h o s p h a t e buffer
6. Fractionation with a m m o n i u m sulphate ( 0 - 5 0 % s a t u r a t i o n ) ; dialysis.
References
1 B. K. Bachhawat, W. G. Robinson & M. J. Coon, J. biol. C h e m . 216, 727 [1955].

2 / . Knappe, Dissertation, University of M u n i c h , 1957.
3 / . R. Stern, S. Ochoa & F. Lynen, J. biol. C h e m . 198, 313 [1955].
4 O. Wieland, G. Loffler, L. Weiss & /. Neufeldt, Biochem. Z. 333, 10 [I960].
6 / . Knappe, E. Ringelmann & F. Lynen, Biochem. Z . 332, 195 [1959].
7 H. Tabor, A. H. Mehler & E. R. Stadtman, J. biol. C h e m . 204, 127 [1953].
8 F. Lynen, U. Henning, C. Bublitz, B. Sorbo & L. Kroplin-Rueff, Biochem. Z. 330, 269 [1958].
9 O. Wieland, personal c o m m u n i c a t i o n .
10 B. D. Ross & O. Wieland, personal c o m m u n i c a t i o n .
11 L.D. Stegink & M. J. Coon, J. biol. C h e m . 243, 5272 [1968].
3-Hydroxypropionyl-Coenzyme A
Principle
(1) 3-Hydroxypropionyl-CoA + N A D P +
3-h drox ropionyi-coA^
y y P Malonylsemialdehyde-CoA
dehydrogenase
+ NADPH + H +
The rate of formation of m a l o n y l s e m i a l d e h y d e - C o A in the presence of excess nicotinamide-adenine

dinucleotide p h o s p h a t e ( N A D P ) a n d 3 - h y d r o x y p r o p i o n y l - C o A d e h y d r o g e n a s e * is directly p r o p o r t i o n a l
to the concentration of 3 - h y d r o x y p r o p i o n y l - C o A u n d e r the conditions described h e r e . 1
At neutral p H the equilibrium of the reaction is to the left, whereas at p H 9.4 it is displaced to the right.
The rate of formation of m a l o n y l s e m i a l d e h y d e - C o A is followed by the increase in extinction at 300 n m .
The reaction c a n n o t be followed by m e a n s of the change in extinction on reduction of N A D P with
sufficient accuracy, because the enzyme is not p u r e .
The stated assay conditions are o p t i m u m .
Equipment
S p e c t r o p h o t o m e t e r for p r e c i s e m e a s u r e m e n t s at 3 0 0 n m .
Reagents
1. 2 - A m i n o - 2 - m e t h y l - l , 3 - p r o p a n e d i o l * * 4. H y d r o c h l o r i c acid, 1 N , A. R.
2. 3-Hydroxypropionyl-CoA 5. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , A . R.,
p r e p a r e d from c o e n z y m e A a n d /?-propiono- KH P0 .
2 4
lactone*** according t o . 1
6. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , A . R . ,
3. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e K HP0 .
2 4
phosphate, NADP 7. 3 - H y d r o x y p r o p i o n y l - C o A dehydrogenase

sodium salt, N A D P - N a H ; commercial p r e p
2 from Clostridium kluyveri , 1
isolation, see Appen
a r a t i o n , see p . 546. dix, p. 2033.
I. 2 - A m i n o - 2 - m e t h y l - l , 3 - p r o p a n e d i o l h y d r o c h l o r i d e buffer ( 1 . 0 M ; p H 9 . 4 ) :
Dissolve 10.51g. 2 - a m i n o - 2 - m e t h y l - l , 3 - p r o p a n e d i o l in 4 0 m l . d i s t i l l e d w a t e r , adjust t o
p H 9.4 with ca. 20 ml. 1 N HC1 and dilute t o 100 ml. with distilled water.
II. 3 - H y d r o x y p r o p i o n y l - C o A (1 m M ; p H 6 ) :
For standardization, dilute the solution prepared according t o . 1
* N o system n u m b e r has yet been assigned by the E n z y m e C o m m i s s i o n . Suggested n a m e : L-3-Hydroxy-

a c y l - C o A : N A D P oxidoreductase.
** e. g. from C o m m e r c i a l Solvents C o r p . , U S A .
'** e. g. E a s t m a n Organic Chemicals, U S A .
III. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( 5 0 m M / ? - N A D P ) :
D i s s o l v e c a . 4 0 m g . N A D P - N a H in 1 m l . d i s t i l l e d w a t e r .
2
IV. P o t a s s i u m p h o s p h a t e (10 m M ; p H 7.5):

a) D i s s o l v e 1.74 g. K H P 0 2 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
b ) D i s s o l v e 1.36 g. K H P 0 2 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l . M i x s o l u t i o n s a)
a n d b) in t h e r a t i o 8 4 : 1 6 v o l u m e s .
V . 3 - H y d r o x y p r o p i o n y l - C o A d e h y d r o g e n a s e (1 m g . p r o t e i n / m l . ) :
Dilute the e n z y m e solution prepared according t o 1
with potassium phosphate buffer
(solution IV) to 1 mg. protein/ml.
The enzyme solution is stable for several m o n t h s at — 20 °C. Solutions II a n d III are stored frozen.
Procedure
T h e m e t h o d d e s c r i b e d h e r e w a s o r i g i n a l l y d e v e l o p e d for t h e d e t e r m i n a t i o n o f t h e activity o f
3 - h y d r o x y p r o p i o n y l - C o A d e h y d r o g e n a s e . It s h o u l d b e a p p l i c a b l e t o a n y m a t e r i a l w h i c h d o e s
1
n o t a b s o r b s t r o n g l y at 3 0 0 n m .
Assay System
W a v e l e n g t h : 3 0 0 n m ; 1 m l . q u a r t z c u v e t t e s , l i g h t p a t h : 1 c m . ; final v o l u m e : 1 m l . T h r e e
experimental cuvettes can be prepared simultaneously. R e a d a g a i n s t a reference cuvette
c o n t a i n i n g distilled w a t e r i n s t e a d o f s a m p l e .
S a m p l e + distilled w a t e r 0.70 ml. 20-150 pU

Buffer (I) 0.10 ml. 0.1 M
N A D P solution (III) 0.10 ml. 5 mM
M i x and read extinction.
Enzyme (V) 0.10 ml. 0.1 m g . / m l .
M i x , read e x t i n c t i o n e v e r y 3 0 sec. N o t e t h e time

t required for a n i n c r e a s e in e x t i n c t i o n o f 0 . 1 0 0 .
T h e a m o u n t o f e n z y m e a d d e d s h o u l d b e s u c h t h a t t = 1 - 3 m i n . M o r e e n z y m e is d e t r i m e n t a l ,
b e c a u s e t h e p r e p a r a t i o n is c o n t a m i n a t e d w i t h a n e n z y m e i n h i b i t o r y t o t h e r e a c t i o n b e i n g
measured.
Calculations
The rate of the reaction is directly p r o p o r t i o n a l to the 3-hydroxypropionyl-CoA concentration of the

assay mixture. T h e concentration is determined by reference to a s t a n d a r d curve, which is prepared
freshly for each new enzyme p r e p a r a t i o n using 0 . 0 2 - 0 . 1 5 ml. 3 - h y d r o x y p r o p i o n y l - C o A solution (II).
3 -Hydroxypropionyl-Co A 2033
S o u r c e s of Error and S p e c i f i c i t y
The 5-fold purified enzyme p r e p a r a t i o n used here is c o n t a m i n a t e d with malonylsemialdehyde-CoA

dehydrogenase, which oxidizes m a l o n y l s e m i a l d e h y d e - C o A to m a l o n y l - C o A . Therefore only the m i n i m u m
a m o u n t of enzyme required to give an o p t i m u m change in extinction should be used.
3-Hydroxybutyryl-CoA reacts like 3-hydroxypropionyl-CoA. 3 - H y d r o x y p r o p i o n y l - p a n t e t h e i n e reacts at a
rate which is not significant in c o m p a r i s o n to the C o A derivative.
Appendix
Isolation of 3-Hydroxypropionyl-CoA Dehydrogenase 1
The enzyme is extracted from Clostridium kluyveri with potassium p h o s p h a t e buffer (solution V) at
0 °C. The purification steps include: p r o t a m i n e sulphate precipitation, a m m o n i u m sulphate fractionation
(0.65 to 0.95 saturation) a n d dialysis. All a t t e m p t s to purify the enzyme further (e. g. by fractionation
with organic solvents, gel a d s o r p t i o n , acid precipitation, c h r o m a t o g r a p h y on D E A E cellulose) have so
far failed.
The resulting enzyme p r e p a r a t i o n is purified ca. 5-fold in c o m p a r i s o n to the c r u d e extract, and is stored
as a solution in 10 m M p o t a s s i u m p h o s p h a t e buffer ( p H 7.5).
References

Malonyl Coenzyme A
Joachim Ziegenhorn and Feodor Lynen
Malonyl coenzyme A is the key substance in the biosynthesis of m a n y n a t u r a l p r o d u c t s to which Birch's

polyacetate r u l e applies. T h e c a r b o n skeleton of these c o m p o u n d s , which include the fatty acids a n d
1
a series of a r o m a t i c c o m p o u n d s , is built u p by successive head-to-tail linkage of acetate units from malonyl

coenzyme A 2 - 6
.
The assay m e t h o d described here is based on the reaction of malonyl coenzyme A with acetyl coenzyme
A and N A D P H by the multienzyme complex, fatty acid synthetase* from yeast, which leads to the form
ation of long-chain acyl coenzyme A d e r i v a t i v e s , . 7 8
Principle
(1) Acetyl-CoA+nMalonyl-CoA+2n N A D P H + 2 n H + f a t t y a c i d s
? n t h e t a s e
)
C H — ( C H — C H ) C O — C o A + n C 0 + n CoA + 2n N A D P + n H 0
3 2 2 n 2
+
2
(n = 7 - 8 )
The decrease in the concentration of N A D P H , which is measured by the change in extinction at 334,
340, or 365 nm, is p r o p o r t i o n a l to the quantity of m a l o n y l - C o A . 2 Moles of N A D P H are c o n s u m e d per
mole of m a l o n y l - C o A .
The reaction equilibrium lies entirely o n the side of the long-chain acyl-CoA c o m p o u n d s , so that all the 5
malonyl-CoA reacts in the presence of excess acetyl-CoA a n d N A D P H . F a t t y acid synthetase exhibits its
o p t i m u m activity at p H values between 6.5 and 7.0 . T h e enzyme c o n c e n t r a t i o n is so chosen that the reaction
8
is complete in 4 - 1 0 min?
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r c a p a b l e o f a c c u r a t e m e a s u r e m e n t s at 3 3 4 ,
340, or 365 n m .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 4. P o t a s s i u m h y d r o x i d e , K O H , A . R.
K H P 0 , A.R.
2 4 5. C y s t e i n e h y d r o c h l o r i d e , m o n o h y d r a t e ,
2. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , A.R.
K HP0 -3H 0,
2 4 2 A.R. 6. B o v i n e s e r u m a l b u m i n , p u r i s s .
3. E t h y l e n e d i a m i n e t e t r a a c e t a t e , E D T A , A . R. 7. A c e t y l - c o e n z y m e A
disodium salt E D T A - N a H -2 H 0 2 2 2 trilithium salt; commercial preparations, see
p. 524.
* N o E n z y m e C o m m i s s i o n system n u m b e r as yet.
Malonyl-CoA 2035
8. R e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o 9. F a t t y a c i d s y n t h e t a s e
tide p h o s p h a t e , N A D P H from yeast, suspension in 3.5 M ammonium
tetrasodium salt NADPH-Na ; 4 commercial sulphate solution. F o r isolation, see Appendix
p r e p a r a t i o n s see p . 547. p . 2037.
M a k e u p all s o l u t i o n s w i t h freshly p r e p a r e d d o u b l y d i s t i l l e d w a t e r .
a) D i s s o l v e 13.61 g. K H P 0 2 4 in w a t e r a n d m a k e u p t o 1 0 0 m l .
b) D i s s o l v e 2 2 . 8 2 g. K H P 0 - 3 H 0 in w a t e r a n d m a k e u p t o 100 m l .
2 4 2
M i x 55 m l . s o l u t i o n a) w i t h 4 4 m l . s o l u t i o n b ) . C h e c k p H w i t h t h e g l a s s e l e c t r o d e .
II. E t h y l e n e d i a m i n e t e t r a - a c e t a t e (0.1 M ) :
D i s s o l v e 3 . 7 2 g. E D T A - N a H 2 2 - 2 H 0 in w a t e r a n d m a k e u p t o 1 0 0 m l .
2
III. P o t a s s i u m h y d r o x i d e s o l u t i o n ( 4 N ) :
D i s s o l v e 2 2 . 4 4 g. K O H in 8 0 m l . o f w a t e r . A f t e r c o o l i n g , m a k e u p t o 100 m l . w i t h w a t e r .
IV. Cysteine hydrochloride (2 M ) :
D i s s o l v e 3.51 g. c y s t e i n e h y d r o c h l o r i d e m o n o h y d r a t e in w a t e r a n d m a k e u p t o 10 m l .
V . C y s t e i n e (1 M c y s t e i n e ; 0 . 2 5 M p h o s p h a t e buffer, p H 6 . 5 ) :
M i x 1 m l . s o l u t i o n I V w i t h 0.5 m l . s o l u t i o n I a n d a d d 0.5 m l . s o l u t i o n III.
VI. Serum albumin (20 m g . / m l . ) :
D i s s o l v e 2 0 m g . s e r u m a l b u m i n in w a t e r a n d m a k e u p t o 1 m l .
VII. Acetyl C o A (7.5 m M ) :
D i s s o l v e 15 m g . a c e t y l C o A - L i 3 in w a t e r a n d m a k e u p to 2 m l .
VIII. R e d u c e d nicotinamide adenine dinucleotide phosphate (20 m M NADPH):
Dissolve 22 mg. N A D P H - N a 4 in w a t e r a n d m a k e u p t o 1 m l .
IX. Fatty acid synthetase (150 /ig./ml.; 2 2 5 - 3 7 5 m U / m l . ) :
D i l u t e s t o c k s u s p e n s i o n o f t h e e n z y m e a s r e q u i r e d w i t h 10 m M p h o s p h a t e buffer, p H 6.5.
Store solution III in a stoppered vessel at r o o m t e m p e r a t u r e , solutions I a n d II at 0 to 4 °C, and solutions

IV, VI, VII, a n d VIII a n d the enzyme suspension at - 1 0 to - 1 5 °C. Solutions I, II, and III can be used
for at least 1 year. Solutions VI a n d VII a n d the enzyme suspension keep for several m o n t h s , solution
IV for a b o u t 1 m o n t h , a n d solution VIII for a b o u t 2 weeks. Solutions V a n d IX must be freshly p r e p a r e d
daily. Store solutions V, VI, VII, VIII, a n d IX in an ice b a t h d u r i n g a series of tests. Solution VIII m u s t
be protected against the action of light.
Procedure
M a l o n y l - C o A c a n b e s t o r e d for s e v e r a l m o n t h s in a q u e o u s s o l u t i o n at p H 5 a n d at - 2 0 ° C
T h e c o m p o u n d is u n s t a b l e in a l k a l i n e o r in s t r o n g l y a c i d i c s o l u t i o n 1 0 , 1 1
.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 o r H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 0 0 m l . ; r o o m
t e m p e r a t u r e ; r e a d a g a i n s t air.
To d e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f t h e s a m p l e , carry o u t a b l a n k
w i t h all a d d i t i o n s e x c e p t t h e e n z y m e ( r e p l a c e this b y w a t e r ) . A d d 0 . 0 2 m l . s a m p l e s o l u t i o n
a n d m i x . U s e t h e r e s u l t i n g c h a n g e in e x t i n c t i o n t o c o r r e c t E^^.
P h o s p h a t e buffer (I) 0.20 ml. 0.1 M

E D T A solution (ID 0.02 ml. 1 mM
Cysteine solution (V) 0.02 ml. 10 m M
Serum albumin solution (VI) 0.03 ml. 0.3 m g . / m l .
Water 1.47 m l .
Acetyl-CoA solution (VII) 0.02 ml. 75 / / M
N A D P H solution (VIII) 0.02 ml. 2 0 0 pM
Enzyme solution (IX) 0.20 ml. 15/xg./ml. = 2 3 - 3 8 m U / m l .
Mix, record extinctions with pen recorder or read

t h r e e t i m e s at i n t e r v a l s o f 1 m i n . , e x t r a p o l a t e t o t h e
time o f addition o f the sample, and determine E x (cf.
p. 308).
Sample 0.02 ml. u p t o a b o u t 8 0 fiM
M i x , r e c o r d e x t i n c t i o n s w i t h p e n r e c o r d e r o r r e a d at
i n t e r v a l s o f 1 m i n . for 4 - 1 0 m i n . , a n d d e t e r m i n e E 2 by
extrapolation to the time o f addition o f the sample
(cf. p. 3 0 8 ) . A E = E j — E is u s e d for t h e c a l c u l a t i o n s .
2
Calculations
The reaction proceeds stoichiometrically under the conditions indicated. F o r m u l a (2) on p. 312 is
therefore valid. Since 2 /miole of N A D P H are c o n s u m e d per /miole of malonyl C o A , the result must be
divided by 2 in the calculation. T h e following relationships are valid for the m a l o n y l - C o A concentration
in the sample.
c= AEx8.20 AEx8.04 z l E x 14.49 [/miole/ml.]
With an average of 4.25 /miole of m a l o n y l - C o A per ml. of sample solution, two s t a n d a r d deviations was
0.17 /miole. T h e coefficient of variation is 2 . 0 % .
Interference in the assay technique: If the final extinction reaches a c o n s t a n t value, solution VIII no longer
has the required content of N A D P H . In this case the test must be repeated with a freshly prepared N A D P H
solution.
Malonyl-CoA 2037
Malonylpantetheine a n d S-malonyl-N-capryloylcysteamine also react, but at a lower r a t e 1 2 1 3

.
Appendix
Isolation of the Fatty Acid Synthetase from Baker's Yeast . 8
T h e purification consists of the following steps:
1. Disintegration of the cells in the cell homogenizer a n d extraction of the enzyme with p h o s p h a t e buffer.
2. F r a c t i o n a t i o n of the extract with a m m o n i u m sulphate between 35 a n d 5 0 % saturation.
3. Dialysis of t h e precipitate
4. A d s o r p t i o n of the enzyme o n calcium p h o s p h a t e gel a n d elution with p h o s p h a t e buffer.
5. F r a c t i o n a t i o n of the eluate with a m m o n i u m sulphate between 35 a n d 5 0 % saturation. Dissolution of
the precipitate in p h o s p h a t e buffer.
6. Sedimentation of the enzyme in the ultracentrifuge (100000 g), this step being carried o u t twice'.
The enzyme is stored at a c o n c e n t r a t i o n of a b o u t 40 mg./ml. in 3.5 M a m m o n i u m sulphate solution (0.1 M

in p h o s p h a t e buffer, p H 6.5; 10 m M in thioglycol; 1 m M in E D T A ) at - 1 0 to - 1 5 °C. A b o u t 250 mg.
pure fatty acid synthetase with a specific activity of 1.5-2.5 U / m g . (25 °C) can be obtained from 2 kg.
b a k e r ' s yeast by 2 to 3 d a y s ' work. F o r determination of activity a n d properties of the multienzyme
complex, s e e 8 1 4
References
1 A. J. Birch, in L. Zechmeister: Fortschritte der Chemie organischer Naturstoffe, Vol. 14, p . 186,
Springer Verlag, Wien 1957.
2 F. Lynen, J. Cellular C o m p a r a t . Physiol. 54, Suppl. 1 p . 33 [1959].
3 F. Lynen, in M. Sela: N e w Perspectives in Biology, p . 132, Elsevier Publishing C o m p a n y , A m s t e r d a m
1964.
4 / . H. Richards & /. B. Hendrickson: T h e Biosynthesis of Steroids, Terpenes a n d Acetogenins, W. A.
Benjamin, Inc., N e w York a n d A m s t e r d a m 1964.
5 F. Lynen, Angew. C h e m . 77, 929 [1965].
6 P. Dimroth, H. Walter & F. Lynen, Eur. J. Biochem. 13, 98 [1970].
7 F. Lynen, in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, Vol. V, p . 443, A c a d e m i c Press,
N e w York a n d L o n d o n 1962.
8 J. Lynen, in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, Vol. X I V , p . 17, A c a d e m i c Press,
N e w York a n d L o n d o n 1969.
9 D. Oesterhelt, Dissertation, University M u n i c h 1967.
10 H. Eggerer & F. Lynen, Biochem. Z. 335, 540 [1962].
11 L. Jdnicke & F. Lynen in P. D. Boyer, H. A. Lardy & K. Myrbdck: T h e Enzymes, Vol. 3, p . 3, A c a d e m i c
Press, N e w York a n d L o n d o n 1960.
12 W. Pirson, Dissertation, University M u n i c h 1970.
13 M. Sumper, Dissertation, University M u n i c h 1970.
14 F. Lynen, K.-I. Arnstadt, M. Sumper, K.-H. Weithmann, W. Winnewisser &/. Ziegenhorn, i n / . Ganguly
& R. M. S. Smellie: C u r r e n t Trends in the Biochemistry of Lipids, p . 5, A c a d e m i c Press, N e w York
a n d L o n d o n 1972.
Malonylsemialdehyde-Coenzyme A
Malonylsemialdehyde-CoA is reduced by 3-hydroxypropionyl-CoA d e h y d r o g e n a s e * and reduced nico

tinamide-adenine dinucleotide p h o s p h a t e ( N A D P H ) .
Principle
(1) Malonylsemialdehyde-CoA + N A D P H + H +
. "
3 h y d r o x y p r o p i o n y l
' C o A
3-Hydroxypropionyl-CoA
v
' J
dehydrogenase
+ NADP +
At neutral p H the equilibrium of the reaction is strongly to the right. The reaction proceeds to completion
and one mole of N A D P H is oxidized for each mole of malonylsemialdehyde-CoA present. The decrease
of the extinction at 340 n m due to the oxidation of N A D P H is m e a s u r e d . 1
The stated conditions are o p t i m u m .
Equipment
(334, 365) n m .
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 4. 3 - H y d r o x y p r o p i o n y l - C o A dehydrogenase
2. S o d i u m h y d r o x i d e , 2 N purified 5-fold from extracts of Clostridium
3. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo kluyveri 1
( p r o t a m i n e sulphate precipitation, a m
tide p h o s p h a t e , NADPH m o n i u m sulphate precipitation, dialysis, see p.
sodium salt, N A D P H - N a ; commercial p r e p
4
2033). The p r e p a r a t i o n is stable for several
a r a t i o n , see p . 547. m o n t h s at - 20 °C.
The enzyme p r e p a r a t i o n obtained according t o is c o n t a m i n a t e d with N A D P H oxidase a n d malonylsemi-

1
aldehyde-CoA dehydrogenase. If sufficiently dilute enzyme solutions are used, these impurities do not
interfere. This must be tested for each new enzyme p r e p a r a t i o n in an assay mixture prepared as described
under "Assay System, experimental c u v e t t e " , except that the sample is replaced by distilled water. A n y
oxidation of N A D P H must be allowed for in the calculations.
* N o system n u m b e r has yet been assigned by the E n z y m e C o m m i s s i o n . Suggested n a m e : L-3-Hydroxy-

a c y l - C o A : N A D P oxidoreductase.
Malonylsemialdehyde-CoA 2039
D i s s o l v e 1 8 . 6 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in a b o u t 5 0 m l . d i s t i l l e d w a t e r , a d j u s t t o
p H 7.5 w i t h 2 N N a O H a n d d i l u t e t o 100 m l . w i t h d i s t i l l e d w a t e r .
II. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e (1 m M / J - N A D P H ) :
Dissolve 4.4 mg. N A D P H - N a 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 5 m l . S t o r e t h e s o l u t i o n at
- 2 0 °C.
III. 3 - H y d r o x y p r o p i o n y l - C o A d e h y d r o g e n a s e (0.1 m g . p r o t e i n / m l . ) :
D i l u t e t h e p r e p a r a t i o n o b t a i n e d a c c o r d i n g t o . S t o r e t h e s o l u t i o n at — 2 0 ° C .
1
Procedure
Experimental Material
M a l o n y l s e m i a l d e h y d e - C o A c a n b e d e t e r m i n e d b y t h e m e t h o d d e s c r i b e d in a n y s a m p l e w h i c h
d o e s n o t a b s o r b s t r o n g l y at 3 4 0 n m .
Assay System
Wavelength: 340 ( H g 334, H g 365) n m ; 1 ml. quartz cuvettes, light p a t h : 1 cm.;'final v o l u m e :

1 m l . ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t a r e f e r e n c e c u v e t t e ( c o n t a i n i n g buffer a n d w a t e r o n l y ) .
Sample + distilled water 0.50 ml. 20-100 pM

Buffer (I) 0.20 ml. 0.2 M
N A D P H solution (II) 0.20 ml. 0.2 m M
Enzyme solution (HI) 0.10 ml. 10 / i g . / m l .
M i x , read e x t i n c t i o n e v e r y 3 0 sec. u n t i l t h e r e a c t i o n is
c o m p l e t e ( 2 - 4 m i n . ) . M u l t i p l y t h e final v a l u e E b y 1.1
2
(dilution factor). A E = El - 1.1 E 2 is u s e d f o r t h e

calculations.
Calculations
The decrease in extinction is strictly p r o p o r t i o n a l to the concentration between 0.01 and 0.15 umole
malonylsemialdehyde-CoA. T h e c o n c e n t r a t i o n is calculated according to e q u a t i o n (2) on p. 312 for
0.2 ml. sample.

c = AE x 0.820 AE x 0.803 AE x 1.449 [^mole/ml.]
The enzyme used is n o t p u r e a n d therefore a control cuvette must be included to determine h o w m u c h

of the N A D P H oxidation is n o t d u e to malonylsemialdehyde-CoA.
Acetoacetyl-CoA reacts like malonylsemialdehyde-CoA. Malonylsemialdehyde-pantetheine and aceto-

acetyl-pantetheine also react u n d e r the conditions of the assay, but very m u c h m o r e slowly than the C o A
derivatives.
References

Succinyl-Coenzyme A
Succinyl-CoA is a n i m p o r t a n t intermediate in the tricarboxylic acid cycle, in the utilization of acetoacetate

by extra-hepatic tissues a n d in the synthesis of p o r p h y r i n s . T h e enzymatic m e t h o d for the determination
of succinyl-CoA is based on the transfer of the C o A S H to acetoacetate with 3-oxoacid CoA-transferase
(Succinyl-CoA :3-oxo-acid CoA-transferase, E C 2.8.3.5), a n d the reduction of the resulting acetoacetyl-
C o A to L-3-hydroxybutyryl-CoA with 3-hydroxyacyl-CoA dehydrogenase, H O A D H (L-3-Hydroxyacyl-
C o A : N A D oxidoreductase, E C 1.1.1.35) a n d N A D H . A 1
fluorimetric modification of this m e t h o d has
been described . 6
Application of the Method: In biochemistry a n d microbiology.
Principle
(1) Succinyl-CoA + Acetoacetate 3

~ °*°* C I D
COA - transferase^ § u c c m a t e _|_ Acetoacetyl-CoA
(2) Acetoacetyl-CoA + N A D H + H +
" O A D H
- L-3-Hydroxybutyryl-CoA + N A D +
T h e decrease in extinction at 340 (334, 365) n m d u e to the oxidation of N A D H is a measure of the succinyl-
C o A present.
T h e conversion of succinyl-CoA to succinate is quantitative at p H 6 . 8 - 7 . 0 a n d with an excess of N A D H

a n d acetoacetate.
Equipment
(334 or 365) n m .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 5. Succinyl-CoA
K H P 0 , A . R.
2 4 p r e p a r e d according t o . 3
2. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , 6. 3 - O x o a c i d CoA-transferase
K H P 0 , A . R.
2 4 prepared from pig heart ,
4
ca. 0.75 U/mg.
3. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo (25 °C). F o r isolation, see p. 2044.
tide, N A D H 7. 3 - H y d r o x y a c y l - C o A dehydrogenase,
disodium salt, N A D H - N a , commercial p r e p
2 HOADH
a r a t i o n , see p . 545. p r e p a r e d from pig h e a r t ; crystalline suspension
4. A c e t o a c e t i c a c i d , l i t h i u m salt in 3.2 M a m m o n i u m sulphate solution; ca. 90
hydrolyze freshly distilled ethyl acetoacetate U / m g . protein. C o m m e r c i a l p r e p a r a t i o n , see
with lithium h y d r o x i d e a n d isolate the crystalline p. 474.
lithium s a l t .2
Purity of Reagents
T h e 3-oxoacid CoA-transferase a n d the H O A D H m u s t be free from 3-hydroxybutyrate dehydrogenase

a n d succinyl-CoA deacylase.
P r e p a r e all s o l u t i o n s w i t h d o u b l e d i s t i l l e d o r d e i o n i z e d w a t e r .
a) D i s s o l v e 13.6 g. K H P 0 2 4 in 1 0 0 0 m l . distilled w a t e r .
b) D i s s o l v e 1 7 . 4 g. K H P 0 2 4 in 1 0 0 0 m l . distilled w a t e r .
M i x e q u a l v o l u m e s o f s o l u t i o n a) a n d b ) .
Check the p H of the mixture with a glass electrode.
II. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (ca. 6 m M / ? - N A D H ) :
D i s s o l v e 10 m g . N A D H - N a 2 in 2 m l . distilled w a t e r .
III. L i t h i u m a c e t o a c e t a t e (0.1 M ) :
D i s s o l v e 108 m g . l i t h i u m a c e t o a c e t a t e in 10 m l . distilled w a t e r .
IV. 3-Oxoacid CoA-transferase (12 m g . p r o t e i n / m l . ) :
U s e the stock solution undiluted.
V . 3 - H y d r o x y a c y l - C o A d e h y d r o g e n a s e , H O A D H (2 m g . p r o t e i n / m l . ) :
U s e t h e s t o c k s u s p e n s i o n in 3.2 M a m m o n i u m s u l p h a t e .
Stability of the Solutions
Store the N A D H solution (II) a n d acetoacetate solution (III) at - 1 5 ° C ; they are stable for several weeks.
T h e 3-oxoacid CoA-transferase p r e p a r a t i o n a n d H O A D H suspension are stable for m o n t h s at 0 ° - 4 °C.
Procedure
T h e m e t h o d has only been tested o n pure solutions o f succinyl-CoA and mixtures o f k n o w n

c o m p o s i t i o n c o n t a i n i n g s u c c i n y l - C o A . S u c c i n y l - C o A is u n s t a b l e at a l k a l i n e p H , b u t is relatively
s t a b l e b e l o w p H 6 a n d at - 1 5 ° C .
Succinyl-CoA 2043
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 1 7 m l . M e a s u r e a g a i n s t
a cuvette containing water.
Sample 1.00 m l . 5 - 5 0 fiM succinyl-CoA

P h o s p h a t e buffer s o l u t i o n (I) 1.00 m l . 0.05 M
N A D H solution (II) 0.05 ml. ca. 0.15 m M
Acetoacetate solution (III) 0.10 ml. ca. 5 m M
M i x w e l l a n d r e a d e x t i n c t i o n E . M i x in
x
3-Oxoacid CoA-transferase c a . 6 0 fig. p r o t e i n / m l . = ca.

0.01 m l .
solution (IV) 50 m U / m l .
H O A D H suspension (V) 0.01 m l . c a . 10 fig. p r o t e i n / m l . = ca.
900 m U / m l .
E x — E 2 = A E is u s e d for t h e c a l c u l a t i o n s .
A c u v e t t e c o n t a i n i n g d i s t i l l e d w a t e r i n s t e a d o f s a m p l e is u s e d t o m e a s u r e t h e c h a n g e in e x t i n c t
ion o n addition o f the enzymes.
Calculations
U n d e r the assay conditions the formation of N A D is stoichiometric with the a m o u n t of succinyl-CoA

present and therefore the calculation formula (2) on p . 312 applies. T h e results are obtained in //mole
succinyl-CoA/ml. sample.

c= AE x 0.356 AE x 0.349 AE x 0.638
T h e coefficient of variation is 6 % .
N o r m a l Values
None known.
C o n t a m i n a t i o n of the 3-oxoacid CoA-transferase p r e p a r a t i o n with o t h e r N A D - l i n k e d dehydrogenases

m a y lead to errors with samples of u n k n o w n composition.
T h e 3-oxoacid CoA-transferase is strictly specific for s u c c i n y l - C o A . 5

Appendix
Isolation of 3-Oxoacid CoA-transferase 4,5
T h e p r o c e d u r e includes the following steps:

Extraction from pig heart. A m m o n i u m sulphate fractionation between 35 a n d 6 5 % . H e a t i n g at 55 °C a n d
acidification with acetic acid to p H 5.8. A m m o n i u m sulphate fractionation between 50 a n d 70 %. C h r o m a t o
graphy on DEAE-cellulose at p H 7.
A n improved purification of the transferase using substrate elution c h r o m a t o g r a p h y has recently been
described .7
References
1 D. H. Williamson, M. W. Bates & H. A. Krebs, unpublished work.

2 L. M. Hall, Analyt. Biochem. 3, 75 [1962].
3 E. J. Simon & D. Shemin, J. A m e r . chem. Soc. 75, 2 520 [1953].
4 L. B. Hersh & W. P. Jencks, J. biol. C h e m . 242, 3468 [1967].
5 / . R. Stern, M. J. Coon, A. del Campillo & M. C Schneider, J. biol. C h e m . 227, 15 [1956].
6 C. M. Smith, J. Bryla, S. Damon, K. F. LaNoue & J. R. Williamson, A n a l . Biochem. 57, 408 [1973].
7 M. R. Edwards, M. Singh & P. K. Tubbs, F E B S Letters 37, 155 [1973].
Nicotinamide-Adenine Dinucleotides
(NAD, NADP, NADH, NADPH)
Spectrophotometric and Fluorimetric Methods
Martin Klingenberg
The nicotinamide dinucleotides, N A D , N A D H a n d nicotinamide dinucleotide p h o s p h a t e s NADP,

N A D P H occur in all living cells. Within cells, the nicotinamide dinucleotides are n o t distributed evenly
over the various subcellular regions, e.g. m i t o c h o n d r i a a n d cytosol. In whole blood, the nicotinamide
dinucleotides occur only within the cell elements (erythrocytes, t h r o m b o c y t e s , leucocytes).
T h e m e t h o d s described below allow the d e t e r m i n a t i o n of the state of reduction of the nicotinamide
dinucleotides by m e a s u r e m e n t of the content of N A D H a n d N A D or N A D P H a n d N A D P . T h e measure
ment of the total content ( N A D + N A D H or N A D P + N A D P H ) , which is sufficient for m a n y p r o b l e m s ,
can be simplified in some cases if N A D H a n d N A D P H are completely oxidized to N A D a n d N A D P
in the biological material before extraction. T h e catalytic m e t h o d (cycling method) also offers a simple
determination of the total nicotinamide dinucleotide content.
N i c o t i n a m i d e dinucleotides can be determined by three different enzymatic m e t h o d s . T h e s p e c t r o p h o t o
metric a b s o r p t i o n allows particularly accurate m e a s u r e m e n t when the c o n t e n t of nicotinamide dinucleo
tides in the extracts is sufficiently high (more t h a n 10 nmole). M e a s u r e m e n t s at lower c o n c e n t r a t i o n s (down
to 0.2 nmole) are possible only with special e q u i p m e n t (high-sensitivity difference s p e c t r o p h o t o m e t e r ) . 1
Lower contents of nicotinamide dinucleotides ( d o w n to 0.05 nmole) can be m e a s u r e d by fluorimetric

m e t h o d s . Both m e t h o d s are stoichiometric substrate assays.
2
Even greater sensitivity is possible in the m e a s u r e m e n t of nicotinamide dinucleotides ( d o w n to 1 0 " nmole) 5
by the catalytic assay (enzymatic cycling) .3,4

Different extraction c o n d i t i o n s are used in this m e t h o d ,
see p . 2063.
Extraction Methods
In addition to the m e t h o d s described in the chapter "Cell a n d Tissue D i s i n t e g r a t i o n " , p . 400, there are a
n u m b e r of additional m e t h o d s available for the extraction of nicotinamide dinucleotides from biological
material. A s in all o t h e r metabolite d e t e r m i n a t i o n s , tissue fixation is necessary t o e n s u r e t h a t the c o n c e n t r a
tions of oxidized a n d reduced nicotinamide dinucleotides t h a t are present in vivo are retained in the ex
tract. T h e fixation rate m u s t be particularly high in this case; for example, local anaerobiosis d u r i n g
quick-freezing first affects t h e n i c o t i n a m i d e dinucleotides of the m i t o c h o n d r i a l c o m p a r t m e n t , so t h a t a
large p r o p o r t i o n of the N A D can be converted into N A D H in less t h a n 50 msec. T h e changes in the o t h e r
metabolites are considerably slower. W h e r e a s quick-freezing is used in the case of tissues, fixation can be
achieved in the case of h o m o g e n a t e s , suspensions of cells, m i t o c h o n d r i a , etc., by rapid mixing of the suspen
sion with the extraction solvent.
T h e extraction of nicotinamide dinucleotides is complicated by the fact t h a t the reduced forms, N A D H
a n d N A D P H , are unstable in acids. N A D H a n d N A D P H are extracted with alkali u n d e r suitable con
ditions, with c o n c o m i t a n t d e c o m p o s i t i o n of the oxidized forms N A D a n d N A D P . F o r the m e a s u r e m e n t
of the metabolic status of n i c o t i n a m i d e dinucleotides, therefore, it is generally necessary to p r e p a r e two
extracts, i. e. a n acid extract for the m e a s u r e m e n t of N A D a n d N A D P (extract S) a n d an alkaline extract
for the m e a s u r e m e n t of N A D H a n d N A D P H (extract A). T h e increased difficulties a n d sources of e r r o r
arise in the m e a s u r e m e n t of N A D H a n d N A D P H in the alkaline extract.
U n d e r certain conditions, b o t h the oxidized a n d the reduced forms can be determined in a single acid ex
tract by measurement of the acid d e c o m p o s i t i o n p r o d u c t s of N A D H , i. e. adenosine d i p h o s p h a t e ribose
( A D P R ) and adenosine d i p h o s p h a t e ribose p h o s p h a t e ( A D P R P ) , by a n i o n exchange c h r o m a t o g r a p h y
together with N A D a n d N A D P ' . 2 5
A particularly g o o d criterion of the successful determination of n i c o t i n a m i d e dinucleotides is the con

stancy of the sums N A D + N A D H a n d N A D P + N A D P H in various metabolic situations. Particularly
with the aid of the c h r o m a t o g r a p h i c m e t h o d , it was possible to establish that only the oxidized and reduced
forms occur in vivo, a n d that there is n o other form present, e.g. p h o s p h o r y l a t e d N A D 5 , 6
. T h e sums
N A D + N A D H a n d N A D P + N A D P H must therefore remain c o n s t a n t .
E x t r a c t i o n o f N A D and N A D P ( " a c i d e x t r a c t " )
Reagents
1. P e r c h l o r i c a c i d , A . R., s p . gr. 1.67; ca. 7 0 % 3. D i p o t a s s i u m h y d r o g e n p h o s p h a t e ,

(w/w) K H P 0 , A . R.
2 4
2. P o t a s s i u m h y d r o x i d e s o l u t i o n , A . R., 3 N
I. P e r c h l o r i c a c i d ( 0 . 6 N ) :
D i l u t e 5.2 m l . H C 1 0 4 t o 100 m l . w i t h d o u b l y d i s t i l l e d w a t e r .
II. P e r c h l o r i c a c i d (3 N ) :
Dilute 26 ml. H C 1 0 4 to 100 ml. with d o u b l y distilled water.
III. D i p o t a s s i u m h y d r o g e n p h o s p h a t e (1 M ) :
D i s s o l v e 17.4 g. K H P 0 2 4 in d o u b l y distilled w a t e r a n d m a k e u p t o 100 m l .
Procedure
Extract N A D and N A D P f r o m animal tissue, b l o o d , or m i t o c h o n d r i a with perchloric acid;

s i n c e t h e p e r c h l o r i c a c i d c o n c e n t r a t i o n in t h e e x t r a c t i o n s y s t e m s h o u l d b e a b o u t 0.5 N , a d d
5 ml. o f 0.6 N H C 1 0 4 p e r 1 g. o f t i s s u e o r 1 m l . o f b l o o d .
C o l l e c t tissue b y t h e " q u i c k - f r e e z e m e t h o d " (refer t o p . 4 0 0 ) , a n d p o w d e r finely in t h e f r o z e n
state in a m o r t a r . I n t r o d u c e 0.6 N p e r c h l o r i c a c i d s o l u t i o n (I) i n t o c e n t r i f u g e t u b e s o r s m a l l
b e a k e r s for a w e i g h t o f r a t i o t i s s u e : H C 1 0 = 1 : 5 , w e i g h , a d d f r o z e n t i s s u e p o w d e r w i t h v i g o r o u s
4
stirring ( m a g n e t i c stirrer), a n d w e i g h a g a i n . A d j u s t r a t i o o f w e i g h t o f t i s s u e : H C 1 0 t o a b o u t 1 :54
by a d d i t i o n o f m o r e t i s s u e o r H C 1 0 . 4
Inject blood f r o m the s y r i n g e d i r e c t l y i n t o t h e p e r c h l o r i c a c i d s o l u t i o n (I).

D e p r o t e i n i z e s u s p e n s i o n s o f mitochondria w i t h 3 N p e r c h l o r i c a c i d s o l u t i o n (II) t o a v o i d e x c e s s i v e
d i l u t i o n . A l l o w 0.2 m l . o f 3 N H C 1 0 s o l u t i o n (II) t o run in per 1 m l . o f m i t o c h o n d r i a s u s p e n s i o n
4
w i t h v i g o r o u s stirring.
T o r e m o v e p r o t e i n , c e n t r i f u g e for 5 m i n . at 3 0 0 0 t o 5 0 0 0 g ; c a r e f u l l y r e m o v e a b o u t 1 m l . o f t h e
s u p e r n a t a n t fluid w i t h a p i p e t t e w i t h o u t d i s t u r b i n g t h e p r o t e i n , p i p e t t e 0.2 m l . o f K HP0
2 4
s o l u t i o n (III) i n t o this p o r t i o n w i t h c o o l i n g in ice, a n d a d d 3 N K O H f r o m a fine c a p i l l a r y w i t h

i n t e n s i v e stirring ( m a g n e t i c stirrer) until t h e p H is 7.2 t o 7.4. A l l o w K C 1 0 4 to sediment, and
p i p e t t e s a m p l e s o f t h e s u p e r n a t a n t fluid for t h e m e a s u r e m e n t s .
N i c o t i n a m i d e - a d e n i n e Dinucleotides 2047
E x t r a c t i o n o f N A D H and N A D P H ( " a l k a l i n e e x t r a c t " )

Reagents
1. E t h a n o l , a b s o l u t e , A . R. 5. D i p o t a s s i u m h y d r o g e n p h o s p h a t e ,
2. P o t a s s i u m h y d r o x i d e , A . R. K H P 0 , A . R.
2 4
3. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 6. A m m o n i u m s u l p h a t e , A . R.
4. P o t a s s i u m d i h y d r o g e n p h o s p h a t e ,
K H P 0 , A . R.
2 4
I. A l c o h o l i c p o t a s s i u m h y d r o x i d e s o l u t i o n (0.5 N ) :
D i s s o l v e 2.8 g. K O H in a m i x t u r e o f e q u a l v o l u m e s o f e t h a n o l a n d d o u b l y d i s t i l l e d w a t e r ,
a n d m a k e u p t o 100 m l .
II. A l c o h o l i c p o t a s s i u m h y d r o x i d e s o l u t i o n (1 N ) :
D i s s o l v e 5.6 g. K O H in 100 m l . o f e t h a n o l .
III. T r i e t h a n o l a m i n e H C l - p h o s p h a t e m i x t u r e ( 0 . 5 M t r i e t h a n o l a m i n e ; 0 . 4 M K H P 0 ; 0.1 M2 4
K HP0 ):
2 4
D i s s o l v e 9 . 3 0 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e + 5 . 4 4 g. K H P 0 2 4 + 1.74 g. K H P 0
2 4 in
d o u b l y distilled water a n d m a k e u p to 100 ml.
IV. T r i e t h a n o l a m i n e H C 1 (1 M ) :
D i s s o l v e 1 8 . 6 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p t o
100 m l .
V. Saturated a m m o n i u m sulphate solution:
S u s p e n d a p p r o x . 8 0 g. ( N H ) S 0 4 2 4 in d o u b l y d i s t i l l e d w a t e r at r o o m t e m p e r a t u r e , m a k e u p
t o 100 m l . , a n d a l l o w t o s t a n d for several h o u r s w i t h o c c a s i o n a l stirring. D o n o t r e m o v e
solid phase.
Procedure
Tissue: C o o l 2 m l . a l c o h o l i c K O H s o l u t i o n (I) in a s u i t a b l e c e n t r i f u g e t u b e t o —17 ° C in a n

ice-salt m i x t u r e . F i n e l y s u s p e n d 2 0 0 - 3 0 0 m g . o f t h e q u i c k - f r o z e n t i s s u e in this c o o l e d KOH
w i t h a s u i t a b l e stirrer. C l o s e t h e c e n t r i f u g e t u b e s ( g l a s s s t o p p e r s ) a n d s h a k e for 5 m i n . in a 9 0 ° C
w a t e r b a t h . C o o l t h e n o w s u b s t a n t i a l l y clear, y e l l o w - b r o w n s o l u t i o n t o 0 ° C as r a p i d l y a s
p o s s i b l y in a n ice b a t h . A f t e r 5 m i n . , n e u t r a l i z e b y s l o w a d d i t i o n o f 1 m l . t r i e t h a n o l a m i n e H C l -
p h o s p h a t e m i x t u r e (III) w i t h c o o l i n g a n d stirring. T h i s s h o u l d b r i n g t h e p H t o 7.8. T h e p H m a y
vary according to the quantity o f tissue, and the quantity o f neutralizing solution s h o u l d be
v a r i e d a c c o r d i n g l y . A l l o w t o s t a n d for 10 m i n . at r o o m t e m p e r a t u r e t o flocculate the denatured
p r o t e i n . T h e n c e n t r i f u g e for 5 m i n . at 2 0 0 0 0 t o 4 0 0 0 0 g w i t h c o o l i n g . U s e t h e a l m o s t c l e a r
s u p e r n a t a n t fluid i m m e d i a t e l y for m e a s u r e m e n t s .
Blood: T o a v o i d e x c e s s i v e d i l u t i o n , u s e t h e c o n c e n t r a t e d a l c o h o l i c K O H ( s o l u t i o n II). P l a c e
0.5 m l . o f 1 N a l c o h o l i c K O H in c e n t r i f u g e t u b e s p e r m l . o f b l o o d . Inject t h e b l o o d w i t h v i g o r o u s
s h a k i n g . A l l o w t o s t a n d for 3 0 m i n . at r o o m t e m p e r a t u r e , t h e n c o o l in a n ice b a t h . N e u t r a l i z e b y
stirring in 0 . 2 m l . t r i e t h a n o l a m i n e . H C 1 s o l u t i o n ( I V ) t o b r i n g t h e p H t o a b o u t 7.8. A d d a m m o n
i u m s u l p h a t e for further p r e c i p i t a t i o n o f h a e m o c h r o m o g e n . C e n t r i f u g e off d e n a t u r e d p r o t e i n
( 1 0 m i n . at 1 0 0 0 0 g). A n e l e g a n t m e t h o d for t h e p r e c i p i t a t i o n o f e r y t h r o c y t e p r o t e i n is t o s h a k e
t h e extract w i t h c h l o r o f o r m . T h e p r o t e i n is p r e c i p i t a t e d at t h e i n t e r f a c e b e t w e e n t h e l o w e r
7
( h e a v i e r ) c h l o r o f o r m p h a s e a n d t h e a q u e o u s extract. T h e p r o c e d u r e is as f o l l o w s . I m m e d i a t e l y
after n e u t r a l i z a t i o n o f t h e e x t r a c t , a d d 0.5 m l . o f c h l o r o f o r m a n d s h a k e v i g o r o u s l y for 3 0 sec.
T h e n c e n t r i f u g e for 2 m i n . at n o t less t h a n 5 0 0 g t o s e p a r a t e t h e p h a s e s a n d t h e p r o t e i n . U s e
t h e s u p e r n a t a n t fluid d i r e c t l y for t h e e n z y m a t i c d e t e r m i n a t i o n o f N A D H a n d N A D P H .
Mitochondria: A d d 0.5 m l . 1 N a l c o h o l i c K O H ( s o l u t i o n II) t o 1 m l . o f m i t o c h o n d r i a l s u s p e n s i o n .

A l l o w t o s t a n d for 3 0 m i n . at r o o m t e m p e r a t u r e , t h e n c o o l in a n ice b a t h . A d d a p p r o x . 1.5 m l .
t r i e t h a n o l a m i n e H C l - p h o s p h a t e m i x t u r e (III) w i t h stirring until t h e p H r e a c h e s 7.8. A l l o w
t o s t a n d for 10 m i n . at r o o m t e m p e r a t u r e for better f l o c c u l a t i o n o f t h e d e n a t u r e d p r o t e i n ,
t h e n c e n t r i f u g e at 2 0 0 0 0 t o 4 0 0 0 0 g w i t h c o o l i n g . U s e t h e s u p e r n a t a n t fluid for t h e m e a s u r e
ments.
N A D H a n d N A D P H are relatively stable at r o o m t e m p e r a t u r e in alkaline media (less t h a n 2 % degradation

after 1 h o u r ) . In the neutralized extract, on the other h a n d , N A D H a n d N A D P H are oxidized at the same
rate by a non-enzymatic reaction (approx. 10% after 1 h o u r ) . This n o n - e n z y m a t i c oxidation can be in
8
hibited by exclusion of most of the atmospheric oxygen. This involves gentle gassing of the alkaline
solution and of the neutralized extract with nitrogen or argon. T h e extract v o l u m e lost by evaporation
must be taken into account. This relatively expensive m e t h o d would be used only in exceptional cases.
T h e interference due to oxidation of N A D H a n d N A D P H can be decreased if the time between neutraliza
tion and the spectrophotometric assay is kept as short as possible. This is m a d e difficult by the fact that
some time is required for the flocculation of the protein from the alkaline extract. As far as possible, the
indicated time of 10 min. between neutralization a n d centrifugation should n o t be exceeded. T h e measure
ment on the extract is carried out immediately after centrifugation.
T h e interfering oxidation of N A D H a n d N A D P H can be completely suppressed by a n o t h e r m e t h o d (see
below), in which N A D H a n d N A D P H in the extract are enzymatically oxidized t o N A D a n d N A D P
immediately after the neutralization, a n d the oxidation p r o d u c t s can then be determined u n d e r stable
conditions.
Spectrophotometric Assay Methods

NAD
Principle
(1) Ethanol + N A D +
Acetaldehyde + N A D H + H +
A D H = alcohol dehydrogenase ( A l c o h o l : N A D oxidoreductase, E C 1.1.1.1).
T h e equilibrium constant
K [Acetaldehyde] x [ N A D H ]
P
" ~ [H ] ~
+
[Ethanol] x [ N A D ] +
is K 7 = 1 0 " (at 25 °C a n d p H 7) a n d K
4
8 8 = 1 0 " at p H 8.8. T h e equilibrium favours the left-hand side
2
of equation (1).
The reduction of N A D can be m a d e quantitative by the use of high p H values a n d high ethanol concentra
tions and by the use of semicarbazide to bind the acetaldehyde f o r m e d . A D H from yeast should be used
9
for this determination of N A D , since A D H from liver reacts with N A D P at 1% of the activity with N A D . 1 0
A D H preparations from yeast contain varying quantities of an NADP-specific A D H . T h e quantity of 1 1

this impurity should be < 0 . 0 5 % . Interference by this c o n t a m i n a t i n g activity can be avoided by using
only a small quantity of A D H . In an extract that contains N A D a n d N A D P , it is then possible to determine
N A D alone.
Reagents
1. S o d i u m p y r o p h o s p h a t e , N a P 0 - 1 0 H O
4 2 7 2 4. A l c o h o l d e h y d r o g e n a s e , ADH
2. S e m i c a r b a z i d e h y d r o c h l o r i d e crystalline from yeast, suspension in 3.2 M
3. E t h a n o l , a b s o l u t e , A . R. ammonium sulphate s o l u t i o n ; ^200 U/mg.
(25 ° C ) ; c o m m e r c i a l p r e p a r a t i o n s , see p . 428.
T h e p r e p a r a t i o n must be substantially free from any NADP-specific d e h y d r o g e n a s e activity ( < 0.05 % ) .
D i s s o l v e 4.5 g. N a P 0 4 2 7 • 10 H 0 a n d 0.5 g. o f s e m i c a r b a z i d e h y d r o c h l o r i d e in d o u b l y
2
distilled w a t e r a n d m a k e u p t o 100 m l .
II. A l c o h o l d e h y d r o g e n a s e , A D H ( 1 . 2 m g . p r o t e i n / m l . ) :
Dilute stock suspension as required with 3.2 M a m m o n i u m sulphate solution.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 0 2 m l . R o o m t e m p e r
ature. M e a s u r e a g a i n s t air o r w a t e r .
Sample [neutralized a n d deproteinized

extract (S)] 1 ml. u p t o 100 / / M N A D
P y r o p h o s p h a t e buffer (I) 1 ml. 50 m M
Ethanol 0.01 m l . 100 m M
M i x , f o l l o w c h a n g e in e x t i n c t i o n u n t i l a constant
v a l u e is r e a c h e d ( 1 0 - 1 5 m i n . ) . T h e n r e a d e x t i n c t i o n
A D H suspension (II) 0.01 m l . 6 jug./ml. = c a . 1 U / m l .
M i x . A f t e r 6 m i n . , r e a d final v a l u e o f e x t i n c t i o n E . 2
AE = E 2 — E t is u s e d in t h e c a l c u l a t i o n s .
Calculations
T h e concentration in the sample u n d e r investigation is
AE x 2.02 (. , V 2 \ A , V 4 + V \ .
5 . . , _
d = light p a t h of cuvette (cm.)

e = extinction coefficient for N A D H ( c m / / m i o l e ) ; 6.22 at 340 n m ; 3.4 at 365 n m ; 6.1 at 334 n m .
2
Vj = volume of sample = weight/density

V 2 = ml. H C 1 0 required for deproteinization
4
V 3 = s u p e r n a t a n t fluid (ml.) r e m o v e d after deproteinization

V 4 = ml. K H P 0 2 4 solution a d d e d
V 5 = ml. of K O H required for neutralization
2.02 = volume of assay m i x t u r e (ml.)
T h e quantity of N A D in the cell is ^ ^ X

^ . This is multiplied by dilution factors t o obtain the
d x£
quantity of N A D per unit v o l u m e of the sample.
The content per g. of fresh weight is found by taking into account the density p of the tissue:
//mole N A D / g . = //mole N A D / m l . x p
NADP
N A D P is determined in the same buffered extract as N A D . Since the N A D P c o n c e n t r a t i o n in the extract

is generally low, use the extract undiluted for the m e a s u r e m e n t a n d o p e r a t e at the greatest possible sensi
tivity of the assay system (longer light p a t h ) .
Principle
(2) Glucose-6-phosphate + N A D P + G 6 p
- p H
, 6-Phosphogluconate + N A D P H + H +
G 6 P - D H = glucose-6-phosphate d e h y d r o g e n a s e , zwischenferment ( D - G l u c o s e - 6 - p h o s p h a t e : N A D P 1-
oxidoreductase, E C 1.1.1.49).
T h e equilibrium constant
K - K
_ [6-Phosphogluconate] x [NADPH]
p H
~ [H ] +
~ [Glucose-6-phosphate] x [NADP ] +
is 1 2
K 7 = 6 (at 25 °C a n d p H 7). T h e equilibrium is largely symmetrical. A large excess of g l u c o s e s -
p h o s p h a t e a n d high p H values are therefore required for the complete reduction of N A D P .
Reagents
1. M a g n e s i u m s u l p h a t e M g S 0 - 7 H 0 , A . R. 4 2 3. G l u c o s e - 6 - p h o s p h a t e dehydrogenase,
2. G l u c o s e - 6 - p h o s p h a t e , G-6-P G6P-DH
disodium salt, c o m m e r c i a l p r e p a r a t i o n s , see from yeast, suspension in 3.2 M ammonium
p. 538. sulphate s o l u t i o n ; ^ 1 4 0 U / m g . (25 ° C ) ; c o m
mercial p r e p a r a t i o n s , see p . 458.
G 6 P - D H m a y contain g lutathione reductase. In an extract containing glutathione, this enzyme causes

reoxidation of N A D P H , so t h a t the N A D P H value obtained is t o o low. T h e glutathione reductase c o n t e n t
of the G 6 P - D H must therefore be < 0 . 2 % (with respect t o the specific activity of the G 6 P - D H ) .
I. M a g n e s i u m s u l p h a t e (1 M ) :
D i s s o l v e 2 . 4 g. M g S 0 - 7 H 0 in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
4 2
II. G l u c o s e - 6 - p h o s p h a t e ( a p p r o x . 0 .2 M G-6-P):
D i s s o l v e 0 . 6 2 g. G - 6 - P - N a 2 in d o u b l y d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
III. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , G 6 P - D H ( 2 m g . p r o t e i n / m l . ) :
Assay System
W a v e l e n g t h : 3 4 0 , H g 3 3 4 n m ( n o t H g 3 6 5 n m ) ; light p a t h : 1 c m . ; final v o l u m e : 4 . 1 4 m l . ;
r o o m t e m p e r a t u r e ; m e a s u r e a g a i n s t air o r w a t e r .
S a m p l e ( n e u t r a l i z e d e x t r a c t S) 4 ml.
MgS0 4 solution (I) 0.02 ml. 5 mM
G-6-P solution (II) 0.1 ml. 5 mM
M i x well. F o l l o w c h a n g e in e x t i n c t i o n until c o n s t a n t
v a l u e is r e a c h e d ( 1 0 - 1 5 m i n . ) . T h e n r e a d e x t i n c t i o n
Ei •
G 6 P - D H suspension (III) 0.02 ml. 4 0 /xg./ml. = 5.6 U / m l .
M i x . R e a d e x t i n c t i o n after 15, 2 0 , a n d 25 m i n . ; d e t e r
mine extinction E 2 by extrapolation o f these values to
the time of addition o f G 6 P - D H .
AE = E 2 — E j is u s e d in t h e c a l c u l a t i o n s .
D e t e r m i n e t h e c h a n g e in e x t i n c t i o n d u e t o a d d i t i o n o f G 6 P s o l u t i o n b y a d d i n g a f u r t h e r
0 . 0 2 m l . o f s u s p e n s i o n at t h e e n d o f t h e r e a c t i o n .
S u b t r a c t t h e r e s u l t i n g c h a n g e in e x t i n c t i o n f r o m E . 2
Calculations
Calculation formula as for N A D . Use 4.14 ml. for assay volume. Extinction coefficient for N A D P H
( c m . / ^ m o l e ) ; 6.22 at 340 n m ; 3.45 at 365 n m ; 6.1 at 334 n m .
2
O t h e r M e t h o d s for t h e D e t e r m i n a t i o n o f N A D P
T h e reaction catalysed by isocitrate d e h y d r o g e n a s e (//*ra>-D -isocitrate: N A D P oxidoreductase, d e c a r b -

s
oxylating, E C 1.1.1.42) can also be used for the d e t e r m i n a t i o n of N A D P : 1
(3) Isocitrate + N A D P +
^ 2-Oxoglutarate + N A D P H + C 0 2 + H +
T h e equilibrium is again strongly in favour of the f o r m a t i o n of N A D P H . F o r c o m m e r c i a l p r e p a r a t i o n s ,

see p . 479.
NADH
N A D H is determined in the alkaline extract buffered after neutralization (extract A). In principle, any
NAD-specific dehydrogenase reaction t h a t allows the complete oxidation of N A D H m a y be used. T h e
oxidation of N A D H by d i h y d r o x y a c e t o n e p h o s p h a t e ( D A P ) , which is catalysed by glycerol-3-phosphate
dehydrogenase, G D H (s/?-Glycerol-3-phosphate: N A D 2-oxidoreductase, E C 1.1.1.8), is the basis of the
m e t h o d described b e l o w . 11
Principle
(4) NADH + H +
+ D A P ^± N A D +
-f Glycerol-3-phosphate
T h e equilibrium c o n s t a n t
„ _ K _ [Dihydroxyacetone p h o s p h a t e ] x [NADH]
p H
~ [H ] ~
+
[Glycerol-3-phosphate] x [ N A D ] +
is 13
K 7 = 5.7 x 1 0 " (at 25 °C a n d p H 7). T h e equilibrium of the reaction is strongly in favour of the
5
right-hand side, so that complete oxidation of N A D H is achieved even with a relatively small excess
of substrate.
Reagents
1. D i h y d r o x y a c e t o n e p h o s p h a t e 2. G l y c e r o l - 3 - p h o s p h a t e d e h y d r o g e n a s e ,
dimethyl k e t a l ; c o m m e r c i a l p r e p a r a t i o n s , see GDH
p. 531. crystalline, from skeletal muscle, suspension in
3.2 M a m m o n i u m sulphate solution; ^ 40 U / m g .
(25 °C); c o m m e r c i a l p r e p a r a t i o n s , see p . 468.
I. D i h y d r o x y a c e t o n e p h o s p h a t e ( 2 0 m M ) :
D i s s o l v e 5 0 m g . d i h y d r o x y a c e t o n e p h o s p h a t e d i m e t h y l k e t a l in a c c o r d a n c e w i t h t h e m a n u
facturer's i n s t r u c t i o n s t o g i v e a final v o l u m e o f 5 m l .
II. G l y c e r o l - 3 - p h o s p h a t e d e h y d r o g e n a s e , G D H (1 m g . p r o t e i n / m l . ) :
Assay System
W a v e l e n g t h : 3 4 0 , H g 3 3 4 n m , for s t r o n g l y c o l o u r e d e x t r a c t s H g 3 6 5 n m ; light p a t h : 1 o r 2 c m . ;
measure against reference cuvette containing dilute p o t a s s i u m d i c h r o m a t e solution to c o m
p e n s a t e for c o l o u r a n d t u r b i d i t y o f t h e e x t r a c t ; a s s a y v o l u m e : 2 . 0 5 5 m l .
S a m p l e (extract A , undiluted) 2 ml. u p t o 1 0 0 fiM NADH

D i h y d r o x y a c e t o n e p h o s p h a t e s o l u t i o n (I) 0.05 ml. 0.5 m M
M i x w e l l , f o l l o w c h a n g e in e x t i n c t i o n for u p t o 15 m i n .
A perfectly c o n s t a n t v a l u e is f r e q u e n t l y u n a t t a i n a b l e ;
in s u c h c a s e s , d e t e r m i n e r i s i n g b a s e l i n e ( E j ) .
G D H suspension (II) 0.005 ml. 5 //g./ml. = 200 m U / m l .
M i x . R e a d e x t i n c t i o n s after 2, 5, a n d 10 m i n . For
further c o n s t a n t i n c r e a s e in e x t i n c t i o n E , e x t r a p o l a t e
2
to t = 0 (addition o f G D H ) with the aid o f baseline

( E ) (parallel shift) a n d r e a d A E = E
t x - E 2 at t = 0.
D e t e r m i n e i n c r e a s e in e x t i n c t i o n d u e t o a d d i t i o n o f G D H s u s p e n s i o n b y a d d i n g a f u r t h e r
0 . 0 0 5 m l . o f s u s p e n s i o n at t h e e n d o f t h e r e a c t i o n . S u b t r a c t t h e r e s u l t i n g c h a n g e in e x t i n c t i o n
from E . 2
Calculations
Calculation formula as for N A D .
O t h e r M e t h o d s for t h e D e t e r m i n a t i o n o f N A D H
A n o t h e r m e t h o d t h a t is frequently used for the d e t e r m i n a t i o n of N A D H is the oxidation of N A D H with

acetaldehyde which is catalysed by alcohol dehydrogenase, A D H (Alcohol:NAD oxidoreductase,
E C 1.1.1.1):
(5) NADH + H +
+ Acetaldehyde NAD +
+ Ethanol
However, alcohol d e h y d r o g e n a s e m a y contain a small quantity of an NADP-specific alcohol d e h y d r o

genase, which varies from p r e p a r a t i o n to p r e p a r a t i o n , so t h a t N A D P H is also oxidized if the A D H
concentration is t o o high.
T h e L D H reaction is also used for the d e t e r m i n a t i o n of N A D H .
(6) NADH + H +
+ Pyruvate ^ E ^ N A D +
+ Lactate
However, lactate d e h y d r o g e n a s e , L D H ( L - L a c t a t e : N A D oxidoreductase, E C 1.1.1.27) from muscle

reacts not only with N A D H b u t also to a small extent with N A D P H . T h e rate of reaction with N A D P H
decreases rapidly with increasing p H , so t h a t at p H 7.8, lactate d e h y d r o g e n a s e reacts a b o u t 2000 times
as rapidly with N A D H as with N A D P H . At this p H value, by the use of only a small quantity of L D H ,
1 4
it is possible to determine N A D H quantitatively without interference by a d d i t i o n a l oxidation of N A D P H .

N A D H and N A D P H — M e t h o d I
N A D P H is determined in the s a m e extract (extract A ) as N A D H . T h e o x i d a t i o n of N A D H (see N A D H

assay) must be carried out before the d e t e r m i n a t i o n of N A D P H in the assay with G I D H ; the simplest
m e t h o d being to carry out the N A D H a n d N A D P H assays successively.
T h e reaction with 2-oxoglutarate catalysed by g l u t a m a t e dehydrogenase, G I D H ( L - g l u t a m a t e : N A D ( P )
oxidoreductase, deaminating, E C 1.4.1.3) is used for the d e t e r m i n a t i o n of N A D P H . 1 1
Principle
(7) 2-Oxoglutarate + N H 4
+
+ NADPH Glutamate + NADP +
T h e equilibrium c o n s t a n t
[2-Oxoglutarate] x [ N H ] x 4
+
[NADPH]
~~ [Glutamate] x [NADP ] +
is 1 5
K = 1 0 " l./mole at 25 °C. T h e equilibrium is strongly in favour of the oxidation of N A D P H . G l u t a
6
mate dehydrogenase reacts at roughly the same rates with N A D P H a n d N A D H . F o r the determination 1 5
of N A D P H , therefore, N A D H m u s t first be oxidized with glycerol p h o s p h a t e dehydrogenase a n d di

hydroxyacetone p h o s p h a t e .
Reagents
A l l t h e r e a g e n t s n e c e s s a r y for t h e d e t e r m i n a t i o n o f N A D H . Also:
3. S o d i u m h y d r o x i d e s o l u t i o n , A . R., 3 N 6. G l u t a m a t e d e h y d r o g e n a s e , GIDH
4. 2 - O x o g l u t a r i c a c i d , O x o G crystalline from liver, suspension in 2 M a m
commercial p r e p a r a t i o n s , see p . 548. m o n i u m sulphate solution. 90 U / m g . (25 °C,
5. A m m o n i u m c h l o r i d e A . R. measured with 2-oxoglutarate a n d NADH);
I. S u b s t r a t e m i x t u r e ( 2 0 m M D A P ; 0.1 M O x o G ; 0 . 2 M N H 4
+
):
D i s s o l v e 5 0 m g . d i h y d r o x y a c e t o n e p h o s p h a t e d i m e t h y l k e t a l a c c o r d i n g t o the m a n u
facturer's i n s t r u c t i o n s a n d m a k e u p t o 4 m l . M i x 0.1 g. 2 - o x o g l u t a r a t e a n d 0.1 g. o f
N H C 1 in 0.5 m l . o f d o u b l y d i s t i l l e d w a t e r , a d j u s t t o p H 7 w i t h 3 N N a O H , m a k e u p
4
to 1 ml. with d o u b l y distilled water a n d mix with 4 ml. d i h y d r o x y a c e t o n e phosphate

solution.
II. G l y c e r o l p h o s p h a t e d e h y d r o g e n a s e , G D H (1 m g . o f p r o t e i n / m l . ) :
III. G l u t a m a t e d e h y d r o g e n a s e , G I D H (2 m g . o f p r o t e i n / m l . ) :
Assay System
W a v e l e n g t h : 3 4 0 , ( H g 3 3 4 , H g 3 6 5 ) n m ; l i g h t p a t h : 1 o r 2 c m . ; final v o l u m e : 2 . 0 6 m l .
S a m p l e (extract A ) 2 ml. u p t o 1 0 0 ,uM N A D P H

Substrate mixture (I) 0.05 ml. 0.5 m M DAP
2.5 m M OxoG
5 mM NH 4
+
M i x w e l l , a n d r e a d e x t i n c t i o n E after 2 , 5, a n d 10 m i n .
x
A p e r f e c t l y c o n s t a n t v a l u e is o f t e n u n a t t a i n a b l e ; in
such cases, determine rising baseline.
G D H suspension (II) 0.005 ml. 5 jUg./ml. = approx.

200 m U / m l .
Read extinction E . U s e E 2 2 — E t for e v a l u a t i o n o f

the N A D H content (see N A D H )
G 1 D H suspension (III) 0.005 ml. 10 / / g . / m l . = approx.

900 m U / m l .
M i x , r e a d e x t i n c t i o n E , after a b o u t 10 m i n .
3
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f G 1 D H s u s p e n s i o n b y a d d i n g a
further 0 . 0 0 5 m l . o f s u s p e n s i o n at t h e e n d o f t h e r e a c t i o n . S u b t r a c t t h e r e s u l t i n g c h a n g e in
extinction from E . 3
Calculations
Calculation formula for N A D , p . 2049; N A D P , p . 2051.
N A D H a n d N A D P H — M e t h o d II
T h e slow n o n - e n z y m a t i c oxidation of N A D H a n d N A D P H in the alkaline extract after neutralization

(see " E x t r a c t i o n " ) c a n be avoided by the following m e t h o d . This gives the m o s t a c c u r a t e values for N A D H
and N A D P H ' . 6 8
Immediately after the neutralization of the alkaline extract, N A D H a n d N A D P H are oxidized enzymatic-
ally in accordance with eqn. (4) a n d (7). S p e c t r o p h o t o m e t r i c m e a s u r e m e n t is ruled o u t by the rapid increase
in the turbidity of the extract d u r i n g t h e first 10 min. All the protein is precipitated by subsequent acid
deproteinization, and the stable N A D and N A D P are d e t e r m i n e d enzymatically in the clear extract in
accordance with eqn. (1) a n d (2).
Reagents
All the reagents required for the d e t e r m i n a t i o n o f N A D , N A D P , N A D H , a n d NADPH.

2056 M e t a b o l i t e s : Nucleic Acids, Purines, Pyrimidnes, Nucleosides, Coenzymes
A l l t h e s o l u t i o n s for t h e d e t e r m i n a t i o n o f N A D a n d N A D P ( p . 2 0 4 9 a n d 2 0 5 1 ) , a n d a l s o :
I. G l u t a m a t e d e h y d r o g e n a s e , G I D H ( 2 m g . p r o t e i n / m l . ) :
II. B u f f e r / s u b s t r a t e m i x t u r e (5 m M O x o G ; 0.5 M t r i e t h a n o l a m i n e ; 3 0 m M N H 4
+
; 0.5 M
phosphate):
D i s s o l v e 73 m g . 2 - o x o g l u t a r i c a c i d + 1 5 0 m g . N H C 1 + 9 . 3 0 g. t r i e t h a n o l a m i n e - H C l
4 -f
5 . 4 4 g. K H P 0 2 4 + 1.74 g. o f K H P 0
2 4 in d o u b l y distilled water and m a k e u p to 100 ml.
Procedure
Extract and Preliminary Treatment
T h e a l k a l i n e e x t r a c t ( e x t r a c t A ) is p r e p a r e d as d e s c r i b e d o n p. 2 0 4 7 , w i t h t h e f o l l o w i n g c h a n g e s :
H e a t all e x t r a c t s , i n c l u d i n g t h o s e o f m i t o c h o n d r i a a n d o f b l o o d , for 5 m i n . at 9 0 ° C . T h e n
c o o l t o 0 °C in a n ice b a t h . N e u t r a l i z e b y s l o w a d d i t i o n o f a p p r o x . 1 m l . o f s o l u t i o n II, w i t h
c o o l i n g a n d stirring, u n t i l p H 7.8 is r e a c h e d . I m m e d i a t e l y a d d 0 . 0 0 5 m l . G I D H suspension
per m l . o f e x t r a c t for t h e e n z y m a t i c o x i d a t i o n o f N A D H a n d N A D P H . A l l o w t o s t a n d for
15 m i n . at r o o m t e m p e r a t u r e , t h e n a d d 0.2 m l . 3 M H C 1 0 / m l . o f extract. C e n t r i f u g e t o
4
r e m o v e p r o t e i n , p i p e t t e off s u p e r n a t a n t fluid, a n d adjust t o p H 7.2 w i t h 1 M K O H in a n ice

bath. A l l o w K C 1 0 4 t o s e d i m e n t , a n d t a k e s a m p l e s for t h e m e a s u r e m e n t s f r o m t h e s u p e r
n a t a n t fluid (extract A ) .
Assay System
W a v e l e n g t h : 3 3 4 , 3 4 0 n m ; light p a t h : 2 o r 4 c m . D e t e r m i n e t h e N A D H c o n v e r t e d i n t o N A D
a n d the N A D P H c o n v e r t e d i n t o N A D P b y t h e m e t h o d s g i v e n f o r N A D a n d N A D P (p. 2 0 4 8
and 2050).
S o u r c e s o f Error and S p e c i f i c i t y
N A D P H decomposes m o r e readily t h a n N A D H in the alkaline extract, particularly when heated (adhere

exactly to heating time specified). After oxidation of N A D H , the m e t h o d is specific for N A D P H .
O t h e r M e t h o d s for the D e t e r m i n a t i o n o f N A D P H
The enzymatic oxidation of N A D P H by glutathione with glutathione reductase, G R (NAD(P)H):

oxidized-glutathione oxidoreductase, E C 1.6.4.2) can be used for the d e t e r m i n a t i o n of N A D P H 1 , 1 6
:
GSSG + N A D P H + H +
2 GSH + NADP+
T h e equilibrium is again strongly in favour of N A D P formation. T h e enzyme is absolutely specific for

N A D P H , and N A D P H can therefore be determined in the presence of N A D H . A purified enzyme pre
p a r a t i o n can be obtained relatively easily e. g. from peas. However, the specific activity of these p r e p a r a t i o n s
is low in c o m p a r i s o n with g l u t a m a t e dehydrogenase. F o r commercial p r e p a r a t i o n s from yeast, see p . 465.
Nicotinamide-adenine Dinucleotides 2057
Fluorimetric Assay Methods
T h e fluorimetric m e t h o d s described here for the d e t e r m i n a t i o n of N A D , N A D P , N A D H , a n d N A D P H

m a k e use of the same extracts as the s p e c t r o p h o t o m e t r i c m e t h o d s . W i t h a suitable fluorimeter, these
m e t h o d s are m o r e sensitive t h a n the s p e c t r o p h o t o m e t r i c m e t h o d s , a n d they are therefore particularly
suitable for the d e t e r m i n a t i o n of small quantities of nicotinamide dinucleotides. Their disadvantage is
that the reading is m a d e in arbitrary units, a n d standardization by a d d i t i o n of a n N A D solution to the
extract is always necessary. It should be noted that the fluorescence yield is strongly influenced by the various
dissolved substances in the extract. A n example of a suitable fluorimeter for the fluorimetric m e t h o d is
the P h o t o m e t e r E p p e n d o r f with fluorimeter a t t a c h m e n t . R e c o r d i n g facilities are a d v a n t a g e o u s . T h e re
corder a d a p t o r should have extensive zero suppression to eliminate the " z e r o fluorescence" due to foreign
substances a n d scattered light, as well as the possibility of scale expansion. T h e " z e r o fluorescence" is
particularly p r o n o u n c e d in samples extracted with alkali.
Reagents
A s for the c o r r e s p o n d i n g s p e c t r o p h o t o m e t r i c m e t h o d . A l s o N A D , N A D P , NADH, and

N A D P H a s s t a n d a r d s . F o r c o m m e r c i a l p r e p a r a t i o n s , see p . 5 4 5 - 5 4 7 .
N A D solution (50 /JM):

D i s s o l v e 7 m g . N A D in 2 m l . o f w a t e r . D i l u t e 0.1 m l . o f this s o l u t i o n t o 10 m l . w i t h w a t e r .
N A D H solution (50 / i M ) :
Dissolve 8 mg. N A D H - N a 2 in 2 m l . o f w a t e r . D i l u t e 0.1 m l . o f t h i s s o l u t i o n t o 10 m l . w i t h
water.
N A D P solution (50 fiM):
D i s s o l v e 8 m g . N A D P - N a H in 2 m l . o f w a t e r . D i l u t e 0.1 m l . o f t h i s s o l u t i o n t o 10 m l . w i t h
2
water.
N A D P H solution (50 fiM):
Dissolve 8 mg. N A D P H - N a 4 in 2 m l . o f w a t e r . D i l u t e 0.1 m l . o f this s o l u t i o n t o 10 m l . w i t h
water.
Assay System N A D
E x c i t a t i o n o f f l u o r e s c e n c e 365 o r ( 3 1 3 + 3 6 5 ) n m . E m i s s i o n o f f l u o r e s c e n c e > 4 2 0 n m . A s s a y
v o l u m e : 2 . 0 2 m l . A d j u s t t h e f l u o r e s c e n c e scale t o t h e s e n s i t i v i t y c o r r e s p o n d i n g t o the e x t r a c t .
S a m p l e ( e x t r a c t S) 1 ml. u p t o 10 /xM
P y r o p h o s p h a t e buffer 1 ml. 50 m M
Ethanol 0.01 m l . 100 m M
M i x , adjust fluorescence scale to approximately zero,

a n d f o l l o w until c o n s t a n t . R e a d .
A D H suspension 0.01 m l . 6 /xg./ml. = a p p r o x . 1 U / m l .
M i x ; after 6 m i n . , r e a d final v a l u e o f fluorescence F . 2
Standardization o f scale by addition o f N A D solution.
N A D s o l u t i o n ( 5 0 pM) e. g. 10 fil 0.5 pM
M i x ; after 6 m i n . , r e a d final v a l u e F . T h e c o n c e n t
3
r a t i o n o f t h e s t a n d a r d s o l u t i o n in t h e a s s a y m i x t u r e
should be roughly equal to the N A D concentration
to be measured.
Calculations
In the assay mixture,
p p
c =-=r ~ - x [NAD S t a n d a r d s o l m i o n ] [^mole/ml.]
and in the material u n d e r investigation,
c = p| I x
[ N A D , ,„,„.]
S + ^ v / 5
) fomole/ml.]
F o r the definition of V etc., refer to the determination of N A D by the s p e c t r o p h o t o m e t r i c m e t h o d (p. 2049).

x
Assay System N A D P
S i m i l a r t o t h a t for t h e d e t e r m i n a t i o n o f N A D . F o r a s s a y s y s t e m , refer t o t h e d e t e r m i n a t i o n
of N A D P by the spectrophotometric m e t h o d , p. 2050.
Assay System N A D H and N A D P H
S i m i l a r t o t h a t for t h e d e t e r m i n a t i o n o f N A D . F o r a s s a y s y s t e m , refer t o t h e d e t e r m i n a t i o n
of N A D H and N A D P H by the spectrophotometric m e t h o d (p. 2052 and 2054). D e c r e a s e
in fluorescence is o b s e r v e d in t h e d e t e r m i n a t i o n o f N A D H a n d N A D P H .
References
1 M. Klingenberg & W. Slenczka, Biochem. Z. 331, 486 [1959]

2 H. W. Heldt & Af. Klingenberg in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, Vol. X,
p. 3, Academic Press, N e w Y o r k / L o n d o n 1967.
3 H. B. Burch, O. H. Lowry & P. von Dippe, J. biol. C h e m . 238, 2838 [1963].
4 O. H. Lowry, J. V. Passonneau, D. W. Schulz & M. K Rock, J. biol. C h e m . 236, 2746 [1961].
5 H. W. Heldt, M. Klingenberg & K Papenberg, Biochem. Z. 342, 508 [1965].
6 H. W. Heldt, N. GreifSc M. Kingenberg, J. biol. C h e m . 240, 4659 [1965].
7 H. Reinauer & F. H. Bruns, Hoppe-Seylers Z. Physiol. C h e m . 337, 93 [1964].
8 N. Greif: Dissertation, M a r b u r g 1964.
9 H. Holzer, S. Goldschmidt, W. Lamprecht & E. Helmreich, Hoppe-Seylers Z. Physiol. Chemie 297, 1
[1954].
10 M. E. Pullman, S. P. Colowick & N. O. Kaplan: J. biol. C h e m . 194, 593 [1952].
11 H. Holzer, D. Busch & H. Kroger, Hoppe-Seylers Z. Physiol. C h e m . 313, 184 [1958].
12 L. Glaser & D. H Brown, J. biol. C h e m . 216, 67 [1955].
13 H J. Hohorst, F. H. Kreutz & Th. Bucher, Biochem. Z. 332, 18 [1959].
14 F. Navazio, B. B. Ernster & L. Ernster, Biochim. Biophys. Acta 26, 416 [1957].
16 M. M. Ciotti & N. O. Kaplan in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c
Press, N e w York 1967, Vol. I l l , p . 890.
Measurement by Enzymatic Cycling

Janet V. P a s s o n n e a u and Oliver H. L o w r y
P y r i d i n e n u c l e o t i d e s p a r t i c i p a t e e x t e n s i v e l y in b i o l o g i c a l r e a c t i o n s a s c o e n z y m e s . T h e y p l a y
a n i m p o r t a n t p a r t in m e t a b o l i s m a s c o e n z y m e s in s y n t h e s e s a n d in oxidation-reduction
reactions. This importance has led to the development o f m e t h o d s that allow the separate
determination o f each individual c o e n z y m e ( N A D , N A D H , N A D P , and N A D P H ) . The
q u a n t i t i e s p r e s e n t in t i s s u e are s m a l l ; m e t h o d s t h a t a l l o w t h e d e t e r m i n a t i o n o f 1 0 " 1 4
moles of
c o e n z y m e are t h e r e f o r e d e s c r i b e d b e l o w . S i n c e m a n y m e t a b o l i t e s are d e t e r m i n e d in p y r i d i n e
c o e n z y m e - d e p e n d e n t d e h y d r o g e n a s e r e a c t i o n s o r in r e a c t i o n s t h a t are c o u p l e d w i t h t h e s e
p r o c e s s e s , t h e s e c a n a l s o b e d e t e r m i n e d in v e r y l o w c o n c e n t r a t i o n s .
In t h e s e r e a c t i o n s , t h e n u c l e o t i d e s a c t a s c a t a l y s t s for a n e n z y m a t i c d i s m u t a t i o n b e t w e e n t w o
s u b s t r a t e s . T h e n u c l e o t i d e c o n c e n t r a t i o n s in this r e a c t i o n a r e far b e l o w t h e i r K M values, and
t h e r e a c t i o n rates are c o n s e q u e n t l y p r o p o r t i o n a l t o t h e n u c l e o t i d e c o n c e n t r a t i o n . O n e o f t h e
p r o d u c t s is d e t e r m i n e d after s e v e r a l t h o u s a n d c y c l e s .
T w o different c y c l i n g m e t h o d s are u s e d . T h e m e t h o d for t h e d e t e r m i n a t i o n o f N A D P and
N A D P H makes use of glucose-6-phosphate dehydrogenase, G 6 P - D H (D-Glucose-6-phosphate:
N A D P 1-oxidoreductase, E C 1.1.1.49) and glutamate d e h y d r o g e n a s e , G 1 D H ( L - G l u t a m a t e :
N A D ( P ) o x i d o r e d u c t a s e , d e a m i n a t i n g , E C 1 . 4 . 1 . 3 ) ; for t h e d e t e r m i n a t i o n o f N A D a n d N A D H ,
we use lactate d e h y d r o g e n a s e , L D H (L-Lactate: N A D oxidoreductase, E C 1.1.1.27) a n d
glutamate dehydrogenase.
Application of Method: In biochemistry a n d in clinical biochemistry. F o r the m e a s u r e m e n t of pyridine

coenzyme concentrations in tissues or for d e t e r m i n a t i o n of substances t h a t can be coupled with the oxid
ation or reduction of a pyridine coenzyme. T h e oxidized coenzymes can be determined after destruction
of the reduced coenzymes (and the interfering enzymes) by brief t r e a t m e n t with acid. T h e reduced coenzymes
can be determined after destruction of the oxidized coenzymes (and interfering enzymes) by mild a l k a l i . 1
Determination of NADP and NADPH

Principle
Cycling System:
(1) NADP +
+ Glucose-6-P G 6 P
~ D H
> Gluconate-6-P + N A D P H + H +
T I
(2) NADP +
+ G l u t a m a t e <— 2-Oxoglutarate + N A D P H + H +
+ NH 3
Determination of the p r o d u c t :
(3) Gluconate-6-P + N A D P + 6
" P G
" D H
* > Ribulose-5-P + C 0 2 + NADPH + H +
The quantity of 6-phosphogluconate formed in the cycle per unit time is p r o p o r t i o n a l to the N A D P con
centration.
The quantities of G 6 P - D H a n d G I D H indicated give m a x i m u m rates in the cycle. Higher concentrations

do not increase the quantity of 6-phosphogluconate, since the enzyme c o n c e n t r a t i o n is higher t h a n the
nucleotide concentration. However, the enzyme concentrations m a y be decreased; the rate in the cycle is
then p r o p o r t i o n a l the enzyme concentration.
The temperature coefficient is 7.7% per degree between 25 °C a n d 38 ° C ; 2.5 times as m u c h reaction p r o
duct therefore accumulates at 38 °C as at 25 °C. It is d a n g e r o u s to increase the t e m p e r a t u r e further, since
denaturation of the enzyme m a y occur. T h e concentration of glucose-6-phosphate m a y be increased to
such a degree that 3 x 1 0 ~ nucleotide is cycled. However, the increased G-6-P concentration increases
7
the blank value.

The 2-oxoglutarate concentration c a n n o t be altered without reducing the rate in the cycle. Lower concen
trations reduce the conversion of N A D P H to N A D P . Higher c o n c e n t r a t i o n s inhibit the G 6 P - D H . A D P
is added to stabilize the G I D H . T h e N H 2
4 concentration is not critical, but the rate in the cycle is reduced
at 0.1 M , since the a p p a r e n t K M value of G 6 P - D H for N A D P is increased.
Equipment
Filter f l u o r i m e t e r w i t h p r i m a r y filter for e x c i t a t i o n at 3 6 0 n m ( C o r n i n g n o . 5 8 4 0 ) a n d s e c o n d a r y

filter for e m i t t e d light at a b o u t 4 6 0 n m ( C o r n i n g filter n o . 3 3 8 7 in c o m b i n a t i o n w i t h 4 3 0 3
c l o s e t o t h e p h o t o e l e c t r i c cell). M e a s u r e m e n t s c a n b e carried o u t in 3 m l . test t u b e s (10 m m . x
x 75 m m . ) w i t h t h e Farrand filter fluorimeter; the tubes must have a uniform diameter and
m u s t b e free f r o m s c r a t c h e s . U s e c o n s t r i c t i o n p i p e t t e s .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , 2. H y d r o c h l o r i c a c i d , A . R . , 12 N
A . R . , tris 3. S o d i u m h y d r o x i d e s o l u t i o n , A . R . , 1 N
and 6 N
* 6-Phosphogluconate dehydro genase ( 6 - P h o s p h o - D - g l u c o n a t e : N A D P 2-oxidoreductase, decarboxy-

lating, E C 1.1.1.44).
4. S o d i u m c a r b o n a t e , N a C 0 2 3 15. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E D T A
5. S o d i u m h y d r o g e n c a r b o n a t e , N a H C 0 3
d i s o d i u m salt, E D T A - N a H • 2 H 0
2 2 2
6. A s c o r b i c a c i d 16. A l b u m i n
7. S u l p h u r i c a c i d , A . R . , 2 0 m M from bovine p l a s m a
8. S o d i u m s u l p h a t e , A . R . , 0.1 M 17. 6 - P h o s p h o g l u c o n a t e d e h y d r o g e n a s e ,
9. 2 - O x o g l u t a r i c a c i d 6PG-DH
commercial p r e p a r a t i o n s , see p . 548. crystalline, from yeast, suspension in 3.2 M a m
10. G l u c o s e - 6 - p h o s p h a t e , G - 6 - P m o n i u m sulphate s o l u t i o n ; ^ 1 2 U / m g . (25 ° C ) ;
d i s o d i u m salt, G - 6 - P - N a ; commercial p r e p
2
commercial p r e p a r a t i o n s , see p. 500.
arations, see p . 538. 18. 6 - P h o s p h o g l u c o n a t e , 6 - P G
11. A m m o n i u m acetate, N H C H C O O , 4 3
(gluconate-6-phosphate) crystalline trisodium
A.R. salt 6 - P G - N a • 2 H 0 ; commercial p r e p a r a t i o n s ,
3 2
12. A d e n o s i n e - 5 ' - d i p h o s p h a t e , ADP see p . 535.

disodium salt, A D P - N a ; commercial preparat- 19. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e
2
ions, see p . 525. phosphate NADP

13. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , disodium salt, N A D P - N a H ; commercial pre
2
G6P-DH p a r a t i o n s , see p . 546.

from yeast, suspension in 3.2 M a m m o n i u m sul- 2 0 . R e d u c e d n i c o t i n a m i d e - a d e n i n e
p h a t e solution, ^ 1 4 0 U / m g . (25 °C); c o m m e r dinucleotide phosphate, N A D P H
cial p r e p a r a t i o n s , see p . 458. disodium salt, N A D P H - N a ; commercial p r e p
4
14. G l u t a m a t e d e h y d r o g e n a s e , GIDH a r a t i o n s , see p . 547.

crystalline, from bovine liver, as solution in 50%
glycerol, ^ 90 U / m g . (25 °C); commercial pre
p a r a t i o n s , see p . 4 6 1 .
Purity of Reagents
The enzymes and all other reagents must be free from N A D P . M o s t enzyme p r e p a r a t i o n s that have been
tested satisfy requirements of the m e t h o d . Certain A D P p r e p a r a t i o n s are c o n t a m i n a t e d with N A D P ; this
can be easily removed. H e a t the A D P solution at p H 12 for 10 min. at 60 °C, a n d neutralize to p H 7. A D P is
not significantly destroyed d u r i n g this t r e a t m e n t . M o s t G-6-P p r e p a r a t i o n s are free from N A D P . N o inter
fering impurities have been found in 2-oxoglutarate, tris, or a m m o n i u m acetate.
S o m e G 6 P - D H p r e p a r a t i o n s contain a protein t h a t binds N A D P a n d N A D P H m o r e strongly t h a n the
enzyme. This protein (probably an enzyme t h a t requires pyridine coenzymes) has not yet been identified or
separated. In this presence of this protein, low nucleotide concentrations ( 1 0 ~ M ) give relatively smaller 9
quantities of p r o d u c t in the cycle t h a n high c o n c e n t r a t i o n s ( 1 0 ~ M ) . If deviations are observed from the

8
linear relation between the nucleotide concentration a d d e d a n d the reaction p r o d u c t found, use a n o t h e r
enzyme p r e p a r a t i o n .
U s e only fresh, d o u b l y distilled w a t e r .

I. T r i s b u f f e r (1 M ; p H 8 . 0 ) :
D i s s o l v e 12.1 g. t r i s i n d i s t i l l e d w a t e r a n d 4 m l . 12 N H C 1 a n d m a k e u p t o 1 0 0 m l . ; k e e p
frozen.
II. 2 - O x o g l u t a r i c acid (1.0 M ) :
D i s s o l v e 146 m g . 2 - o x o g l u t a r i c a c i d in 1 m l . distilled w a t e r ; s t o r e f r o z e n .
III. G l u c o s e - 6 - p h o s p h a t e , G - 6 - P ( 0 . 1 M ) :
D i s s o l v e 180 mg. G - 6 - P - N a 2 in 5 m l . d i s t i l l e d w a t e r ; s t o r e f r o z e n .
I V . A m m o n i u m a c e t a t e (5 M ) :
D i s s o l v e 3 8 . 5 g. N H C H C O O in distilled w a t e r a n d m a k e u p t o 100 m l . ; c a n b e s t o r e d
4 3
V . A d e n o s i n e - 5 ' - d i p h o s p h a t e , A D P ( c a . 0.1 M ) :
Dissolve 54 mg. A D P - N a 2 in 1 m l . d i s t i l l e d w a t e r ; k e e p f r o z e n .
VI. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , G 6 P - D H (2.5 m g . p r o t e i n / m l . ) :
C e n t r i f u g e s t o c k s u s p e n s i o n , d e c a n t s u p e r n a t a n t , a n d d i s s o l v e s e d i m e n t in a n e q u a l
v o l u m e of a m m o n i u m acetate solution (IV).
VII. Glutamate dehydrogenase, G 1 D H (10 mg. protein/ml.):
Use stock solution.
V I I I . E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E D T A (0.1 M ) :
D i s s o l v e 3.7 g. E D T A - N a H - 2 H 0 in w a t e r a n d m a k e u p t o 100 m l . ; c a n b e s t o r e d
2 2 2
IX. Albumin (10% w/v):
D i s s o l v e 1 g. a l b u m i n in w a t e r a n d m a k e u p t o 10 m l .
X . 6 - P h o s p h o g l u c o n a t e d e h y d r o g e n a s e , 6 - P G - D H (1 m g . p r o t e i n / m l . ) :
D i l u t e 1 m l . s t o c k s o l u t i o n w i t h 9 m l . 2 0 m M tris buffer ( s o l u t i o n I d i l u t e d 1 : 5 0 , 0 . 0 2 %
in a l b u m i n ) .
X I . 6 - P h o s p h o g l u c o n a t e , 6 - P G (0.1 M ) :
D i s s o l v e 3 4 . 2 m g . 6 - P G - N a - 2 H 0 in 1 m l . w a t e r . D i l u t e 1 m l . o f t h i s s o l u t i o n t o 100 m l .
3 2
with water, and determine concentration as f o l l o w s :

A d d 5 0 pi. o f t h i s s o l u t i o n a n d 5 fil. N A D P s o l u t i o n ( X I I ) t o 5 0 0 fil. 6 - p h o s p h o g l u c o n a t e
r e a g e n t , see p . 2 0 6 4 , m e a s u r e initial e x t i n c t i o n at 3 4 0 n m , a n d a d d \fil. 6-PG-DH
s o l u t i o n ( X ) ; t h e i n c r e a s e in e x t i n c t i o n m u s t b e c a . 0 . 5 6 0 . C o r r e c t s o l u t i o n a c c o r d i n g l y .
X I I . N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e (0.1 M ) :
D i s s o l v e 8 0 m g . N A D P - N a H in 1 m l . d i s t i l l e d w a t e r ; k e e p f r o z e n . D i l u t e 0.1 m l . o f
2
this s o l u t i o n t o 10 m l . w i t h w a t e r , a n d d e t e r m i n e c o n c e n t r a t i o n a s f o l l o w s :
A d d 5 0 0 fil. tris buffer I d i l u t e d t o 0.1 M (1 m M G - 6 - P ) t o 5 0 fil. o f this s o l u t i o n , m e a s u r e
e x t i n c t i o n at 3 4 0 n m , a d d 1 fil. G 6 P - D H s o l u t i o n ( V I ) , a n d m e a s u r e i n c r e a s e in e x t i n c t i o n ,
w h i c h s h o u l d b e c a . 0 . 5 6 0 . C o r r e c t s o l u t i o n a c c o r d i n g l y . B o t h s o l u t i o n s (0.1 M a n d 1 m M )
k e e p for several m o n t h s at —20 ° C .
X I I I . R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e (5 m M ) :
Dissolve 4 mg. N A D P H - N a 4 in 5 m l . c a r b o n a t e buffer ( 8 0 m M N a C 0 , 2 0 2 3 mM
N a H C 0 ; p H 1 0 . 6 ) s t a b l e for 1 w e e k at 4 ° C . T h i s s o l u t i o n s h o u l d n o t b e f r o z e n .
3
H i g h e r c o n c e n t r a t i o n s are less s t a b l e . S t a n d a r d i z e as f o l l o w s for u s e as s t a n d a r d s o l u t i o n :

A d d 5 0 0 fil. 0.1 M tris buffer ( c o n t a i n i n g 1 m M 2 - o x o g l u t a r a t e a n d 25 m M a m m o n i u m
a c e t a t e ) t o 10 fil. o f this s o l u t i o n , r e a d initial e x t i n c t i o n at 3 4 0 n m , a d d 2 fil. G 1 D H
s o l u t i o n ( V I I ) , a n d m e a s u r e d e c r e a s e in e x t i n c t i o n , w h i c h s h o u l d b e ca. 0 . 6 1 5 . C o r r e c t
solution accordingly.
The solutions are stable u n d e r the conditions indicated.

Procedure
T i s s u e s a m p l e s n e e d n o t b e d e p r o t e i n i z e d , s i n c e t h e y are u s e d in s u c h a l o w c o n c e n t r a t i o n for
t h e a s s a y . T h e p r e p a r a t i o n o f t i s s u e a c c o r d i n g t o Burch et a l . a v o i d s o x i d a t i o n o f N A D P H b y
3
h a e m o g l o b i n d u r i n g t h e a c i d i f i c a t i o n . T h i s is p a r t i c u l a r l y critical w h e n [ N A D P H ] > [NADP].

L i g h t l y a n a e s t h e t i z e rats, freeze t i s s u e , or in t h e c a s e o f b r a i n freeze t h e e n t i r e h e a d , w i t h i n
1 - 5 sec. after s u p p r e s s i o n o f t h e b l o o d s u p p l y in F r e o n 12 ( C C 1 F ) , c h i l l e d t o its f r e e z i n g
2 2
p o i n t ( — 150 ° C ) w i t h l i q u i d n i t r o g e n .
P r e p a r e t h e t i s s u e in a r o o m k e p t at — 2 0 ° C . G r i n d o r g a n s a m p l e s w i t h a m o r t a r a n d p e s t l e
t h a t h a v e b e e n c o o l e d t o t h e t e m p e r a t u r e o f l i q u i d n i t r o g e n . R a p i d l y h o m o g e n i z e 50 m g .
s a m p l e s in 5 m l . 4 0 m M N a O H ( c o n t a i n i n g 0.5 m M c y s t e i n e ) at 0 ° C . C a r r y o u t all further s t e p s
at 0 ° C .
To determine the s u m N A D P + N A D P H , dilute part o f the h o m o g e n a t e by a factor o f 20
w i t h N a O H / c y s t e i n e s o l u t i o n ; u s e for t h e a s s a y w i t h i n 3 0 m i n . T o d e t e r m i n e N A D P H , h e a t
part o f t h e d i l u t e d h o m o g e n a t e for 10 m i n . at 6 0 ° C t o d e s t r o y N A D P . T o d e t e r m i n e N A D P ,
a d d 5 pi 1.2 M a s c o r b i c a c i d t o 2 0 0 / d . o f t h e u n d i l u t e d h o m o g e n a t e (final c o n c e n t r a t i o n
30 m M ) . T h i s a d d i t i o n p r e v e n t s t h e o x i d a t i o n o f N A D P H t o N A D P b y h a e m o g l o b i n in t h e
tissue. A c i d i f y this s a m p l e w i t h 2 m l . 2 0 m M H S O / 0 . 1 M N a S 0
2 4 2 4 'solution a n d h e a t for 3 0
m i n . at 6 0 ° C . U s e for a s s a y w i t h i n 3 0 m i n .
F o r e a c h series o f m e a s u r e m e n t s , d e t e r m i n e s t a n d a r d s h a v i n g s i m i l a r c o n c e n t r a t i o n s : e . g . for
liver a d d 2 0 a n d 4 0 / d . 0.1 m M N A D P s o l u t i o n ( X I I ) o r 2 0 a n d 4 0 pi 1.0 m M N A D P H s o l u t i o n
( X I I I ) t o the 5 m l . N a O H / c y s t e i n e s o l u t i o n .
T h e t i s s u e d i l u t i o n u s e d in t h e c y c l i n g test is c o n s i d e r a b l e ; t h e f o l l o w i n g d i l u t i o n s are g e n e r a l l y
used:
Homogenate dilution (in the 100 ul. cycling mixture)

for the determination of
Tissue NADPH or NA DP
NADP+ NADPH
Liver 100000 25000

Kidney 25000 8000
Heart 6000 8000
Brain 6000 2000
Blood 6000 1000
Assay System
Cycling:
V o l u m e : 0 . 1 2 m l . ; 38 °C.
U s e 2 pi o f s a m p l e for t h e a s s a y in t h e d e t e r m i n a t i o n o f N A D P H o r N A D P + N A D P H , a n d
4 pi in the d e t e r m i n a t i o n o f N A D P .
F o r e a c h series o f m e a s u r e m e n t s , a n a l y s e s t a n d a r d s h a v i n g s i m i l a r c o n c e n t r a t i o n s (see a b o v e ) ,
as well as a b l a n k t h a t h a s u n d e r g o n e t h e e n t i r e p r e t r e a t m e n t t o w h i c h t h e s a m p l e is s u b j e c t e d .
Determination of 6-Phosphogluconate:
Primary w a v e l e n g t h : 360 n m ; secondary w a v e l e n g t h : 460 n m ; light p a t h : 1 c m . (standardized

test t u b e s ) ; final v o l u m e : 1.12 m l . ; r o o m t e m p e r a t u r e .
F o r e a c h series o f m e a s u r e m e n t s , a l s o a n a l y s e a 6 - p h o s p h o g l u c o n a t e s t a n d a r d .
6-Phosphogluconate reagent
Cycling reagent
M a k e u p fresh r e a g e n t d a i l y ; k e e p s for 3 - 4 hr.
K e e p s for t w o w e e k s at — 2 0 ° C o r t w o
at r o o m t e m p e r a t u r e
m o n t h s at - 85 ° C
Pipette into 100 ml. mixing cylinder: Pipette into 100 ml. mixing cylinder:
Tris buffer (I) 1 0 . 0 0 m l . Tris buffer (I) 2 . 0 0 0 m l .

2-Oxoglutarate soln. (II) 0.50 ml. NH 4 acetate soln. (IV) 0.600 ml.
G-6-P solution (III) 1.00 m l . E D T A solution (VIII) 0.100 ml.
A D P solution (V) 0.10 ml. Albumin solution (IX) 0.200 ml.
Water t o 100 m l . N A D P solution (XII) 0.020 ml.
6 - P G - D H suspension ( X ) 0.005 ml.
o f this m i x t u r e 5.00 ml.
Water to 100 ml.
G 6 P - D H solution (VI) 0.05 ml.
G 1 D H solution (VII) 0.05 m l .
Water 0.05 ml.
P i p e t t e i n t o 3 m l . test t u b e s (in ice b a t h ) : C o n c e n t r a t i o n in a s s a y m i x t u r e
Cycling r e a g e n t 0.100 ml. 81 m M tris, 0.8 m M G - 6 - P

4 0 m M N H , 8 0 pM
4
+
ADP
4 m M 2-oxoglutarate
c a . 5 0 pg. G6P-DH/ml.
c a . 0.1 m g . G l D H / m l . ^ 7 U
G6P-DH/ml., 9 U GIDH/ml.
S a m p l e (or s t a n d a r d s o l u t i o n X I I 0.020 ml. 1-50 n M
or XIII) + water
P l a c e test t u b e h o l d e r in 38 ° C w a t e r b a t h , i n c u b a t e for
e x a c t l y 6 0 m i n . a n d p l a c e in b o i l i n g w a t e r b a t h for
2 min.
6-Phosphogluconate reagent 1.00 ml. 18 m M tris, 2 7 m M NH 4

+
9 0 pM E D T A , 18 pM
NADP
0.018% albumin
0.45 pg. 6 - P G - D H / m l . = 6 m U / m l .
A l l o w t o s t a n d for 3 0 m i n . at r o o m t e m p e r a t u r e t h e n
read fluorescence.
T h e p h o s p h o g l u c o n a t e v a l u e is a p p r o x i m a t e l y 1 5 0 0 0 t o 2 0 0 0 0 t i m e s t h e n u c l e o t i d e v a l u e .
It s h o u l d n o t e x c e e d 10 pM in t h e a s s a y , s i n c e a b o v e this level t h e N A D P H f l u o r e s c e n c e is n o
l o n g e r linearly p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n .
Modifications
T h e i n c u b a t i o n t i m e in t h e c y c l e c a n b e v a r i e d b e t w e e n 15 a n d 6 0 m i n . ; t h e q u a n t i t y o f 6 - p h o s
p h o g l u c o n a t e is p r o p o r t i o n a l t o t h e t i m e . T h e rate in t h e c y c l e , a n d h e n c e t h e q u a n t i t y o f
p r o d u c t , c a n a l s o b e d e c r e a s e d , e . g . b y t h e u s e o f 1/10 o f t h e i n d i c a t e d q u a n t i t i e s o f e n z y m e ;
t h e r e s u l t i n g q u a n t i t y o f 6 - P G is t h e n o n l y 1 5 0 0 t o 3 0 0 0 t i m e s t h e q u a n t i t y o f n u c l e o t i d e . In
suitably small tubes, moreover, the v o l u m e can be reduced to 1 /d., and the sensitivity thus in
creased.
If a b o u t 2 x 1 0 - 1 1
to 5 x 1 0 ~ 1 0
m o l e o f 6 - p h o s p h o g l u c o n a t e are f o r m e d , t h e c o r r e s p o n d i n g
N A D P H c a n b e m e a s u r e d as a f l u o r e s c e n c e p r o d u c t in s t r o n g a l k a l i . In t h i s c a s e p r o c e e d as
f o l l o w s : A f t e r h e a t i n g in t h e b o i l i n g w a t e r b a t h , i n c u b a t e t h e m i x t u r e for 3 0 m i n . w i t h 3
t o 10 t i m e s its v o l u m e o f 6 - p h o s p h o g l u c o n a t e r e a g e n t ; a d d t w i c e t h e cycling volume of
0.3 M N a P O / 0 . 3 M K H P 0 , a n d h e a t for 10 m i n . at 6 0 ° C t o d e s t r o y e x c e s s N A D P . A d d 1 m l .
3 4 2 4
6 N N a O H ( c o n t a i n i n g 0 . 0 3 % H 0 ) t o a n a l i q u o t o f this s o l u t i o n in a f l u o r i m e t e r t u b e .
2 2
1
H e a t a g a i n for 10 m i n . at 6 0 ° C , c o o l , a n d m e a s u r e fluorescence. Protect the alkaline solution

a g a i n s t direct light, s i n c e t h e a l k a l i fluorescence p r o d u c t is u n s t a b l e in light.
Calculations
Determine the results o n the basis of the N A D P or N A D P H s t a n d a r d s .
0.9 x 1 0 ~ 1 3
mole N A D P ± 0.1 x 1 0 ~ 1 3
mole can be d e t e r m i n e d ; the coefficient of variation ( C V ) is
2 . 5 % . It is possible to m e a s u r e 2.6 x 1 0 " 1 6
± 0.2 x 1 0 ~ m o l e , CV = 5 % . O n average, 7.2 ± 0.25//mole
1 6
N A D P / k g . ( C V = 3.5%) a n d 22.3 ± 0.21 /rniole N A D P H / k g . ( C V = 0.94%) were found in rat brain.
Normal Values
The following values were found by Burch et a l . with the aid of this m e t h o d in rat o r g a n s (//mole per kg.
3
fresh weight);
NADP NADPH
Liver 63 486
Kidney 19 135
Heart 4.5 93
Brain 5.3 26
Blood 5.4 15
The main source of error a n d of difficulty in obtaining reproducible values lies in the blank values. In the
m e t h o d described here, the blank values should not exceed 2 x 1 0 - 9
M N A D P in the cycle; there are three
main sources of the b l a n k :
1. The cycling reagent c o n t a i n s substances t h a t fluoresce at p H 8.0; these substances a c c o u n t for a p p r o x

imately one third of the b l a n k value. If the cycle takes place in a smaller v o l u m e , this c o m p o n e n t of the
blank value is correspondingly smaller.
2. T h e 6-phosphogluconate reagent m a y have a fluorescence blank value of 2 x 1 0 ~ M N A D P H (cor 7
responding to 5 x 1 0 _ 7
M N A D P H in the cycle); this is partly due to water a n d partly to N A D P in
the reagent. N o w a d a y s commercial p r e p a r a t i o n s of 6 - P G - D H from yeast d o not c o n t r i b u t e measurably
to the blank value in the quantities used. Higher values point to dirty tubes or c o n t a m i n a t e d solutions.
It is advisable to heat all tubes in half-concentrated H N 0 , then in water for 15 min. each at 100 °C,
3
a n d then to rinse in d o u b l y distilled water. They should be dried in a i r ; drying at 100 °C increases the
fluorescence.
3. Even in the absence of sample, small quantities of 6-phosphogluconate are formed in the cycle d u r i n g
incubation if the reagents are c o n t a m i n a t e d with N A D P . This c o m p o n e n t of the blank value should not
exceed 5 x 1 0 " 1 0
M in the cycle.
Avoid c o n t a m i n a t i o n of the reagents a n d pipettes with coenzyme. Pipettes used for the p r e p a r a t i o n of the
solutions or the cycling should not be used for pipetting concentrated coenzyme s o l u t i o n s ; keep a p a r t from
other pipettes, a n d rinse inside a n d outside with 0.1 N N a O H a n d then with 0.1 N HC1 before use.
T h e cycling m e t h o d is specific for N A D P ( H ) . O n addition of a 100-fold excess of N A D , the N A D P value

increased by 0 . 0 5 % ; with a 1000-fold excess of N A D , the value is found to be reduced by 0 . 0 3 % .
Determination of NAD and NADH

Principle
Cycling system:
(1) NAD +
+ Lactate Pyruvate + N A D H + H +
T i
(2) NAD +
+ Glutamate 2-Oxoglutarate + N A D H + H +
+ NH 3
M e a s u r e m e n t of p r o d u c t
Lactate + N A D +
The quantity of pyruvate formed per unit time in the cycle of equations (1) and (2) is p r o p o r t i o n a l to the
pyridine nucleotide concentration. Pyruvate is determined fluorimetrically in a c c o r d a n c e with eqn. (3).
T h e o p t i m u m p H v a l u e for t h e c y c l e is 8 . 4 , t h o u g h t h e rate is o n l y 2 % a n d 7% l o w e r at p H 8.8

a n d p H 8.0, r e s p e c t i v e l y . T h e t e m p e r a t u r e c o e f f i c i e n t is 7% p e r d e g r e e b e t w e e n 0 ° C a n d 25 ° C ,
o n l y 3 % b e t w e e n 2 5 ° C a n d 32 ° C , a n d 2 . 2 % b e t w e e n 3 2 ° C a n d 38 ° C . T h e r e is t h e r e f o r e o n l y
a m o d e s t g a i n b y w o r k i n g at t e m p e r a t u r e s a b o v e 2 5 ° C . H o w e v e r , t h e m e a s u r e m e n t s m a y b e
c a r r i e d o u t a t 3 8 ° C , s i n c e t h e a s s a y s y s t e m is s t a b l e . A s in t h e N A D P s y s t e m , t h e c o e n z y m e s
h e r e are s a t u r a t e d w i t h e n z y m e s , s o t h a t h i g h e r e n z y m e c o n c e n t r a t i o n s d o n o t i n c r e a s e t h e rate.
S i n c e t h e rate in t h e c y c l e at 0 ° C is a b o u t 2 0 % o f t h a t at 2 5 ° C , a n i n c u b a t i o n t i m e o f l e s s t h a n
* L-Lactate dehydrogenase from bovine heart (L-Lactate: N A D oxidoreductase, E C 1.1.1.27).

3 0 m i n . is n o t r e c o m m e n d e d . If c y c l i n g is a l l o w e d t o p r o c e e d for 6 0 m i n . at 2 5 ° C a s i n d i c a t e d ,
t h e q u a n t i t y o f p r o d u c t is 6 0 0 0 t i m e s t h e initial n u c l e o t i d e c o n c e n t r a t i o n . T h e p y r u v a t e y i e l d
c a n b e r e d u c e d if t h e q u a n t i t i e s o f r e a g e n t s are r e d u c e d t o 5 0 m M l a c t a t e , 0.1 m g . G I D H / m l .
a n d 5 pg. L D H / m l . T h e s e quantities give o n l y 600 m o l e s o f pyruvate per m o l e o f nucleotide
p e r h o u r . L D H s h o u l d b e d e c r e a s e d m o r e t h a n G I D H , in o r d e r t o k e e p t h e r a t i o N A D +
:NADH
h i g h a n d t o k e e p t h e p y r u v a t e p r o d u c t i o n linear.
T h e rate in t h e c y c l e d e c r e a s e s w i t h i n c r e a s i n g a c c u m u l a t i o n o f p y r u v a t e a n d w e h a v e set a
p y r u v a t e c o n c e n t r a t i o n o f 0.1 m M a s a n arbitrary limit. To m i n i m i z e t h e i n h i b i t o r y effect o f
pyruvate w e use the high lactate concentration. Because even the best c o m m e r c i a l lactate
p r e p a r a t i o n s c o n t a i n p y r u v a t e in a p r o p o r t i o n o f 1 : 4 0 0 0 0 t h e r e is a n a d v a n t a g e in d e c r e a s i n g
lactate in t h e d e t e r m i n a t i o n o f v e r y s m a l l q u a n t i t i e s o f N A D (1 t o 5 n M ) , s i n c e t h e initial rate
w i t h 5 0 m M l a c t a t e is t h e s a m e a s w i t h 1 0 0 m M . T h e c o n c e n t r a t i o n s o f 2 - o x o g l u t a r a t e a n d N H /
are less critical h e r e t h a n in t h e N A D P c y c l e . A c o n s i d e r a b l e d e c r e a s e in t h e o x o g l u t a r a t e
c o n c e n t r a t i o n w o u l d l o w e r t h e G I D H a c t i v i t y , b u t t h e r a t e - d e t e r m i n i n g s t e p is t h e o x i d a t i o n
o f l a c t a t e . A s in t h e N A D P c y c l e , A D P is a d d e d t o s t a b i l i z e t h e G I D H . 2
L a c t a t e is u s e d in a h i g h c o n c e n t r a t i o n t o i m p r o v e t h e u n f a v o r a b l e e q u i l i b r i u m i n t h e c y c l e ;
h o w e v e r , t h i s m a k e s t h e s u b s e q u e n t p y r u v a t e d e t e r m i n a t i o n m o r e difficult. T h e l o w p H v a l u e
favours the c o n v e r s i o n o f pyruvate. A t p H 6.5, [pyruvate] [ N A D H ] / [ l a c t a t e ] [ N A D +
] =
= 1 x 1 0 ~ . A n y u n r e a c t e d p y r u v a t e c a u s e s a n e g a t i v e error. T h e error a s a f r a c t i o n o f t h e t o t a l
5
initial p y r u v a t e is e q u a l t o t h e final e q u i l i b r i u m v a l u e for [ p y r u v a t e ] / [ N A D +

]. W i t h 5 0 m M
l a c t a t e after d i l u t i o n in t h e s e c o n d s t e p , t h e e q u i l i b r i u m c o n s t a n t for p y r u v a t e : N A D +
is
e q u a l t o 5 x 10~~ : N A D H . T h i s m e a n s t h a t t o a v o i d a " n e g a t i v e " error o f > 5%, N A D H m u s t
7
n o t b e less t h a n 1 0 " 5
M , i.e. 0.02% o f the lactate concentration. The provision o f adequate
N A D H c o n c e n t r a t i o n s is a p r o b l e m o n l y in t h e m e a s u r e m e n t o f v e r y s m a l l q u a n t i t i e s o f N A D .
F o r e x a m p l e , after c y c l i n g 2 0 0 0 t i m e s , 1 0 " 9
M N A D gives 1 0 ~ 6
M p y r u v a t e in t h e s e c o n d
s t e p o f t h e a n a l y s i s at p H 6 . 5 ; 10 t i m e s t h i s q u a n t i t y o f N A D H m u s t t h e r e f o r e b e a d d e d .
Equipment
A s in t h e N A D P ( H ) d e t e r m i n a t i o n .
Reagents
1. Tris-hydroxymethyl-aminomethane, 11. 2-Oxoglutaric acid

A . R . , tris commercial preparations, see p. 548.
2. H y d r o c h l o r i c a c i d , A . R., 12 N a n d 5 N 12. S o d i u m p y r u v a t e
3. S o d i u m h y d r o x i d e s o l u t i o n , A . R., 6 N commercial preparations, see p 550.
The fluorescence may be considerably reduced 13. C a l c i u m l a c t a t e
if the solution is exposed to sunlight for a few 14. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA
hours. disodium salt, E D T A - N a H • 2 H 0 2 2 2
4. S o d i u m c a r b o n a t e , N a C 0 , a n h y d r o u s
2 3 15. A d e n o s i n e - 5 ' - d i p h o s p h a t e , ADP
5. S o d i u m h y d r o g e n c a r b o n a t e , N a H C 0 3 disodium salt, A D P - N a ; commercial preparat
2
6. Imidazole ions, see p. 525.

low fluorescence, e.g. from Sigma Chem. Co., 16. A m m o n i u m a c e t a t e , N H C H C O O , 4 3
Grade III A.R.

7. Ethanol 17. G l u t a m a t e d e h y d r o g e n a s e , G I D H
8. C y s t e i n e crystalline, from bovine liver, as solution in 50%
9. S u l p h u r i c a c i d , A . R . , 2 0 m M glycerol, ^ 9 0 U / m g . (25 °C); commercial prep
10. S o d i u m s u l p h a t e , A . R . , 0.1 M arations, see p. 461.
18. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , 21 . L a c t a t e d e h y d r o g e n a s e , L D H
NAD crystalline, from rabbit m u s c l e ; suspension in
commercial p r e p a r a t i o n s , see p. 546. 3.2 M a m m o n i u m sulphate solution, ^ 360 U /
19. R e d u c e d n i c o t i n a m i d e - a d e n i n e mg. (25 °C); commercial p r e p a r a t i o n s , see p.481.
dinucleotide, N A D H 22. A l c o h o l d e h y d r o g e n a s e , A D H
2
crystalline, from yeast, suspension in 3.2 M
arations, see p . 545. a m m o n i u m sulphate solution; ^ 200 U/ml.
20. L a c t a t e d e h y d r o g e n a s e , L D H (25 °C); commercial p r e p a r a t i o n s , see p. 428.
crystalline, from bovine heart, suspension in 3.2
M a m m o n i u m sulphate solution; ^ 2 5 0 U / m g .
(25 °C); commercial p r e p a r a t i o n s , see p . 482.
Purity of Reagents
A m o n g several products, calcium lactate from the California Biochemical Corp. contains the least pyruvate.
T h e commercial heart L D H contains t r o u b l e s o m e quantities of N A D ; for removal see under " P r e p a r a t i o n
of Solutions". T h e other reagents d o not c o n t r i b u t e appreciably to the b l a n k value.
P r e p a r e all s o l u t i o n s w i t h f r e s h l y p r e p a r e d , d o u b l y d i s t i l l e d o r d e i o n i z e d w a t e r .
I. Tris b u f f e r (1 M ; p H 8 . 4 ) :
D i s s o l v e 12.1 g. t r i s in d i s t i l l e d w a t e r a n d 5.8 m l . 12 N H C 1 a n d m a k e u p t o 100 m l . ;
keep frozen.
II. 2 - O x o g l u t a r i c acid (1.0 M ) :
D i s s o l v e 146 m g . 2 - o x o g l u t a r i c acid in 1 m l . distilled w a t e r ; k e e p frozen.
III. S o d i u m lactate (ca. 1 M ) :
S u s p e n d 5 g. c a l c i u m l a c t a t e i n 30 m l . d i s t i l l e d w a t e r , a d d 11 m l . 2 M N a C 0 2 3 solution,
s h a k e v i g o r o u s l y , a n d filter. A d j u s t f i l t r a t e t o p H 7.0 w i t h c a . 1 m l . 5 N H C 1 . If n e c e s s a r y ,
determine concentration fluorimetrically a s d e s c r i b e d o n p . 1 4 6 8 ; it is s l i g h t l y less t h a n
1 M . K e e p frozen.
I V . A d e n o s i n e - 5 ' - d i p h o s p h a t e , A D P ( c a . 0.1 M ) :
D i s s o l v e 54 m g . A D P - N a 2 in 1 m l . d i s t i l l e d w a t e r ; k e e p f r o z e n .
V . A m m o n i u m a c e t a t e (5 M ) :
D i s s o l v e 3 8 . 5 g. N H C H C O O in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l . S t o r e a t r o o m
4 3
temperature.
VI. G l u t a m a t e d e h y d r o g e n a s e , G 1 D H (10 m g . p r o t e i n / m l . ) :
Use stock solution.
V I I . N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (0.1 M / ? - N A D ) :
D i s s o l v e 70 m g . N A D in 1 m l . distilled w a t e r ; k e e p frozen. D i l u t e o n e p a r t b y a f a c t o r
o f 100 a n d d e t e r m i n e c o n c e n t r a t i o n a s f o l l o w s . A d d 5 0 0 p\. 0.1 M t r i s b u f f e r ( w i t h 4 %
e t h a n o l a n d 1 m M E D T A ) a n d 6 p\. = 6 pg. a l c o h o l d e h y d r o g e n a s e f r o m y e a s t (in 2 0 m M
t r i s b u f f e r , p H 8 w i t h 0 . 0 2 % a l b u m i n ) t o 50 p\. o f t h e a b o v e s o l u t i o n , a n d m e a s u r e t h e
i n c r e a s e i n e x t i n c t i o n a t 3 4 0 n m ; it s h o u l d b e c a . 0 . 5 6 0 . B o t h s o l u t i o n s (0.1 M a n d 1 m M )
k e e p f o r s e v e r a l m o n t h s a t —20 ° C .
V I I I . R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (5 m M / ? - N A D H ) :
D i s s o l v e 3.5 m g . N A D H - N a 2 in 1 ml. c a r b o n a t e buffer ( X I I ) . B e f o r e u s e h e a t for 15 m i n .
at 6 0 ° C t o d e s t r o y N A D ; s t a b l e for several w e e k s at + 4 ° C ; d o n o t freeze. H i g h e r
c o n c e n t r a t i o n s are less s t a b l e . A n a l y s e b e f o r e u s e a s s t a n d a r d s o l u t i o n : a d d 10 / d . N A D H
s o l u t i o n t o 5 0 0 fil. i m i d a z o l e buffer X I ( c o n t a i n i n g 1 m M p y r u v a t e ) , r e a d e x t i n c t i o n
at 3 4 0 n m , a n d a d d 2 ^1 = 0.5 fig. m u s c l e L D H ( 1 : 2 0 d i l u t i o n o f a 5 m g . / m l . s u s p e n
s i o n ) ; r e a d d e c r e a s e in e x t i n c t i o n , w h i c h s h o u l d b e c a . 0 . 6 0 0 . C o r r e c t s o l u t i o n a c c o r
dingly.
IX. Lactate dehydrogenase, L D H (20 mg. protein/ml.):
To r e m o v e N A D f r o m t h e b o v i n e heart p r e p a r a t i o n , d i l u t e a 2 % s u s p e n s i o n o f e n z y m e
to 0.4 % protein with water, and add 2% N o r i t e charcoal; centrifuge and bring superna
tant fluid t o 3 . 2 M ( N H ) S 0 . C e n t r i f u g e off e n z y m e a n d r e s u s p e n d in 2.5 M a m m o n i u m
4 2 4
s u l p h a t e s o l u t i o n at c a . 2 0 m g . / m l . D e t e r m i n e p r o t e i n c o n c e n t r a t i o n b e f o r e u s e f o r
cycling. This treatment reduces the N A D concentration from 8 x 1 0 " m o l e to 1 x 1 0 " 5 5
m o l e per kg. p r o t e i n .
X . I m i d a z o l e buffer (1 M ; p H 6 . 2 ) :
D i s s o l v e 6.8 g. i m i d a z o l e in distilled w a t e r a n d 6.7 m l . 12 N H C 1 , a n d m a k e u p t o 100 m l .
X L I m i d a z o l e buffer (0.1 M ; p H 7 . 0 ) :
D i s s o l v e 6 8 0 m g . i m i d a z o l e in distilled w a t e r a n d 3.8 m l . 1 N H C 1 . a n d m a k e u p t o 100 m l .
X I I . C a r b o n a t e buffer (0.1 M ; p H 1 0 . 6 ) :
D i s s o l v e 0 . 8 5 g. N a C 0
2 3 a n d 0 . 1 7 g. N a H C 0 3 in distilled w a t e r a n d m a k e u p t o 100 m l .
The solutions are stable u n d e r the conditions indicated.
Procedure
T h e t r e a t m e n t o f t h e s a m p l e differs f r o m t h a t for t h e d e t e r m i n a t i o n o f N A D P ( H ) in t h a t t h e
ratio N A D H : N A D is m u c h s m a l l e r t h a n N A D P H : N A D P . T h e o x i d a t i o n b y t i s s u e h a e m o
g l o b i n d u r i n g t h e e x t r a c t i o n o f t h e t i s s u e c o n s e q u e n t l y d o e s n o t p r o d u c e s u c h large errors.
N e v e r t h e l e s s , t h e p r o c e d u r e d e s c r i b e d o n p. 2 0 6 3 m a y b e u s e d here.
F o r t h e N A D a n a l y s i s , h o m o g e n i z e 5 0 m g . f r o z e n t i s s u e at O ° C in 5 ml. 2 0 m M H S0 /
2 4
0.1 M N a S 0 2 4 s o l u t i o n a n d h e a t for 4 5 m i n . at 6 0 ° C . F o r t h e N A D H a n a l y s i s , h o m o g e n i z e
50 m g . f r o z e n t i s s u e in 5 m l . 2 0 m M N a O H ( w i t h 0.5 m M c y s t e i n e ) , a n d h e a t for 10 m i n . at
60 ° C . B e f o r e u s e for t h e a s s a y , d i l u t e w i t h h o m o g e n i z a t i o n s o l u t i o n a c c o r d i n g t o t h e f o l l o w i n g
scheme:
Dilution of homogenate (in the 100ul. cycling mixture) for the
determination of
Tissue NAD NADH
Liver 100000 5000
Kidney 50000 15000
Heart 50000 5000
Brain 30000 10000
Blood 10000 10000
T h e f r o z e n t i s s u e are s t a b l e at - 8 5 ° C . A f t e r h o m o g e n i z a t i o n t h e y s h o u l d b e a n a l y s e d o n t h e
same day.
Assay System
Cycling:
V o l u m e : 0 . 1 2 m l . ; 2 5 ° C ( o r 38 ° C ) . U s e 1 t o 2 0 fil sample.
F o r e a c h series o f m e a s u r e m e n t s , a n a l y s e a l s o N A D a n d N A D H s t a n d a r d s p r e p a r e d b y d i l u
t i o n o f 1 m M N A D s o l u t i o n (1 : 1 0 0 d i l u t i o n o f s o l u t i o n V I I ) t o 0.1 ^ M « w i t h d i s t i l l e d w a t e r a n d
a d d i n g 1 pi o f this s o l u t i o n t o 100 pi cycling m i x t u r e ( 1 0 - 9
M).
Pyruvate Determination :
P r i m a r y r a d i a t i o n : 3 6 0 n m ; s e c o n d a r y r a d i a t i o n : 4 6 0 n m ; light p a t h : 1 c m . ( s t a n d a r d i z e d
test t u b e s ) ; final v o l u m e : 1.245 m l . ; r o o m t e m p e r a t u r e .
Cycling reagent Pyruvate reagent
K e e p s for t w o w e e k s at — 2 0 ° C o r P r e p a r e 15 m i n . b e f o r e u s e .
t w o m o n t h s at - 8 5 ° C .
Pipette into 100 ml. mixing cylinder: P i p e t t e i n t o 100 m l . m i x i n g c y l i n d e r :

Tris buffer (I) 20.00 ml. I m i d a z o l e buffer (X) 8.00 ml.
Oxoglutarate soln. (II) 0.50 ml. N A D H solution (VIII) 0 . 1 - 1 . 0 0 ml.*
Lactate solution (III) 10.00 ml. L D H suspension (IX) 1.6 pi**
A D P solution (IV) 0.30 ml. Water to 10.00 ml.
NH solution
4
+
(V) 1.00 m l .
W a t e r t o 100 m l .
M i x at 0 ° C i m m e d i a t e l y b e f o r e u s e :
o f this s o l u t i o n 5.00 ml.

L D H suspension (IX) 0.012 ml.
G 1 D H solution (VI) 0.100 ml.
Water 0.100 ml.
* 3 to 10-fold excess in accordance with p . 2066.

** 1 mg. L D H / m l . in 20 m M tris buffer, p H 8 with 0.02% a l b u m i n .
P i p e t t e i n t o 3 m l . test t u b e s (in ice b a t h ) C o n c e n t r a t i o n in a s s a y m i x t u r e
Cycling reagent 0.100 ml. 167 m M tris, 8 3 m M l a c t a t e

4 m M oxoglutarate
0.25 m M ADP
42 m M N H 4
+
4 2 fig. L D H / m l . = 15 U / m l .
165 pg. G I D H / m l . = 15 U / m l .
Sample (or standard solution VII 0.020 ml. 1-30 n M
or VIII) + water
P l a c e test t u b e h o l d e r in 25 ° C w a t e r b a t h , i n c u b a t e
for e x a c t l y 6 0 m i n . , a n d p l a c e in b o i l i n g w a t e r b a t h
for 2 m i n . C o o l in ice b a t h .
Pyruvate reagent 0.100 ml. 36 m M i m i d a z o l e

0.023-0.23 m M NADH
0.07 fig. L D H / m l . = 25 m U / m l .
A l l o w t o s t a n d at r o o m t e m p e r a t u r e ( 2 0 - 3 0 ° C ) f o r
15 m i n . , t h e n c o o l in ice b a t h .
5NHC1 0.025 ml. 0.5 N
S h a k e v i g o r o u s l y . E x c e s s N A D H is d e s t r o y e d .
6 N NaOH 1.000 m l . 4.8 N
M i x i m m e d i a t e l y , h e a t for 10 m i n . at 6 0 ° C , c o o l t o
room temperature, dry outside of tubes, and
m e a s u r e f l u o r e s c e n c e . A v o i d direct a c t i o n o f light.
T h e f l u o r e s c e n c e is s t a b l e for s e v e r a l h o u r s in t h e d a r k . A d j u s t t h e f l u o r i m e t e r s o t h a t t h e
m e a s u r e m e n t is c a r r i e d o u t w i t h t h e l e a s t p o s s i b l e e x c i t a t i o n r a d i a t i o n .
Calculations
Determine results on the basis of the s t a n d a r d values.
2.02 x 1 0 - 1 3
mole N A D can be determined with a s t a n d a r d deviation of 0.08 x 1 0 " 1 3
m o l e ; the coefficient
of variation ( C V ) is 4 % . O n average, 322 ± 10 /rniole N A D / k g . ( C V = 8 % ) a n d 95 ± 10 //mole N A D H / k g .
( C V = 11%) are found in m o u s e brain.
Normal Values
The following values were found by Burch et a l . for rat organs with this m e t h o d (/rniole per kg. fresh
4
weight)
NAD NADH
Liver 741 41
Kidney 357 131
Heart 325 45
Brain 282
Brain 5
321 95
Blood 5
110 97
The main sources of error in N A D cycling are c o n t a m i n a t i o n with N A D . See also u n d e r " O p t i m u m
Conditions for M e a s u r e m e n t s " and " P r e p a r a t i o n of Solutions". T h e N A D H used for the pyruvate
determination gives a N A D blank value of 1-2% of the quantity of N A D H used after acidification. The
fluorescence from the alkali used in the last step should not exceed 1 - 2 x 1 0 " M N A D (direct reading).
8
The blank values are m u c h m o r e erratic t h a n in N A D P cycling, u n d o u b t e d l y because of c o n t a m i n a t i o n

with N A D . F o r precautions for the prevention of c o n t a m i n a t i o n , see p . 2066.
Specificity
The cycling m e t h o d is specific for N A D . O n addition of 50 x 10 9

M N A D P to the cycle, the error is 0.1 %.
References
1 O. H. Lowry, N. R. Roberts & J. I. Kapphahn, J. biol. C h e m . 224, 1047 [1957].

2 C. Frieden, J. biol. C h e m . 234, 815 [1959].
3 H. B. Burch, M. E. Bradley & O. H. Lowry, J. biol. C h e m . 242, 4546 [1967].
5 O. H. Lowry, J. V. Passonneau, D. W. Schulz & M. K. Rock, J. biol. C h e m . 236, 2746 [1961].
Nicotinamide Mononucleotide
Marianne Grassl and Hans Mollering
Nicotinamide m o n o n u c l e o t i d e ( N M N ) can be converted to n i c o t i n a m i d e - a d e n i n e dinucleotide ( N A D )

by the action of nucleotide p y r o p h o s p h o r y l a s e . Until n o w the d e t e r m i n a t i o n of the N A D was carried
1
out by formation of a cyanide complex which has a m a x i m u m a b s o r p t i o n at 327 n m . By the action of N A D

nucleosidase a n d nicotinamide ribosidase any N A D or nicotinamide riboside initially present can be
destroyed a n d thus the m e t h o d is relatively specific . 2
N A D - p y r o p h o s p h o r y l a s e ( A T P : N M N adenylyltransferase, E C 2.7.7.1), which forms N A D a n d p y r o

p h o s p h a t e from N M N a n d a d e n o s i n e - 5 ' - t r i p h o s p h a t e ( A T P ) , coupled with alcohol d e h y d r o g e n a s e , A D H
(Alcohol: N A D oxidoreductase, E C 1.1.1.1) allows the resulting N A D , a n d hence the N M N , to be d e t e r m i n
ed quantitatively. To displace the equilibrium of the reaction in favour of N A D , inorganic p y r o p h o s
phatase, PPase ( P y r o p h o s p h a t e p h o s p h o h y d r o l a s e , E C 3.6.1.1) is a d d e d to hydrolyse the p y r o p h o s p h a t e
formed. As N A D - p y r o p h o s p h o r y l a s e rapidly hydrolyses A T P to A D P a n d Pj even in the absence of N M N ,
the A D P is r e p h o s p h o r y l a t e d with p h o s p h o e n o l p y r u v a t e (PEP) a n d p y r u v a t e kinase, P K ( A T P : 2 - 0 -
pyruvate phosphotransferase, E C 2.7.1.40).
Principle
(1) NMN + ATP NAD-pyrophosphorylase } N A D + + 2 p _
(2) NAD +
+ Ethanol ^ • NADH + H +
+ Acetaldehyde
T h e increase in N A D H , as m e a s u r e d by the change in extinction at 340 (334, 365) n m , is p r o p o r t i o n a l t o

the a m o u n t of N M N present.
T h e equilibrium of reaction (2) at p H 8.7 is far o n the side of N A D H . By a d d i t i o n of semicarbazide t o

remove the acetaldehyde there is virtually quantitative reduction of N A D . T h e c o n c e n t r a t i o n s of A T P ,
P E P a n d P K suitable for the assay mixture were found empirically . They apply for a m o u n t s of samples
3
giving 0.01 to 0.05 /rniole N M N / m l . assay mixture (corresponding to ca. 0 . 0 6 - 0 . 3 /rniole N M N in the
incubation mixture). T h e incubation time with N A D - p y r o p h o s p h o r y l a s e a n d P P a s e should n o t exceed
30 min., otherwise some of the N A D formed m a y be b r o k e n d o w n .
Equipment
3 3 4 o r 3 6 5 n m ; w a t e r b a t h 37 ° C a n d 1 0 0 ° C ; b e n c h c e n t r i f u g e .
Reagents
1. S o d i u m h y d r o x i d e , 2 N , A . R. 4. A d e n o s i n e - 5 ' - t r i p h o s p h a t e , A T P
2. G l y c y l g l y c i n e , p u r e d i s o d i u m salt, A T P - N a H - 3 H 0 ; commercial
2 2 2
2 2
5. P h o s p h o e n o l p y r u v a t e , P E P 8. P y r u v a t e k i n a s e , P K
tricyclohexylammonium salt; commercial prep from rabbit muscle, crystalline suspension in
aration, see p. 548. 3.2 M a m m o n i u m sulphate solution; ^200
6. NAD-pyrophosphorylase U / m g . (25 °C); c o m m e r c i a l p r e p a r a t i o n , see
from hog liver, lyophilized; ^ 0 . 1 5 U/mg. pro p . 509.
tein (37 °C); commercial preparation, see p. 487. 9. S o d i u m p y r o p h o s p h a t e ,
7. I n o r g a n i c p y r o p h o s p h a t a s e , P P a s e Na P O 10H O,
4 2 7 2 A.R.
from yeast, crystalline suspension in 3.2 M 10. S e m i c a r b a z i d e h y d r o c h l o r i d e , A . R .
ammonium sulphate solution; ^ 200 U/mg. 11. Glycine, A . R .
(25 °C); commercial preparation, see p. 508. 12. E t h a n o l , 9 6 %
13. A l c o h o l d e h y d r o g e n a s e , A D H
from yeast crystallized; lyophilized; ^300
U / m g . protein (25 °C); commercial p r e p a r a t i o n ,
see p . 428.
Purity of Reagents
T h e enzymes should be as free as possible from p h o s p h a t a s e s ( N A D - p y r o p h o s p h o r y l a s e acts like an

A T P a s e ) . In addition, they should be free from c o n t a m i n a n t s which b r e a k d o w n N A D .
I. G l y c y l g l y c i n e buffer ( 5 0 m M ; p H 7 . 4 ) :
D i s s o l v e 0 . 6 5 g. g l y c y l g l y c i n e in c a . 8 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7 . 4 w i t h 2 N
N a O H a n d dilute t o 100 ml. with distilled water.
D i s s o l v e 2 . 0 3 g. M g C l - 6 H 0 in 1 0 0 m l . d i s t i l l e d w a t e r .
2 2
III. A d e n o s i n e - 5 ' - t r i p h o s p h a t e ( 1 6 m M ) :
D i s s o l v e 10 m g . A T P - N a H - 3 H 0 in 1 m l . buffer I.
2 2 2
I V . P h o s p h o e n o l p y r u v a t e (21 m M ) :
D i s s o l v e 10 m g . P E P - ( C H A ) 3 in 1 ml. distilled water.
V. N A D - p y r o p h o s p h o r y l a s e (10 m g . protein/ml.):
D i s s o l v e 3 0 m g . l y o p h i l i z a t e ( c o n t a i n i n g 1 0 m g . N A D - p y r o p h o s p h o r y l a s e ) in 1 m l .
distilled water.
V I . I n o r g a n i c p y r o p h o s p h a t a s e , P P a s e ( 5 0 pg. p r o t e i n / m l . ) :
Dilute the stock suspension accordingly with 3.2 M a m m o n i u m sulphate solution.
VII. Pyruvate kinase, P K (10 mg. protein/ml.):
U s e the stock s u s p e n s i o n undiluted.
V I I I . S o d i u m p y r o p h o s p h a t e buffer ( 7 5 m M p y r o p h o s p h a t e ; 7 5 m M s e m i c a r b a z i d e ; 32 m M
glycine; 165 m M e t h a n o l ) :
D i s s o l v e 3 . 3 3 g. N a P O 1 0 H O , 0 . 8 4 g. s e m i c a r b a z i d e h y d r o c h l o r i d e , 0 . 1 7 g. g l y c i n e
4 2 7 2
a n d 1.00 m l . e t h a n o l ( 9 6 % ) i n c a . 6 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 8.7 w i t h 2 N N a O H
and dilute to 100 ml. with distilled water.
IX. Alcohol dehydrogenase, A D H (30 mg. protein/ml.):
D i s s o l v e 5 0 m g . l y o p h i l i z a t e ( c o n t a i n i n g 3 0 m g . p r o t e i n ) in 1 m l . distilled w a t e r .
Nicotinamide Mononucleotide 2075
Store all solutions a n d suspensions, stoppered, in a refrigerator. Solutions I - I V a n d V I I I should be pre

pared freshly every 2 weeks, P K a n d P P a s e are stable for 6 - 1 2 m o n t h s , t h e lyophilizate of A D H a n d
N A D - p y r o p h o s p h o r y l a s e are stable for ca. 6 m o n t h s a n d the solutions of these enzymes are stable for
a b o u t 2 weeks at 4 ° C . T h e A D H solution is usable for u p to 2 m o n t h s if a d r o p of toluene is a d d e d .
Procedure
S o far N M N h a s o n l y b e e n d e t e r m i n e d in p r o t e i n - f r e e , a q u e o u s s o l u t i o n s , b u t t h e m e t h o d
s h o u l d b e a p p l i c a b l e t o b i o l o g i c a l m a t e r i a l , if t h e c o n c e n t r a t i o n is i n t h e right r a n g e . S o l u t i o n s
of samples should be analysed within a few hours.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 1 m l . or 3.01 m l . ; 37 ° C

o r r o o m t e m p e r a t u r e . R e a d a g a i n s t air.
Pipette into centrifuge tubes: Blank Sample
assay mixture
G l y c y l g l y c i n e buffer (I) 0.20 ml. 0.20 ml. 10 m M

MgCl 2 solution (II) 0.10 ml. 0.10 ml. 10 m M
A T P solution (III) 0.12 ml. 0.12 ml. 1.9 m M
PEP solution (IV) 0.20 ml. 0.20 ml. 4.2 m M
D o u b l y distilled water 0.30 ml. 0.20 ml.
NAD-pyrophosphorylase
solution (V) 0.05 ml. 0.05 ml. 0.5 m g . / m l . = 75 m U / m l .
PPase suspension (VI) 0.02 ml. 0.02 ml. 1 /zg./ml. = 2 0 0 m U / m l .
P K suspension (VII) 0.01 m l . 0.01 m l . 0.1 m g . / m l . = 2 0 U / m l .
Sample 0.10 ml. 60 to 300 ^ M NMN
M i x , i n c u b a t e for 3 0 m i n . at 37 ° C , t h e n h e a t for 1 m i n . i n a
b o i l i n g w a t e r b a t h . C e n t r i f u g e for 5 m i n . at c a . 3 0 0 0 r p m . T a k e
0.5 m l . o f t h e s u p e r n a t a n t fluid f o r t h e N A D a s s a y
Pipette into cuvettes: Blank Sample
assay mixture
P y r o p h o s p h a t e buffer (VIII) 2.50 ml. 2.50 ml. 62.5 m M pyrophos

phate ;
62.5 m M semi
carbazide ;
26.6 m M glycine
137 m M e t h a n o l
Supernatant fluid 0.50 ml. 0.50 ml. 1 0 - 5 0 fiM NAD
M i x and read extinction E. 1
A D H solution (IX) 0.01 m l . 0.01 m l . 0.1 m g . / m l . = 3 0 U / m l .
M i x a n d o n c o m p l e t i o n o f the reaction (ca. 5 min.) read extinction

E .
2 (E 2 - Example - (E 2 - E^bunk = ^ E is used for the
calculations.
T h e increase in extinction due to addition of A D H (IX) alone is determined by a further addition of 0.01 ml.
A D H solution (IX) o n completion of the reaction. T h e extinction change is used to correct E . 2
Calculations
U n d e r the above conditions the reaction proceeds stoichiometrically and therefore the calculation formula
(2) on p . 312 applies. T h e results are obtained as jumole N M N / m l . sample. T h e following relationships
hold:

c = AE x 9.87 AE x 9.68 AE x 17.71 [/^mole/ml.]
c = AE x 3.00 AE x 3.23 AE x 5.92 [mg./ml.]
Nicotinamide Mononucleotide 2077
Insufficient purity of the reagents (see p . 2074), in particular the enzymes, can result in false results. A check
of the reagents can be m a d e by testing the recovery of N A D . F o r this an a p p r o p r i a t e a m o u n t of N A D is
a d d e d instead of s a m p l e ; the a m o u n t of N A D is determined in a separate assay according t o the p r o c e d u r e
on p . 2048.
N o c o m p o u n d is k n o w n which can also react u n d e r the present assay c o n d i t i o n s .

4
References
1 G. W. E. Plaut & K. Armstrong in D. Shemin: Biochemical P r e p a r a t i o n s , vol. 5, p . 55, J o h n Wiley a n d

Sons, N e w York 1957.
2 N. O. Kaplan & F. E. Stolzenbach in C P. Colowick & N. 0 . Kaplan: M e t h o d s in E n z y m o l o g y vol. I l l ,
p . 899, Academic Press, N e w York 1957.
3 M. Grass I, unpublished.
4 H. Mollering, unpublished.
Analytical Differentiation of
Purine and Pyrimidine Nucleotides
Determination of ADP, ATP, and Sum of GTP + ITP in Biological
Material
Wolfgang Gruber, Hans Mollering and Hans Ulrich Bergmeyer
Specific enzymatic d e t e r m i n a t i o n s of A T P , e. g. using luciferase, have been k n o w n for some time, refer t o
p. 2112. In routine work, however, A T P has been determined in the past mainly by two non-specific m e t h o d s :
a) with hexokinase ( H K ) * a n d glucose-6-phosphate d ehydrogenase ( G 6 P - D H ) * , see p . 2101,
b) with phosphoglycerate kinase ( P G K ) * a n d glyceraldehyde p h o s p h a t e dehydrogenase ( G A P D H ) * , see
p . 2097.
H K a n d P G K act on the other nucleoside t r i p h o s p h a t e s as well as on A T P . As far as we know, only
myokinase ( M K ) * is specific for A D P a n d A T P . W h e n nucleoside t r i p h o s p h a t e s are converted n o n -
specifically into the d i p h o s p h a t e s by any kinase, only A D P , out of all the d i p h o s p h a t e s , can be specifically
converted by m y o k i n a s e into A T P a n d A M P . F o r the specific d e t e r m i n a t i o n of the A D P originally present
in the sample, the creatine kinase ( C K ) * reaction is used in the m e t h o d described h e r e . 1
Application oj Method: In biochemistry a n d clinical chemistry.
P r i n c i p l e and T h e o r y
(1) A T P ( + G T P + I T P ) + 3-PG ^ 2
K
+ > 1,3-PG-P ( + I D P + G D P ) + A D P
I
(2) •GAP + N A D +
+ P,
1,3-PG-P + N A D H + H +
-
(3) V 2 AMP + 7
2 ATP
I
ADEN
If A T P , G T P , a n d I T P are converted into the d i p h o s p h a t e s with P G K [equation (1)] as described on p . 2101,
the indicator reaction [equation (2)] leads t o a decrease in extinction at 340, 334, o r 365 n m t h a t corres
p o n d s to the sum A T P + G T P + I T P . If reaction (3) is started with M K in the same cuvette after the
completion o f r e a c t i o n s ( l ) a n d (2), then of the s u m of A D P + G D P + I D P , only A D P reacts to give A M P
a n d A T P , and the latter then undergoes reaction (1) again. This cycle continues until all the A T P has been
converted into A M P . Let the first extinction difference of the coupled reactions (1) and (2) be A and the
second [equation (3)] B (see Fig. 1).
T h e A D P originally present is best p h o s p h o r y l a t e d to A T P in an additional a s s a y ' with C K in accordance
1 2
with e q u a t i o n (4):
(4) A D P + Creatine p h o s p h a t e — ^ — * Creatine + A T P
HK = A T P : D-hexose 6-phosphotransferase, E C 2.7.1.1

CK = A T P : creatine ^ - p h o s p h o t r a n s f e r a s e , E C 2.7.3.2
G6P-DH = D - G l u c o s e - 6 - p h o s p h a t e - N A D P 1-oxidoreductase, E C 1.1.1.49
GAPDH = D - G l y c e r a l d e h y d e - 3 - p h o s p h a t e : N A D oxidoreductase (phosphorylating), E C 1.2.1.12
MK = A T P : A M P phosphotransferase, E C 2.7.4.3
PGK = A T P : 3-phospho-D-glycerate 1-phosphotransferase, E C 2.7.2.3.
Differentiation of P u r i n e a n d Pyrimidine N u c l e o t i d e s 2079
T h o u g h C K reacts in principle with all nucleoside d i p h o s p h a t e s , the reaction rate at p H 9 is practically zero
or negligible except in the case of A D P (Table 1). T h u s if A D P alone is converted into A T P with C K at p H 9
in an additional assay a n d the A T P d e t e r m i n a t i o n carried out in a c c o r d a n c e with e q u a t i o n s (1) to (3)
0.6
l1
U_l
0.4
0.2
Fig. 1. Typical reaction course for the

specific d e t e r m i n a t i o n of A T P , A D P , 5 10 15 20
a n d G T P + 1TP. t min ^
after denaturing the enzyme by heating, one o b t a i n s a decrease in extinction (Fig. 1, b o t h reaction courses
are plotted on the same d i a g r a m with a n a u t o m a t i c recorder) that c o r r e s p o n d s to the s u m A T P + A D P +
G T P + ITP.
Table 1. Percentage conversion of nucleoside d i p h o s p h a t e s into the t r i p h o s p h a t e s o n incubation with C K .

Incubation for 15 min. at 38 °C in 0.1 M t r i e t h a n o l a m i n e buffer or 0.1 M glycine buffer, each with 15 m M
creatine p h o s p h a t e a n d 1 m M M g C l ; 0.2 /miole of nucleoside d i p h o s p h a t e per mixture. D e t e r m i n a t i o n ot
2
the resulting triphosphates, after d e n a t u r a t i o n by heating (3 min., 100 °C), with P G K , G A P D H , G D H , a n d

T I M . Check by d e t e r m i n a t i o n of unreacted d i p h o s p h a t e s with P K a n d L D H .
p H (40 fig. C K per assay) fig. C K per assay ( p H 9.0)
6.5 7.9 8.5 9.0 1 5 10 40

ADP 97 101 103 98 23 86 98 99
GDP 80 48 15 5 — 5 5 5
UDP a b o u t 80* about 45* 0 0 1 2
IDP — — — 2 2
CDP — — — — — 0 0 0
In Figure 1, therefore, the extinction difference

A corresponds to the c o n c e n t r a t i o n s of A T P + G T P + I T P
B corresponds to the c o n c e n t r a t i o n s of A T P + A D P
C corresponds to the c o n c e n t r a t i o n s of A T P + A D P + G T P + I T P .
* Indicator reaction P G K , G A P D H , G D H , a n d T I M is t o o slow, a n d reading therefore taken before end

value was reached.
We couple o u r assay system with the following auxiliary and indicator r e a c t i o n s :
(5) 3-Phosphoglyceraldehyde T 1 M
* > Dihydroxyacetone phosphate
(6) Dihydroxyacetone phosphate + N A D H + H + G P H

* > L-Glycerol-3-phosphate + N A D +
T h u s 2 mole of N A D H are t r a n s f o r m e d per mole of nucleoside t r i p h o s p h a t e . We o b t a i n :
A - 2 [ATP] + 2 [ G T P ] + 2 [ITP]
B ~ 2 [ATP] + 2 [ADP]
C ~ 2 [ATP] + 2 [ A D P ] + 2 [ G T P ] + 2 [ITP]
Hence:
[ATP] ~ B — (C — A) A + B— C
2
C —A
[ADP]
[ G T P ] + [ITP] ~ C B
If A K is used instead of P G K as the kinase, all 5'-triphosphates ( X T P ) are determined, since according t o
Table 2, acetate is p h o s p h o r y l a t e d at c o m p a r a b l e rates by A T P , G T P , I T P , a n d U T P in the presence of A K ,
a n d a b o u t half as rapidly by C T P .
(7) X T P + Acetate A K
* * > Acetylphosphate + XDP
T h e auxiliary a n d indicator reactions in this case are
(8) Acetylphosphate + Glucose A G T

• G l u c o s e - 6 - p h o s p h a t e + Acetate
(9) Glucose-6-phosphate + N A D P + G 6 P
~ P H
> 6-Phosphogluconic acid + N A D P H + H +
B o t h t a k e the place of e q u a t i o n (2) where P G K is used. T h e function of the m y o k i n a s e [equation (3)]

remains the same.
To differentiate further, C T P + U T P can be d e t e r m i n e d specifically in a d d i t i o n to A T P and G T P + I T P by
analysis with the aid of reactions (1) to (3) on the o n e h a n d a n d reactions (7) to (9) on the other.
T h e o p t i m u m p H value for reaction (4) is 9.0 ( C K reaction, refer to Table 1). T h e triphosphates are
advantageously determined at p H 7.6 (Table 2). A T P inhibits the F - 6 - P K reaction, but this inhibition can be
eliminated by the addition of A - 5 - M P .
* GDH = M - G l y c e r o l - 3 - p h o s p h a t e : N A D 2-oxidoreductase, E C 1.1.1.8

TIM = D-Glyceraldehyde-3-phosphate ketol-isomerase, E C 5.3.1.1
** A K = A T P : acetate p h o s p h o t r a n s f e r a s e , E C 2.7.2.1
AGT = Acyl-phosphate:D-hexose p h o s p h o t r a n s f e r a s e , E C 2.7.1.61
Differentiation of Purine a n d Pyrimidine Nucleotides 2081
Table 2. Relative reaction rates of various kinases with nucleoside t r i p h o s p h a t e s and nucleoside diphos
phates.
Assay system: 0.1 M t r i e t h a n o l a m i n e buffer, p H 7.6 (for A K 0.1 M glycine buffer with 0.2 M acetate,
p H 8.5); 25 °C. In the reactions with the t r i p h o s p h a t e s , G 6 P - D H was the indicator enzyme for H K ; for
P G K (yeast) the auxiliary a n d indicator e n z y m e s were G A P D H , G D H , a n d T I M , those for A K were
1
A G T and G 6 P - D H , and those for F-6-PK, G K , G - l - P K , a n d M K were P K and L D H . In the reactions

with the diphosphates, P K was determined by the L D H reaction, and C K a n d M K were determined with
the aid of the auxiliary a n d indicator enzymes P G K , G A P D H , G D H , a n d T I M . T h e indicator enzyme for
gluconate kinase was 6-phosphogluconate dehydrogenase.
HK PGK 1
PGK 4
PGK 4
AK Gluconate
yeast muscle yeast kinase 5
ATP 100 100 100 100 100 100

ITP 20 100 77 70 100 2
GTP 5 100 56 55 100 9
UTP 5 5 <1 <1 92 12
CTP 1 1 0 0 46 18
dTTP — — 0 0 — 2
F-6-PK F-6-PK F-6-PK GK G-l-PK MK

muscle yeast* yeast**
ATP 100 100 100 100 100 100

ITP 36 49 100 12 100 0
GTP 45 83 100 7 100 0
UTP 35 33 86 7 — 0
CTP 8 12 92 4 — 0
PK CK MK
ADP 100 100 100
IDP 25 — 0
GDP 25 0.4 0
UDP 6 — 0
CDP 2 — 0
Equipment
3 3 4 , or 3 6 5 n m (for series o f i n v e s t i g a t i o n s , a p h o t o m e t e r w i t h a n a u t o m a t i c 6 - c u v e t t e c h a n g e r
is r e c o m m e n d e d t o a l l o w m e a s u r e m e n t s o n several s a m p l e s in a s i n g l e o p e r a t i o n ) ; l a b o r a t o r y
c e n t r i f u g e , h o m o g e n i z e r , a n d e q u i p m e n t for q u i c k - f r e e z e o p e r a t i o n . 38 ° C w a t e r b a t h .
Reagents***
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e , 2. C r e a t i n e phosphate
crystalline. disodium s a l t - 6 H 0 , commercial p r e p a r a t i o n ,
2
see p . 529.
* D e t e r m i n a t i o n of F - 6 - P K with P K a n d L D H .
** F-6-PK from yeast was determined by the aldolase reaction, with addition of A - 5 - M P and A T P . 3
* * * Only the reagents a n d details of m e t h o d s that can be considered for use in the specific analysis of adenine
nucleoside p h o s p h a t e s are described b e l o w ; the m e t h o d s used for the analysis of pyrimidine nucleoside
triphosphates are n o t m e n t i o n e d .
3. G l u t a t h i o n e , GSH 10. M y o k i n a s e , M K
Commercial p r e p a r a t i o n , see p . 538. from r a b b i t muscle, suspension in 3,2 M a m
4. 3-Phosphoglycerate, 3 - P G monium sulphate solution; ^ 360 U/mg.
disodium salt, commercial p r e p a r a t i o n , see (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p . 486.
p . 540. 11. Creatine kinase, C K
5. R e d u c e d n i c o t i n a m i d e - a d e n i n e from rabbit muscle, salt-free lyophilizate; ^ 2 5
dinucleotide, 0 - N A D H U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , see
disodium salt, N A D H - N a , commercial p r e p
2
p . 444.
aration, see p . 545. 12. L a c t a t e d e h y d r o g e n a s e , L D H
6. G l y c e r o l - 3 - p h o s p h a t e d e h y d r o g e n a s e , from rabbit muscle, crystalline suspension in
GDH 3.2 M a m m o n i u m sulphate solution; ^ 550 U /
from rabbit muscle, crystalline suspension in mg. (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 481:
3.2 M a m m o n i u m sulphate solution; ^ 40 U / 13. P y r u v a t e k i n a s e , P K
mg. (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 468. from rabbit muscle, crystalline suspension in
7. T r i o s e p h o s p h a t e i s o m e r a s e , T I M 3.2 M a m m o n i u m sulphate solution; ^ 2 0 0 U/
from rabbit muscle, suspension in 3.2 M a m mg. (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 509.
m o n i u m sulphate solution; ^ 5 000 U/mg. 14. P o t a s s i u m h y d r o x i d e s o l u t i o n , A . R., 2 N
(25 °C). C o m m e r c i a l p r e p a r a t i o n , see p . 515. 15. P e r c h l o r i c a c i d , 0.9 M .
8. Glyceraldehyde-3-phosphate 16. M a g n e s i u m s u l p h a t e ,
dehydrogenase, G A P D H M g S 0 - 7 H 0 , A.R.
4 2
from rabbit muscle, crystalline suspension in 17. G l y c i n e

3.2 M a m m o n i u m sulphate solution; ^ 80 U/ 18. S o d i u m h y d r o g e n c a r b o n a t e , N a H C 0 , 3
mg. (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 466. A.R.

9. 3 - P h o s p h o g l y c e r a t e k i n a s e P G K
from yeast, crystalline suspension in 3.2 M
a m m o n i u m sulphate s o l u t i o n ; ^ 4 5 0 U / m g .
(25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 502.
Purity of Reagents
All enzymes should be free from c o n t a m i n a t i n g enzymes t h a t destroy N A D H ( N A D H oxidase) or t h a t

degrade A T P o r A D P (e.g. A T P a s e ) .
M a k e u p all s o l u t i o n s w i t h freshly p r e p a r e d d o u b l y d i s t i l l e d w a t e r . Sterilize c o n t a i n e r s t o

p r e v e n t the g r o w t h o f m i c r o - o r g a n i s m s .
I. B u f f e r / s u b s t r a t e s o l u t i o n (0.1 M t r i e t h a n o l a m i n e ; 5 m M M g S 0 ; 10 m M 3 - p h o s p h o -
4
glycerate; p H 7.6):
D i s s o l v e 1.86 g. t r i e t h a n o l a m i n e - H C l , 1 2 3 m g . M g S 0 - 7 H 0 , a n d 3 0 0 m g . 3 - p h o s p h o -
4 2
g l y c e r a t e - N a in 8 0 m l . H 0 , adjust p H t o 7.6 w i t h 2 N K O H , a n d m a k e u p t o 100 m l .

2 2
w i t h distilled w a t e r .
II. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 1 2 m M j ? - N A D H ) :
III. B u f f e r / c r e a t i n e phosphate solution (14 m M creatine phosphate; 0.1 M glycine;
ImM MgSO ;pH9.0): 4
D i s s o l v e 7 5 0 m g . g l y c i n e , 25 m g . M g S 0 - 7 H 0 , a n d 5 0 0 m g . c r e a t i n e p h o s p h a t e in
4 2
8 0 m l . w a t e r , adjust t o p H 9 . 0 w i t h 2 N K O H , a n d m a k e u p t o 1 0 0 m l . w i t h distilled
water.
IV. Creatine kinase (2 m g . p r o t e i n / m l . ) :

D i s s o l v e 2 m g . l y o p h i l i z e d d r y p o w d e r in 1 m l . s o l u t i o n X .
V. Glycerol-3-phosphate dehydrogenase/triosephosphate isomerase ( G D H / T I M ) :
M i x in r a t i o 1 0 : 1 b a s e d o n p r o t e i n , 1 : 1 2 b a s e d o n a c t i v i t i e s . C o m m e r c i a l p r e p a r a t i o n ,
see p. 468.
VI. Glyceraldehyde-3-phosphate dehydrogenase (10 mg. protein/ml.):
U s e commercial preparation undiluted.
VII. 3-Phosphoglycerate kinase (2 m g . p r o t e i n / m l . ) :
V I I I . M y o k i n a s e (5 m g . p r o t e i n / m l . ) :
IX. Glutathione (50 m M G S H / m l . ) :
D i s s o l v e 15 m g . G S H i n 1 m l . d i s t i l l e d w a t e r a n d n e u t r a l i z e t o c a . p H 7.0 w i t h a little
2 N KOH.
X. N a H C 0 3 solution (0.5%):
D i s s o l v e 0.5 g. N a H C 0 3 in w a t e r a n d m a k e u p t o 100 m l .
T h e enzyme suspensions are stable for several m o n t h s at 0 to + 4 °C but the C K solution lasts for at m o s t
1 week. N A D H and buffer/substrate solutions keep for 8 - 1 4 days.
Procedure
A D P a n d A T P in s a m p l e s w i t h l o w p r o t e i n c o n t e n t s c a n b e d e t e r m i n e d w i t h o u t p r i o r t r e a t m e n t .
T i s s u e s s u c h a s h e a r t , liver, a n d m u s c l e m u s t b e c o l l e c t e d b y t h e f a s t e s t p o s s i b l e m e t h o d
( q u i c k - f r e e z e t e c h n i q u e , s e e p. 4 0 0 ) , d e e p - f r o z e n w i t h l i q u i d n i t r o g e n , a n d g r o u n d t o a p o w d e r
w h i l e still f r o z e n .
Deproteinization
A c c o r d i n g t o , d e p r o t e i n i z a t i o n o f t i s s u e w i t h 7 t i m e s its w e i g h t o f c o l d 0.9 M p e r c h l o r i c a c i d
1
h a s p r o v e d s u i t a b l e * . T h e t i s s u e is p r e f e r a b l y w e i g h e d o u t in t h e d e e p - f r o z e n s t a t e b e f o r e b e i n g
g r o u n d w i t h l i q u i d n i t r o g e n . I m m e d i a t e l y b l e n d t h e p e r c h l o r i c a c i d / t i s s u e m i x t u r e v e r y finely f o r
2 m i n . in a s u i t a b l e h o m o g e n i z e r , c e n t r i f u g e at h i g h s p e e d , a n d n e u t r a l i z e t h e s u p e r n a t a n t
fluid w i t h 2 N K O H . A l l o w t o s t a n d in a n ice b a t h for 10 m i n , t h e n filter t o r e m o v e p r e c i p i t a t e d
K C 1 0 , a n d u s e t h e filtrate f o r t h e n u c l e o t i d e d e t e r m i n a t i o n .
4
S i n c e t i s s u e c o n t a i n s a b o u t 2 0 % d r y m a t e r i a l , 1 g. t i s s u e + 7 m l . p e r c h l o r i c a c i d i n t h e a b o v e
d e p r o t e i n i z a t i o n m e t h o d g i v e s 7.8 m l . e x t r a c t ; after n e u t r a l i z a t i o n w i t h 3.3 m l . 2 N K O H , a
* According to Jaworek et al. (p. 2099), deproteinization with trichloroacetic acid is preferable for the
determination of A T P in blood. Trichloroacetic acid is not suitable for the differentiation of adenine
nucleotides in tissues by the m e t h o d described here, since the p h o s p h o r y l a t i o n of A D P with creatine kinase
does not take place in the neutralized trichloroacetic acid extract. Practically n o destruction of A T P in
perchloric acid occurs ( m a x i m u m 5 % decrease in the unneutralized perchloric acid extract in one h o u r at
4 °C) when tissues are used (rat skeletal muscle and rat liver).
t o t a l o f 11.1 m l . is o b t a i n e d . 1 m l . o f e x t r a c t p r e p a r e d for t h e a s s a y c o r r e s p o n d s t o 0 . 0 9 g.
tissue.
F o r t h e stability o f A D P a n d A T P in b l o o d , s e e T a b l e 4 , p. 166. T h e n u c l e o t i d e d e t e r m i n a t i o n
s h o u l d be carried o u t s o o n after d e p r o t e i n i z a t i o n .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 3 . 0 0 m l . ; 25 ° C . M e a s u r e
a g a i n s t air.
Measurement of the Extinction Differences A and B according to Figure 1
Buffer/substrate solution (I) 1.60 m l . 53 m M triethanolamine

2.7 m M M g 2 +
5.3 m M 3 - P G
G S H solution (IX) 0.20 ml. 3.3 m M G S H
N A D H solution (II) 0.10 ml. 0.4 m M NADH
Sample + H 0
2
1.05 m l . u p t o c a . 1 0 0 pM A T P
G A P D H suspension (VI) 0.02 ml. 5.3 U G A P D H / m l .
G D H / T I M suspension (V) 0.01 m l . 0.23 U G D H / m l .
3.3 U T I M / m l .
M i x ; read extinction E . x
P G K suspension (VII) 0.01 m l . ca. 3 U P G K / m l .
A T P a n d G T P -f I T P p r e s e n t in t h e s a m p l e react (ca.
3 min.) D e p e n d i n g o n the U T P content o f the sample,
the r e a c t i o n c o m e s t o a s t o p o r " c r e e p s " . A f t e r 10
m i n . , read o r e x t r a p o l a t e E . 2 Ej - E = A.
2
Myokinase suspension (VIII) 0.01 m l . ca. 6 U M K / m l .
A D P reacts in ca. 10 m i n . ( A D P c o n t e n t o f t h e s a m p l e
+ reaction product A D P according to eqn. 1 from
measurement A ) . R e a d or extrapolate E . 3
E2 — E 3 = B.
Measurement of the Extinction Difference C According to Figure 1:
A D P is specifically p h o s p h o r y l a t e d t o A T P .
Incubation Mixture:
M i x 2 ml. o f d e p r o t e i n i z e d a n d n e u t r a l i z e d t i s s u e extract w i t h 1.98 m l . b u f f e r / c r e a t i n e p h o s p h a t e

s o l u t i o n (III) in a c e n t r i f u g e t u b e , a d d 0 . 0 2 m l . C K s o l u t i o n ( I V ) , i n c u b a t e for 15 m i n . at
38 ° C , h e a t for 3 m i n . in a b o i l i n g w a t e r b a t h , a n d c e n t r i f u g e . S i n c e t h e s u p e r n a t a n t fluid
c o r r e s p o n d s t o t h e t i s s u e e x t r a c t d i l u t e d b y a f a c t o r o f 2, u s e t w i c e t h e v o l u m e . T h e r e a c t i o n
A D P —• A T P is q u a n t i t a t i v e .
Measurement:
F o r t h e d e t e r m i n a t i o n o f A T P , i n t r o d u c e t h e s o l u t i o n s a s for m e a s u r e m e n t A i n t o n e w c u v e t t e s ,
b u t u s e t w i c e t h e q u a n t i t y o f s a m p l e f r o m t h e i n c u b a t i o n m i x t u r e . Set initial e x t i n c t i o n E x
o n t h e r e c o r d e r t o t h e E v a l u e o f m e a s u r e m e n t A . P r o c e e d further as in t h e m e a s u r e m e n t o f A .
1
A T P + G T P -f I T P react, a s w e l l a s t h e A T P f o r m e d f r o m A D P d u r i n g p r i o r i n c u b a t i o n w i t h
CK. Measure E 4 in t h e s a m e w a y a s E . E j — E 2 4 = C.
Calculations
The reaction proceeds stoichiometrically u n d e r the conditions indicated. T h e general calculation formula
(2) on p . 312 is therefore valid. T h e extinction differences A, B, a n d C are used in this calculation formula.
AE is replaced by the expressions c o r r e s p o n d i n g to the nucleotide c o n c e n t r a t i o n s from p. 2080:
ATP = A + B
~ C
, ADP = G T P + ITP =
2 2 2
The result is obtained as /miole of nucleotide per ml. of sample. However, this value must be multiplied by a
factor if the samples have been deproteinized, neutralized, o r otherwise diluted. W h e n whole blood is used,
its specific gravity (ca. 1.06) a n d the fluid content (ca. 80%) must also be taken into account.
The following relations are found for the nucleotide concentration of the extract.

c = AE x 0.491/v AE x 0.482/v AE x 0.882/v [/miole/ml.]
v = volume of sample
The sensitivity of this m e t h o d for the determination of A T P is 4 times that of the P G K and H K m e t h o d s .
The accuracy has not been investigated, but it must be less t h a n that of the k n o w n m e t h o d s , since errors
can a d d u p .
N o r m a l Values
Tables 3 - 5 are taken from the article by Gruber, Mollering, and Bergmeyer .
1
Table 3. Nucleotide content of rat liver. Values in /rniole per g. fresh weight.
No. AMP ADP ATP G T P + ITP
1 0.24 0.70 3.00 0.48

2 0.25 0.60 2.50 0.30
3 0.24 0.84 2.66 0.24
4 0.20 0.80 3.20 0.30
5 0.20 0.86 2.54 0.54
6 0.26 0.97 2.86 0.46
7 0.26 1.01 2.81 0.42
8 0.28 0.87 2.93 0.50
9 0.23 0.93 3.63 0.53
10 0.31 0.83 3.00 0.24
11 0.28 0.85 2.95 0.41
12 0.18 0.54 3.13 0.21
13 0.15 0.78 3.00 0.31
14 0.15 0.31 3.18 0.10
15 0.25 0.83 2.85 0.25
16 0.23 0.91 3.11 0.23
17 0.21 0.38 2.18 0.04
18 0.24 0.81 2.11 0.33
19 0.19 0.68 3.98 0.11
20 0.20 0.90 3.07 0.35
Mean 0.23 0.77 2.94 0.32
Table 4. Nucleotide content of rat heart. Values in /rniole per g. fresh weight.
1 0.13 0.65 3.95 0.26

2 0.11 0.52 2.82 0.10
3 0.34 0.89 3.40 0.12
4 0.43 0.94 3.45 0.16
5 0.33 0.72 3.34 0.10
6 0.44 0.87 3.35 0.02
7 0.37 1.10 3.30 0.24
Mean 0.31 0.81 3.38 0.14
Table 5. Nucleotide content of rat skeletal muscle. Values in /*mole per g. fresh weight.
1 0.08 0.32 6.36 0.00

2 0.02 0.11 6.28 0.00
3 0.05 0.26 5.95 0.00
4 0.00 0.12 5.95 0.00
5 0.04 0.53 5.30 0.12
6 0.06 1.00 6.10 0.00
7 0.01 0.75 5.25 0.00
8 0.02 0.76 5.25 0.20
Mean 0.03 0.48 5.80 0.04
See under " P u r i t y of R e a g e n t s " . U T P present in the sample also reacts slowly in m e a s u r e m e n t s A and B.
However, extrapolation is possible here. Cysteine or preferably G S H is essential for the determination of
A T P in tissues (particularly in liver extracts), since the enzymes used are otherwise inhibited.
See p . 2081 Table 2. A T P , A D P , a n d G T P + I T P are specifically determined. C T P + U T P can be determin

ed by the glucose-6-phosphate d e h y d r o g e n a s e reaction with a large excess of H K . d T T P can be determined
by the A K / A G T m e t h o d , see p. 2080.
References
1 W. Gruber, H. Mollering & H. V. Bergmeyer, Enzym. biol. clin. 7, 115 [1966].

2 T. Nihei, L. Noda & M. F. Morales, J. biol. C h e m . 236, 3203 [1961].
3 K. Gawehn, 1969, unpublished.
4 W. Krietsch: Dissertation University of M u n i c h , M e d . F a k u l t a t , 1969 "Kristallisation u n d Eigen-
schaften der K a n i n c h e n m u s k e l - P G K " . D i s c o u r s e : H e r b s t t a g u n g der Gesellschaft Biol. C h e m . 1967,
Z. Physiol. C h e m . 348, 1236 [1967].
5 H. Mollering, 1969, unpublished.
Determination of 5'-Nucleotides as
Nucleoside-5'-monophosphates
Dietrich Keppler
A variety of nucleotides c a n n o t be determined by direct a n d specific enzymatic analysis. The hydrolysis of

5'-nucleotides by phosphodiesterase or nucleotide p y r o p h o s p h a t a s e from snake venom, P D E (Oligo-
nucleate 5'-nucleotidohydrolase, E C 3.1.4.1.), however, results in the liberation of nucleotide-5'-mono
p h o s p h a t e s ' , which can then be specifically determined. By this principle it is possible to determine the
1 2
sum of all adenine ( A M P ) , guanine ( G M P ) , cytosine ( C M P ) , or uracil ( U M P ) 5'-nucleotides in tissue

e x t r a c t s ' . Moreover, some nucleotides that are not very a m e n a b l e to direct enzymatic analysis can be
3 4
accurately determined via their n u c l e o s i d e - 5 ' - m o n o p h o s p h a t e residue; this is true e.g. of U D P - g l u c u r o n -

a t e , U D P - N - a c e t y l h e x o s a m i n e s , C D P - c h o l i n e , C T P , G T P , or G D P - f u c o s e .
5 5 6 6 7
A M P is determined with m y o k i n a s e , M K ( A T P : A M P p h o s p h o t r a n s f e r a s e , E C 2.7.4.3), pyruvate kinase,

P K ( A T P : pyruvate 2-O-phosphotransferase, E C 2.7.1.40), a n d lactate d e h y d r o g e n a s e , L D H ( L - L a c t a t e :
N A D oxidoreductase, E C 1.1.1.27) (see p. 2127). G M P is determined by addition of G M P kinase, G M P K
8
( A T P : (d) G M P phosphotransferase, E C 2 . 7 . 4 . 8 ) 9,10

, (see p 2162); the a d d i t i o n of n u c l e o s i d e m o n o p h o s -
phate kinase, N M P K ( A T P : n u c l e o s i d e m o n o p h o s p h a t e p h o s p h o t r a n s f e r a s e , E C 2.7.4.4) allows the de
termination of C M P + U M P 1 1
(see p. 2153). T h e specific d e t e r m i n a t i o n of U M P is carried out with N M P K ,
nucleosidediphosphate kinase, N D P K ( A T P : nucleosidediphosphate p h o s p h o t r a n s f e r a s e , E C 2.7.4.6),
UDP-glucose p y r o p h o s p h o r y l a s e , UDPGP ( U T P : a-D-glucose-1-phosphate uridylyltransferase, EC
2.7.7.9), and U D P - g l u c o s e dehydrogenase, U D P G - D H ( U D P - g l u c o s e : N A D 6-oxidoreductase, EC
1.1.1.22) ' ,(see p. 2172).
3 12
Application of Method: In biochemistry and clinical chemistry.
Principle
(1) Nucleoside-5'-monophosphate-R + H 0 — — — • Nucleoside-5'-monophosphate + R

2
(2) AMP + ATP, M K

- 2 ADP
. I
I
(3) 2 A D P + 2 PEP ^ — • 2 A T P + 2 Pyruvate
(4) GM P 4 ATP , G M P K
» , G D P 4- A D P ,
,
(5) 'GDP + ADP'+2 PEP ^ — • G T P 4 A T P + 2 Pyruvate
(6) (CMP 4 UMP) + ATP v

: J M P K
- (CDP + UDP) + ADP
| I
(7) ' ( C D P + U D P H ADP'+ 2 PEP ^ — • ( C T P 4 U T P ) 4 A T P 4 2 Pyruvate
(8) 2 Pyruvate 4 2 N A D H + 2 H +
— * 2 Lactate 4 2 N A D +
(9) UMP4ATP , N M P K
» UDP4ADP
(10) UDP4ATP , N D P K
> UTP4ADP
D e t e r m i n a t i o n of 5'-Nucleotides as N u c l e o s i d e - 5 ' - m o n o p h o s p h a t e s 2089
(11) U T P + glucose-1 -P t
U P P G P
> U D P - g l u c o s e + PPj
(12) UDP-glucose + H 0 + 2 N A D 2
4 U D P G
" D 1
^ UDP-glucuronate + 2 N A D H + 2 H +
In the hydrolysis reaction (eqn. 1), n u c l e o s i d e - 5 ' - m o n o p h o s p h a t e s are liberated from mononucleotides
and dinucleotides, nucleosidediphosphate sugars, and related c o m p o u n d s . The reaction proceeds q u a n t i
tatively under the conditions i n d i c a t e d . T h e m o n o p h o s p h a t e s formed can be determined specifically in
3
accordance with eqn. (2) to (8) a n d (9) to (12). T h e decrease in the q u a n t i t y of N A D H is measured in the
first series of reaction at 340 (334, 365) n m , and the increase in the quantity of N A D H in the second. A M P
is determined according to eqn. (2) and (3), G M P according to eqn. (4) and (5), a n d C M P -f U M P according
to eqn. (6) and (7). T h e c o m m o n indicator reaction is eqn. (8).
U M P is quantitatively determined in accordance with eqn. (9) to (12). All the reactions proceed quanti
tatively.
Optimum Conditions of Measurements
The p H o p t i m u m of the irreversible reaction (1) is p H 8.9 . P D E requires an excess of divalent cations to
1
obtain full activity. E D T A , citrate, and various reducing substances (cysteine, glutathione) are inhibitors
of P D E 1 , 2
. The equilibria of reactions (7) and (8) lie almost completely on the right at neutral p H . T h e
o p t i m u m p H for the d e t e r m i n a t i o n of U M P is 8.7; the indicator reaction (12) is irreversible.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r for m e a s u r e m e n t at 334, 340, or 365 n m .

L a b o r a t o r y centrifuge. T h e r m o s t a t for i n c u b a t i o n at 40 ° C .
Reagents
1. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , 12. R e d u c e d n i c o t i n a m i d e - a d e n i n e
s p . g r . 1.67 dinucleotide, NADH
2. P o t a s s i u m h y d r o g e n c a r b o n a t e , A . R . disodium salt, /i-N A D H - N a ; commercial prep
2
3. P o t a s s i u m h y d r o x i d e s o l u t i o n , A . R . , 1 N arations, see p. 545.

4. H y d r o c h l o r i c acid, A . R . , 1 N 13. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ,
5. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , t r i s , jff-NAD
A.R. free acid; commercial p r e p a r a t i o n s , see p. 545.
6. T r i e t h a n o l a m i n e h y d r o c h l o r i d e , A . R . 14. P h o s p h o d i e s t e r a s e , P D E
7. Glycine from Crotalus terr. terr., solution in 50% (v/v)
8. M a g n e s i u m a c e t a t e , glycerol: ^50 U/mg. (25 C) with UDP-
Mg(CH C0 ) -4H 0,
3 2 2 2 A.R. glucose as s u b s t r a t e ; commercial preparations,
9. P h o s p h o e n o l p y r u v a t e , P E P see p. 498.
m o n o p o t a s s i u m salt; P E P - K ; commercial prep 15. L a c t a t e d e h y d r o g e n a s e , L D H
arations, see p. 548. from rabbit skeletal muscle, crystalline su
10. G l u c o s e - 1 - p h o s p h a t e , G-l-P spension in 3.2 M a m m o n i u m sulphate solution;
crystalline d i p o t a s s i u m salt, G - 1 - P - K - 2 H 0 : 2 2
^ 5 5 0 U / m g . (25 C ) ; commercial preparations,
commercial p r e p a r a t i o n s , see p. 548. see p. 4 8 1 .
11. A d e n o s i n e - 5 ' - t r i p h o s p h a t e , A T P 16. P y r u v a t e k i n a s e , P K
crystalline d i s o d i u m salt, A T P - N a H - 3 H 0 : 2 2 2
from rabbit skeletal muscle, solution in 50%
commercial p r e p a r a t i o n s , see p. 527. (v/v) glycerol; ^ 200 U / m g . (25 C); com
mercial p r e p a r a t i o n s , see p. 509.
17. M y o k i n a s e , M K 20. N u c l e o s i d e d i p h o s p h a t e kinase, N D P K

from rabbit skeletal muscle, suspension in from bovine liver, solution in 50% (v/v) glycerol,
3.2 M a m m o n i u m sulphate solution; ^ 350 ^ 80 U / m g . (25 °C); commercial preparations,
U/mg. (25 °C); commercial preparations, see s e e p . 488.
p. 486. 21. U D P - g l u c o s e pyrophosphorylase,
18. G u a n o s i n e - 5 ' - m o n o p h o s p h a t e k i n a s e , UDPGP
GMPK from bovine liver, solution in 50% (v/v) glycerol,
from hog brain, solution in 50% (v/v) glycerol; ^ 100 U / m g . (25 °C); commercial preparations,
^ 1 0 U / m g . (25 °C); commercial preparations, see p. 519.
see p. 472. 22. U D P - g l u c o s e dehydrogenase,
19. N u c l e o s i d e m o n o p h o s p h a t e k i n a s e , UDPG-DH
NMPK from bovine liver, suspension in 3.2 M a m
from bovine liver, lyophilized; ^0.5 U/mg. m o n i u m sulphate solution; ;> 0.6 U / m g . (25 °C);
protein (25 °C); commercial p r e p a r a t i o n s , see commercial p r e p a r a t i o n s , see p. 519.
p. 489.
Purity of Reagents
L D H , P K , and M K must be substantially free from N M P K . P D E must be as free as possible from 5'-
nucleotidase and alkaline p h o s p h a t a s e .
D i l u t e 7.8 ml. 7 0 % H C 1 0 4 t o 100 m l . w i t h w a t e r .
II. Tris buffer ( 0 . 2 M t r i s ; p H 8 . 9 ; 1 m M m a g n e s i u m a c e t a t e ) :
D i s s o l v e 2 . 4 2 g. tris a n d 2 1 . 4 m g . M g ( C H C 0 ) - 4 H 0 in a b o u t 80 m l . w a t e r , adjust
3 2 2 2
t o p H 8.9 w i t h 1 N H C 1 , a n d m a k e u p t o 100 m l . w i t h w a t e r .
III. T r i e t h a n o l a m i n e buffer ( 0 . 3 M t r i e t h a n o l a m i n e ; p H 7 . 5 ; 9 m M m a g n e s i u m a c e t a t e ) :
D i s s o l v e 5.6 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e a n d 193 m g . M g ( C H C 0 ) - 4 H 0 3 2 2 2 in
a p p r o x . 8 0 m l . w a t e r , a d j u s t p H t o 7.5 w i t h a p p r o x . 12 m l . 1 N K O H , a n d m a k e u p t o
100 ml. w i t h w a t e r .
IV. G l y c i n e buffer ( 0 . 5 M g l y c i n e ; p H 8 . 7 ; 4 m M m a g n e s i u m a c e t a t e ) :
D i s s o l v e 3 . 7 6 g. g l y c i n e a n d 8 6 m g . M g ( C H C 0 ) - 4 H 03 2 2 2 in a p p r o x . 8 0 m l . w a t e r ,
adjust p H t o 8.7 w i t h a p p r o x . 3.6 m l . 1 N K O H , a n d m a k e u p t o 100 m l . w i t h w a t e r .
V . P h o s p h o d i e s t e r a s e f r o m Crotalus terr. terr., P D E (1 m g . p r o t e i n / m l . ) :
V I . L a c t a t e d e h y d r o g e n a s e , L D H (5 m g . p r o t e i n / m l . ) :
U s e s t o c k s u s p e n s i o n in 3 . 2 M a m m o n i u m s u l p h a t e s o l u t i o n u n d i l u t e d .
V I I I . M y o k i n a s e , M K (2 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n as r e q u i r e d .
IX. G u a n o s i n e - 5 ' - m o n o p h o s p h a t e kinase, G M P K (2 mg. p r o t e i n / m l . ) :
X . N u c l e o s i d e m o n o p h o s p h a t e kinase, N M P K (15 m g . p r o t e i n / m l . ) :
D i s s o l v e a p o r t i o n o f t h e l y o p h i l i z a t e in c o l d w a t e r t o g i v e a p r o t e i n c o n c e n t r a t i o n o f
a p p r o x . 15 m g . / m l . If t h e e n z y m e s o l u t i o n is t o b e u s e d f o r s e v e r a l d a y s , s o l u t i o n in
5 0 % ( v / v ) g l y c e r o l is a d v a n t a g e o u s .
XI. N u c l e o s i d e d i p h o s p h a t e k i n a s e , N D P K (5 m g . p r o t e i n / m l . ) :
X I I . U D P - g l u c o s e p y r o p h o s p h o r y l a s e , U D P G P (5 m g . p r o t e i n / m l . ) :
X I I I . U D P - g l u c o s e d e h y d r o g e n a s e , U D P G - D H (5 m g . p r o t e i n / m l . ) :
C e n t r i f u g e a p o r t i o n o f t h e s u s p e n s i o n in a m m o n i u m s u l p h a t e s o l u t i o n for 15 m i n . at
1 5 , 0 0 0 g , p i p e t t e off t h e s u p e r n a t a n t , a n d d i s s o l v e t h e p r o t e i n in t h e s a m e v o l u m e o f
g l y c i n e buffer ( I V ) .
For the determination o f A M P , G M P , and C M P + U M P :

X I V . R e a g e n t m i x t u r e ( 0 . 3 M t r i e t h a n o l a m i n e ; 8.9 m M M g " " ; 1.38 m M P E P ; 0 . 3 6
2
mM
N A D H ; 0 . 1 6 m M A T P ; 3 0 ^ g . / m l . = 16.5 U / m l . L D H ; 3 0 /xg./ml. = 6 U / m l . P K ) :
D i s s o l v e 3 m g . P E P - K , 3 m g . N A D H - N a , a n d 1 m g . A T P - N a - H - 3 H 0 in 9.91 m l .
2 2 2 2
t r i e t h a n o l a m i n e buffer ( I I I ) ; a d d 6 0 pi L D H ( V I ) a n d 3 0 fil P K (VII) and mix.
F o r the determination o f U M P :
X V . R e a g e n t m i x t u r e ( 0 . 4 8 M g l y c i n e ; 3.8 m M M g 2 +
; 3 . 2 4 m M A T P ; 2.1 m M G-l-P;
2 . 2 2 m M N A D ; 1 5 0 pg./ml = 9 0 m U / m l . U D P G - D H ; 25 pg./ml = 2.5 U / m l . U D P G P ;
25 / i g . / m l . = 2 U / m l . N D P K ) :
Dissolve 20 mg. A T P - N a H - 3 H 0 , 8 mg. G - 1 - P - K - 2 H 0 ,
2 2 2 2 2 16 m g . N A D , a n d c a .
30 m g . K H C 0 3 in 9.6 m l . g l y c i n e buffer ( I V ) ; a d d 3 0 0 fil U D P G - D H ( X I I I ) , 50 fil
U D P G P ( X I I ) , a n d 5 0 fil N D P K (XI) and mix.
Keep all solutions a n d suspensions in stoppered containers at 0 - 4 °C. Solution I keeps indefinitely, solutions
II and III for several m o n t h s , a n d solution IV for a b o u t 1 m o n t h at 0 - 4 °C. T h e reagent mixtures X I V
and XV should be freshly p r e p a r e d o n the day of the analysis.
Procedure
Collection of sample: F o r n u c l e o t i d e d e t e r m i n a t i o n s in t i s s u e s , t h e s a m p l e m u s t b e t a k e n w i t h
" q u i c k - f r e e z e " t o n g s (cf. p. 4 0 0 ) .
Deproteinization: A d d a p p r o x . 5 p a r t s b y w e i g h t o f i c e - c o l d p e r c h l o r i c a c i d (I) t o a p i e c e o f
d e e p - f r o z e n tissue ( 0 . 2 - 1 . 0 g.) a n d h o m o g e n i z e i m m e d i a t e l y . A l l o w h o m o g e n a t e t o s t a n d f o r
1 0 - 1 5 m i n . at 4 ° C , t h e n c e n t r i f u g e for 15 m i n . at a b o u t 2 0 , 0 0 0 g ( 0 ° C ) . D e c a n t off t h e s u p e r
n a t a n t a n d adjust t o a p H o f a b o u t 8 w i t h s o l i d K H C O 3 . A f t e r a b o u t 15 m i n . at 0 - 4 ° C ,
centrifuge t o r e m o v e t h e p r e c i p i t a t e d p o t a s s i u m p e r c h l o r a t e a n d u s e t h e s u p e r n a t a n t for t h e
hydrolysis.
Stability of sample: T h e s t a b i l i t y o f t h e n u c l e o s i d e - 5 ' - m o n o p h o s p h a t e r e s i d u e s t o b e d e t e r m i n

e d is m u c h greater in t h e w e a k l y a c i d i c a n d w e a k l y a l k a l i n e r a n g e s t h a n t h a t o f m o s t n u c l e o t i d e s
from which the n u c l e o s i d e - 5 ' - m o n o p h o s p h a t e s are liberated b y hydrolysis. Protein-free neutral
s o l u t i o n s c a n b e u s e d f o r at least 1 w e e k if s t o r e d b e l o w 4 ° C .
Assay System
Hydrolysis of Nucleotides
Pipette into sealable centrifuge tube

C o n c e n t r a t i o n in i n c u b a t i o n
(e. g. w i t h s c r e w c a p ) :
Sample (deproteinized, neutralized) 0.20 ml. u p t o ca. 0.8 m M

5'-nucleotide
Tris buffer (II) 0.50 ml. 137 m M tris
0.7 m M M g 2 +
P D E solution (V) 0.03 ml. 41 /xg./ml. = 2 U / m l .
M i x , i n c u b a t e s e a l e d t u b e f o r 2 0 m i n . at 4 0 ° C , t h e n
p l a c e in a b o i l i n g w a t e r b a t h f o r 5 m i n . C e n t r i f u g e at
a b o u t 2 0 0 0 g f o r a b o u t 10 m i n . a t 4 ° C . U s e 0 . 2 m l . o f
the s u p e r n a t a n t f o r t h e d e t e r m i n a t i o n .
Determination of AMP, GMP, CMP+ UMP
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 0 . 7 0 5 - 0 . 7 3 5 ml.

( s e m i m i c r o c u v e t t e s ) ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t air.
Concentration
in a s s a y m i x t u r e
Reagent mixture (XIV) 0.50 ml. 0.50 ml. 211 m M triethanol

amine
6.3 mMMg 2 +
1 m M PEP
0.26 m M NADH
0.12 m M A T P
21 pg./ml^
12 U L D H / m l .
21 pg./ml =
4.3 U P K / m l .
Water 0.20 ml.
0.20 ml. up to ca. 0.12 m M
5'-nucleotide
Sample (hydrolysate)
M i x , w a i t until e x t i n c t i o n is c o n s t a n t , a n d r e a d E.t
M K suspension (VIII) 0.005 ml. 0.005 ml. 14,ug./mi. = 5 U / m i .
Mix, and w h e n extinction has reached a constant value, read E ; 2
if a c r e e p r e a c t i o n is o b s e r v e d , e x t r a p o l a t e in a c c o r d a n c e w i t h
p. 3 0 8
(E -E )
1 2-(E -E )
S a m p l e 1 2 B l a n k = JE 1 corresponds to the AMP
content o f the sample.
G M P K solution (IX) 0,02 ml. 55 /xg./ml. = 0 . 5 5 U / m l .
Mix, and w h e n extinction has reached a constant value, read E . 3
E - E = z l E corresponds to the G M P content of the sample.

2 3 2
N M P K solution (X) 0.01 m l . 2 0 4 / i g . / m l . = 0.1 U / m l .
M i x , and w h e n extinction has reached a constant value, read E . 4
E - E = zl E c o r r e s p o n d s t o t h e c o n t e n t o f C M P + U M P in t h e
3 4 3
sample.
Calculations
Two moles of N A D H are oxidized per mole of A M P a n d / o r G M P a n d / o r C M P + U M P . Since the assay

was preceded by hydrolysis with P D E , the sums of the c o r r e s p o n d i n g acid-soluble 5'nucleotides (I A M P ,
£ G M P , I ( C M P + U M P ) ) are found. In the analysis of tissue extracts, the result must be multiplied by
the dilution factor due to deproteinization. T h e following relationships are valid for the concentration
in the sample solution before hydrolysis
TAMP c = J E i X 1.055 AE xx 1.034 AE xx 1.892 [jumole/ml.]

I GUP c=AE x 2 1.085 AE x2 1.064 ^ E x 1.946
2
[/imole/ml.]
I (CMP+UMP) c = ^ E x 1.099
3 AE,x 1.078 J E , x 1.973 [/miole/ml.]
Calculation of Z C M P , see below.
Determination of UMP
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 0.71 ml. ( s e m i m i c r o

c u v e t t e s ) ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t air.
Reagent mixture (XV) 0.50 ml. 338 m M glycine

2.7 m M M g 2 +
2.3 m M A T P
1.5 m M G - l - P
1.6 m M N A D
106 Mg/ml. =
63 m U U D P G - D H / m l .
18 Mg./ml. = 1.8 U U D P G P / m l .
18 Mg./ml. = 1.4 U N D P K / m l .
Sample (hydrolysate) 0.20 ml. u p t o ca. 0.15 m M U M P
M i x and read extinction E.x
N M P K solution (X) 0.01 m l . 211 /ig./ml. = 106 m U / m l .
M i x a n d f o l l o w i n c r e a s e in e x t i n c t i o n u n t i l v a l u e b e
c o m e s c o n s t a n t ( a p p r o x . 15 m i n . ) ; t h e n r e a d e x t i n c t i o n
E . E - E = zlE corresponds to the U M P content of
2 2 1
sample.
A t the e n d o f t h e r e a c t i o n , d e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n r e s u l t i n g f r o m the a d d i t i o n o f
the N M P K s o l u t i o n ( X ) b y a further a d d i t i o n a n d s u b t r a c t t h e result f r o m E . It is a l s o p o s s i b l e
2
t o read the e x t i n c t i o n i m m e d i a t e l y after m i x i n g in the N M P K s o l u t i o n ( X ) .
Calculations
Two moles of N A D are reduced per mole of U M P . Since the m e a s u r e m e n t was preceded by hydrolysis
with P D E , the sum of all acid-soluble uracil-5'-nucleotides (I" U M P ) is found. In the analysis of tissue
extracts, the result must be multiplied by the dilution factor due to deproteinization. The following relation
ships are valid for the I U M P concentration in the sample solution before hydrolysis.
ZUMP c = J E x 1.062 ,d E x 1.042 A Ex 1.906 [/miole/ml.]
The concentration of I C M P is calculated as follows:
I (CMP + UMP) - 1 U M P = I CMP.

Solutions of nucleosidediphosphates a n d nucleosidetriphosphates a n d of nucleosidediphosphate sugars

were hydrolysed for multiple d e t e r m i n a t i o n s , a n d the n u c l e o s i d e - 5 ' - m o n o p h o s p h a t e residue was analysed
as described. T h e recovery after hydrolysis was 9 8 - 1 0 4 % . T h e coefficients of variation of the determin
ations were less t h a n 2 % when 8 5 - 1 1 0 n m o l e of 5'-nucleotide was used for the hydrolysis.
Normal Values
The sums of all acid-soluble adenine, guanine, cytosine, a n d uracil 5'-nucleotides were determined in
rat liver; the contents (//mole per g. of liver ± s t a n d a r d deviation) a r e : 2 J A M P 3.90 + 0.38 (n = 1 8 ) ; 3
£ G M P 0.47 + 0.06 ( n - 1 2 ) ' ; C M P 0.25 + 0.02 ( n = 4 ) ; I U M P 1.24 + 0.06 (n = 3 6 ) ' . In rat brain, a
4 7 6 3 7
value of 3.23 + 0.27 /miole per g. was found for T A M P , a n d 0.52 + 0.10 /miole per g. for T U M P ; in rat
skeletal muscle, I AMP is 7.51 ± 0.44 a n d T U M P is 0.27 + 0.03 /miole per g . . 3
C o n t a m i n a t i o n of the P D E with 5'-nucleotidase o r p h o s p h a t a s e s m a y lead t o loss of nucleoside-5'-

m o n o p h o s p h a t e s ; this can be checked by addition of a s t a n d a r d (e.g. U D P - g l u c o s e or A T P ) , which
also indicates the completeness of t h e hydrolysis. C o n t a m i n a t i o n of the reagents with pyruvate o r A M P ,
or an unusually high c o n t e n t of adenine-5'-nucleotides in the sample, m a y lead to an excessively low
result for the c o n c e n t r a t i o n of N A D H ; in this case, N A D H is a d d e d again before the addition of G M P K
or N M P K .
T h e nucleotide contents in t h e liver are greatly increased by a d m i n i s t r a t i o n of the corresponding nucle
osides. Uridine doubles the I U M P value in 1 h o u r , a n d o r o t a t e , ureidosuccinate, a n d c a r b a m o y l p h o s p h a t e ,
and even D-galactosamine, also lead t o an i n c r e a s e . D - F r u c t o s e causes a decrease in Z A M P , I G M P ,
7
2 X M P , a n d T U M P in the liver.
Spe c ific ity o f M e t h o d
T h e enzymes used allow a specific d e t e r m i n a t i o n of the s u m of adenine, g u a n i n e , a n d uracil 5'-nucleotides;

however, the c o r r e s p o n d i n g 2 ' - d e o x y - 5 ' - n u c l e o s i d e m o n o p h o s p h a t e s also r e a c t 3 , 1 0
. T h e m e t h o d is largely
specific for c y t o s i n e - 5 ' - n u c l e o t i d e s , a n d c h r o m a t o g r a p h i c separation of C M P before its d e t e r m i n a t i o n
11 6
is unnecessary.
T h e m e t h o d described can be used for the enzymatic d e t e r m i n a t i o n of individual n u c l e o t i d e s 5 - 7

. For
the analysis of these nucleotides in tissue extracts, previous c h r o m a t o g r a p h i c separation of 5'-nucleotides
having the same base residue, possibly in c o m b i n a t i o n with isotope dilution, is necessary. This m e t h o d
has been used a n d described for t h e d e t e r m i n a t i o n of the following nucleotides (in rat liver): U D P -
hexosamines, U D P - N - a c e t y l h e x o s a m i n e s , U D P - g l u c u r o n a t e , C T P , C D P - c h o l i n e , a n d G T P .
5 6 7
References
1 W. E. Razzell in S. P. Colowick & N. O. Kaplan: M e t h o d s in E n z y m o l o g y , A c a d e m i c Press, N e w York

1963, Vol. VI, p . 236.
2 W. E. Razzell & H. G. Khorana, J. biol. C h e m . 234, 2105 [1959].
3 D. Keppler, J. Rudiger & K. Decker, Analyt. Biochem. 38, 105 [1970].
4 D. Keppler & K. Decker, Scand. J. clin. L a b . Invest. 29, Suppl. 126, 30. 8 [1972].
5 D. Keppler, J. Rudiger, E. Bischoff& K. Decker, E u r o p e a n J. Biochem. 17, 246 [1970].
6 W. Domschke, D. Keppler, E. Bischoff & K. Decker, Z, Physiol. C h e m . 352, 275 [1971].
7 D. Keppler, J. Pausch & K. Decker, J. biol. C h e m . 249, 211 [1974].
8 H. Adam in H. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag Chemie, Weinheim/
BergstraBe, 1st. edn., 1962, p . 577.
9 R. P. Miech & R. E. Parks, Jr., J. biol. C h e m . 240, 351 [1965].
10 M. Grassl in H. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag Chemie, Weinheim/
BergstraBe, 2nd edn., 1970, p . 2084.
11 M. Grafil in H. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag Chemie, Weinheim/
BergstraBe, 2nd edn., 1970. p . 2075.
12 D. Keppler, K. Gawehn & K. Decker in H. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse.
Verlag Chemie, Weinheim/BergstraBe, 2nd edn., 1970, p . 2094.
Adenosine-5 -triphosphate
Of all the naturally occurring p h o s p h a t e s adenosine-5'-triphosphate is the m o s t widely distributed. As an
energy-rich p h o s p h a t e it is formed in energy-yielding reactions by p h o s p h o r y l a t i o n of adenosine-5'-
d i p h o s p h a t e a n d in energy-consuming reactions it is reconverted to a d e n o s i n e - 5 ' - d i p h o s p h a t e with the
liberation of energy. T h e energy-rich nucleotides serve as an "energy p o o l " t o meet the energy require
ments of cellular metabolism.
In pathological situations where m e t a b o l i s m is altered the d e t e r m i n a t i o n of A T P is of diagnostic value a n d
is increasingly used in heart, liver a n d kidney diseases.
T h e enzymatic d e t e r m i n a t i o n of A T P with P G K or H K is preferable to the existing c h r o m a t o g r a p h i c
m e t h o d s , especially with serial m e a s u r e m e n t s , because of the simplicity a n d speed of the assay.
Determination with 3-Phosphoglycerate Kinase

Dieter Jaworek, Wolfgang Gruber and Hans Ulrich Bergmeyer
The enzymatic d e t e r m i n a t i o n of adenosine-5'-triphosphate with 3-phosphoglycerate kinase, P G K ( A T P :

3-phospho-D-glycerate-l-phosphotransferase, E C 2.7.2.3) was first described by Biicher . 1
T h e 1,3-di-
phosphoglycerate formed in the reaction is determined by the indicator reaction with glyceraldehyde-3-
p h o s p h a t e dehydrogenase, G A P D H (D-glyceraldehyde-3-phosphate: N A D oxidoreductase, p h o s p h o r y l a t -
ing, E C 1.2.1.12).
Principle
(1) Glycerate-3-phosphate + A T P , P G K
- Glycerate-1,3-P 2 + ADP
, I
r
(2) Glycerate-1,3-P + N A D H + H
2
+
Glyceraldehyde-3-P + N A D +
+ Pi
The oxidation of N A D H , as measured by the change of extinction at 340 (334, 365) n m , is p r o p o r t i o n a l

the a m o u n t of A T P present.
The equilibrium of reaction (1) is to the left, but t h a t of reaction (2) is to the r i g h t ; s a t u r a t i o n of P G K
1
with glycerate-3-phosphate results in a sufficiently rapid formation of g l y c e r a t e - l , 3 - d i p h o s p h a t e and its

further reaction in e q u a t i o n (2).
Equipment
3 3 4 o r 365 n m ; b e n c h c e n t r i f u g e .
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 8. P h o s p h o g l y c e r a t e k i n a s e , P G K
2. P o t a s s i u m c a r b o n a t e , A . R . from yeast, crystalline suspension in 3.2 M
3. M a g n e s i u m s u l p h a t e , M g S 0 - 7 H 0 , 4 2
a m m o n i u m sulphate solution: ^ 400 U / m g .
A . R. (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 502.
4. Ethylenediaminetetra-acetate 9. G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e
d i s o d i u m salt, E D T A - N a H 2 2 2H 0 2 nase, G A P D H
5. Glycerate-3-phosphate from rabbit muscle, crystalline suspension in
tricyclohexylammonium salt. C o m m e r c i a l prep 3.2 M ammonium sulphate solution: ^80
aration, see p . 540. U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , see
6. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o p . 466.
tide, N A D H 10. P e r c h l o r i c a c i d , A . R . , 70% (w/w), sp.
disodium salt N A D H - N a . C o m m e r c i a l p r e p
2 gr. 1.67
a r a t i o n , see p . 545. 1 1 . T r i c h l o r o a c e t i c a c i d , A . R.
7. S o d i u m h y d r o g e n c a r b o n a t e , A . R . 12. P o t a s s i u m h y d r o x i d e , A . R .
I. T r i e t h a n o l a m i n e b u f f e r / g l y c e r a t e - 3 - p h o s p h a t e (0.1 M buffer, p H 7 . 6 ; 6 m M g l y c e r a t e - 3 -
phosphate, 4 m M M g S 0 ) : 4
D i s s o l v e 9 3 0 m g . triethanolamine hydrochloride, 169.5 m g . K C 0 , 50 m g . 2 3 MgS0 - 4
• 7 H 0 , 2 0 m g . E D T A - N a H H 0 a n d 165 m g . g l y c e r a t e - 3 - p h o s p h a t e in distilled w a t e r
2 2 2 2
and m a k e u p to 50 ml.
II. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 1 4 m M / ? - N A D H ) :
D i s s o l v e 12 m g . N A D H - N a 2 i n 1 m l . 5% N a H C 0 3 solution.
III. P h o s p h o g l y c e r a t e k i n a s e / g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e ( 2 m g . P G K / m l . ;
10 m g . G A P D H / m l . ) :
Dilute the stock suspensions accordingly with a m m o n i u m sulphate solution. Pipette
0.6 m l . P G K a n d 0 . 4 m l . G A P D H t o g e t h e r a n d m i x .
IV. Perchloric acid:
a) 0.2 M : D i l u t e 1.7 m l . p e r c h l o r i c a c i d t o 100 m l . w i t h d i s t i l l e d w a t e r .
b ) 0 . 9 M : D i l u t e 7.7 m l . p e r c h l o r i c a c i d t o 100 m l . w i t h d i s t i l l e d w a t e r .
V. Trichloroacetic acid (3 M ) :
D i s s o l v e 4 9 g. t r i c h l o r o a c e t i c a c i d i n d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
VI. Potassium hydroxide (2 M ) :
D i s s o l v e 1 1 . 2 g. K O H in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
VII. Potassium carbonate (3.75 M ) :
D i s s o l v e 4 3 . 4 g. K C 0
2 3 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
Store all solutions, stoppered, in a refrigerator at 0 - 4 °C. Solutions I, IV, V a n d VI are stable indefinitely;
the separate enzyme suspensions are stable for ca. one year. P r e p a r e the N A D H solution freshly each week.
Adenosine-5 '-triphosphate 2099
Procedure
D r o p b l o o d d i r e c t l y f r o m a v e i n i n t o stirred p e r c h l o r i c a c i d . C o l l e c t t i s s u e s w i t h f r e e z e - s t o p
t o n g s (see " C e l l a n d T i s s u e D i s i n t e g r a t i o n " , p . 4 0 0 ) .
Deproteinization :
B l o o d : In c o n t r a s t t o t h e d e p r o t e i n i z a t i o n o f t i s s u e s ( s e e b e l o w ) t h e u s e o f p e r c h l o r i c a c i d f o r
t h e d e p r o t e i n i z a t i o n o f b l o o d h a s n o t p r o v e d s u i t a b l e . W i t h p e r c h l o r i c a c i d t h e r e is r a p i d
h y d r o l y s i s o f t h e A T P i n b l o o d e v e n if t h e b l o o d is d r o p p e d d i r e c t l y i n t o t h e p e r c h l o r i c a c i d .
B y i m m e d i a t e c e n t r i f u g a t i o n o f t h e p r e c i p i t a t e d b l o o d t h e s u p e r n a t a n t fluid o n l y c o n t a i n s
c a . 7 0 % o f t h e A T P p r e s e n t , t h e o t h e r 3 0 % is f o u n d a s A D P a n d A M P . A n o t e w o r t h y o b s e r v a t
i o n is t h e finding t h a t t h i s r a p i d h y d r o l y s i s o f A T P a l s o o c c u r s if t h e p r e c i p i t a t e is w a s h e d
several t i m e s w i t h p e r c h l o r i c a c i d a n d t h e n A T P is a d d e d t o a s u s p e n s i o n o f t h e p r e c i p i t a t e in
p e r c h l o r i c a c i d . T h e r e is n o significant h y d r o l y s i s o f A T P i n t h e c l e a r s u p e r n a t a n t fluid. O n
deproteinization with trichloroacetic acid the rapid d e c o m p o s i t i o n o f A T P by constituents o f
t h e b l o o d c a k e is n o t o b s e r v e d .
P i p e t t e s u c c e s s i v e l y i n t o a c e n t r i f u g e t u b e 1 m l . o f freshly c o l l e c t e d b l o o d a n d 2 m l . i c e - c o l d
trichloroacetic acid solution (V). M i x thoroughly with a thin glass rod and immediately
c e n t r i f u g e for 10 m i n . at c a . 3 0 0 0 r p m . P i p e t t e off 2 m l . o f t h e s u p e r n a t a n t fluid, n e u t r a l i z e
w i t h 2 N K O H ( V I ) a n d d i l u t e w i t h d i s t i l l e d w a t e r t o a final v o l u m e o f 5 m l . ; t a k e 0 . 4 m l . o f
this solution for the assay.
Tissue: Powder frozen t i s s u e 2 , 3
in a p r e - c o o l e d mortar. In a s e c o n d mortar deep-freeze m o r e
than d o u b l e the tissue weight o f 0.9 M perchloric acid s o l u t i o n ( I V b ) a n d p o w d e r with c o o l i n g .
W e i g h t h e t i s s u e p o w d e r (1 p a r t b y w t . ) a n d t h e a c i d ( 2 p a r t s b y w t . ) i n t h e f r o z e n s t a t e , m i x i n
a d e e p - c o o l e d mortar and grind. A l l o w the p o w d e r e d mixture to s l o w l y w a r m u p from —18 °C
t o 0 ° C o v e r a p e r i o d o f 2 hr. T h e n h o m o g e n i z e a n d c e n t r i f u g e f o r 1 0 m i n . at 2 ° C a n d 3 0 0 0 g.
E x t r a c t t h e s e d i m e n t w i t h 0 . 2 M p e r c h l o r i c a c i d ( I V a) u s i n g a t h i r d o f t h e v o l u m e o f p e r c h l o r i c
a c i d u s e d for t h e first e x t r a c t i o n . A d j u s t t h e c o m b i n e d s u p e r n a t a n t fluids t o p H 6 . 0 - 6 . 5 w i t h
2 M K O H or 3.75 M K C 0 2 3 ( p r e f e r a b l e w i t h v i s c o u s e x t r a c t s ) a n d c a r e f u l , r a p i d stirring.
A l l o w t o s t a n d f o r 1 hr. i n a n i c e b a t h , r e m o v e t h e p r e c i p i t a t e o f K C 1 0 4 and use the solution
for the a s s a y .
A n y c h a n g e o f the physiological state causes i m m e d i a t e c h a n g e s in the A T P content. Therefore

a l l o w b l o o d t o d r o p d i r e c t l y i n t o t h e a c i d , freeze o r g a n s in situ w i t h d e e p - c o o l e d m e t a l b l o c k s
and store at — 30 ° C until w o r k e d up.
T h e A T P c o n t e n t o f b l o o d e x t r a c t s is m a i n t a i n e d at — 3 0 ° C f o r at l e a s t 8 d a y s . 4
Assay System

ature. R e a d a g a i n s t b l a n k .
assay mixture
Buffer/glycerate-3-P (I) 2.5 ml. 2.5 ml. 5.05 m M g l y c e r a t e -

3-phosphate;
3.33 m M M g S 0 ; 4
0.9 m M E D T A
N A D H solution (II) 0.05 ml. 0.05 ml. 0.23 m M NADH
neutralized) 0.4 ml. u p to 0.08 m M A T P
M i x and read E . x
P G K / G A P D H suspension (III) 0.05 ml. 0.05 ml. 8 U PGK/ml.

5 U GAPDH/ml.
M i x , after 5 m i n . r e a d e x t i n c t i o n E 2
AE = (E l - ^2)sample
- (E t - E )
2 b l 9 n k .
F o r an a c c u r a t e e n d - p o i n t d e t e r m i n a t i o n t a k e 3 - 5 further r e a d i n g s at 1 m i n . intervals a n d
extrapolate E 2 t o t h e t i m e o f a d d i t i o n o f s u s p e n s i o n III.
Calculations
(2) on p . 312 applies. This value m u s t be multiplied by a factor if the sample has been deproteinized,
neutralized or diluted in any way.
In the case of b l o o d the specific gravity (ca. 1.06) a n d the water content (ca. 80%) must be t a k e n into account.
F o r this m e t h o d the factor for blood is 7.12 (see p. 315) and therefore the following relationships hold
for the A T P concentration in blood.

c = AE x 8.75 z l E x 8.59 AE x 15.71 foimole/ml.]
c = AE x 4.44 AE x 4.35 AE x 7.97 [mg./ml.]
With a mean value of 0.447 mg. A T P / m l . blood a s t a n d a r d deviation of 0.0013 mg. A T P / m l . was found.
The coefficient of variation is 3 % .
N o r m a l Values
T h e published n o r m a l values in b l o o d are 1 0 - 2 6 mg/100 m l . ' , 1 9 - 3 2 mg./lOO m l . a n d 3 0 - 5 4 mg./

5 6 7
100 m l . . T h e values vary considerably even for the same individual over a period of m o n t h s . T h e values
8
for rat uterus have been given as 0 . 9 5 - 1 . 6 5 /miole/g. . 9

Interference in the assay technique: Insufficient purity of the reagents, especially the enzymes (presence of
ATPases) results in low A T P values.
I T P , G T P and U T P also react with P G K , but C T P does n o t . T h e rates of the reactions are not very
1 0
different . These t r i p h o s p h a t e s occur only in very low concentration in b l o o d .

11
References
1 Th. Bucher, Biochem. Biophys. Acta / , 292 [1947].

2 H. Adam, Biochem. Z. 325, 25 [1961].
3 H. Adam in H.-U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag C h e m i e , Weinheim,
BergstraBe, 1st edn. 1962, p . 541.
4 G. Michal, unpublished results.
5 H. A. Hoetzl, Arztl. Wochenschrift 13, 850 [1958].
6 G. Laudahn, Klin. Wochenschrift 37, 850 [1959].
7 H. Dennemann, Z. Ges. Exp. M e d . 134, 335 [1961].
8 M. V. Buell, J. biol. C h e m . 108, 273 [1935].
9 P. M. Carrol & J. C. Graham, C a n a d . J. Biochem. 44, 529 [1966].
10 W. Gruber, H. Mollering & H.-U. Bergmeyer, Enzym. Biol. Clin. 7, 115 [1966].
11 A. Kornberg, J. biol. C h e m . 182, 805 [1950].
Determination with Hexokinase and G lucose-6-phosphate Dehydrogenase

W a l t h e r L a m p r e c h t a n d Ivar T r a u t s c h o l d
The enzymatic d e t e r m i n a t i o n of adenosine t r i p h o s p h a t e ( A T P ) by the s p e c t r o p h o t o m e t r i c m e t h o d invol

ving pyridine nucleotides has c o m e into general use because of its simplicity c o m p a r e d with paper or c o l u m n
c h r o m a t o g r a p h i c m e t h o d s , especially over a series of analyses. T h e question of the specificity of the enzymes
used m u s t be examined for w o r k o n special p r o b l e m s . In recent years, in a d d i t i o n to the m e t h o d of Bucher
et al. (see p . 2097), the use of hexokinase a n d glucose-6-phosphate d e h y d r o g e n a s e , G 6 P - D H , (discovered
by O. Warburg et a l . 1 - 3
) has p r o v e d of value in the enzymatic d e t e r m i n a t i o n of A T P . 4
Hexokinase, H K ( A T P : D-hexose 6-phosphotransferase, E C 2.7.1.1) p h o s p h o r y l a t e s glucose* with A T P

in the presence of M g 2 +
to glucose-6-phosphate*, G - 6 - P " . G 6 P - D H ( D - G l u c o s e - 6 - p h o s p h a t e : N A D P 1-
5 7
oxidoreductase, E C 1.1.1.49) catalyses the d e h y d r o g e n a t i o n of G-6-P with N A D P to give 6-phosphoglu-

cono-<5-lactone**.
* Pyranose form = a - D - ( + ) - g l u c o p y r a n o s e o r a - D - ( + ) - g l y c o p y r a n o s e - 6 - p h o s p h a t e ( " R o b i s o n - e s t e r " )

(cf. C. S. Hudson, Advances in C a r b o h y d r a t e Chemistry 3, 1 [1948]).
** The lactone is hydrolysed to the free carboxylic acid if the hexokinase or glucose-6-phosphate d e h y d r o
genase contains g l u c o n o - ^ - l a c t o n a s e ' . 8 9
Principle
(1) A T P + Glucose — — • G-6-P + A D P
(2) G-6-P + N A D P + G 6 P
~ D H
> 6-Phosphoglucono-<5-lactone + N A D P H -f H +
F o r each mole of A T P , therefore, 1 mole of N A D P H is f o r m e d ; this p r o d u c t is determined by m e a s u r e m e n t

of the extinction at 340 (334 o r 365) n m .
A reversal o f the e x t i n c t i o n c h a n g e is o f t e n o b s e r v e d after 2 0 - 2 5 m i n . T h i s is d u e t o n o n - s p e c i f i c
b r e a k d o w n o r o x i d a t i o n o f t h e N A D P H ; it d o e s n o t interfere w i t h t h e d e t e r m i n a t i o n o f A E A T P .
W i t h very p u r e H K a n d G 6 P - D H p r e p a r a t i o n s , c o n t a m i n a t e d o n l y e x t r e m e l y slightly w i t h
p h o s p h o g l u c o s e isomerase ( P G I ) a n d p h o s p h o g l u c o m u t a s e ( P G l u M ) , the extinction difference
E 2 - E ! is d u e a l m o s t e x c l u s i v e l y t o g l u c o s e - 6 - p h o s p h a t e (AE _ _ ), G 6 P w i t h o n l y very s m a l l
contributions from fructose-6-phosphate and glucose-1-phosphate.
If F - 6 - P a n d G - l - P are p r e s e n t in h i g h e r c o n c e n t r a t i o n s t h a n , for e x a m p l e , in s e r u m , t h e a d d i t i o n
o f P G I a n d P G l u M ( c o m m e r c i a l p r e p a r a t i o n s , s e e p . 5 0 1 , 4 9 9 ) a s w e l l as G 6 P - D H is n e c e s s a r y
for t h e d e t e r m i n a t i o n o f all h e x o s e m o n o p h o s p h a t e s .
T h e s e n s i t i v i t y o f t h e A T P d e t e r m i n a t i o n c a n b e d o u b l e d if r e a c t i o n s (1) a n d (2) ( a b o v e ) are
coupled with the 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e ( 6 - P G D H ) reaction (3).
(3) 6-Phosphogluconate + N A D P +
- > Ribulose-5-P + N A D P H + H +
+ C0 2
2 m o l e s o f N A D P H are t h e n f o r m e d p e r m o l e o f A T P .
W i t h suitable glucose a n d M g 2 +
concentrations A T P is virtually quantitatively converted to A D P by hexo
k i n a s e . W h e n the enzyme is saturated with substrate, 13000 mole glucose/10 g. enzyme are esterified per
10 5
minute (30 ° C ; p H 7 . 5 ) . T h e values given for the Michaelis c o n s t a n t v a r y : values of 1.5 x 1 0 " M , 1 x
11 4 1 2
10 _ 3
M 1 3
a n d 5 x 10" M 4 1 4 1 5
have been found for glucose, 9.5 x 1 0 " M 5 1 2
a n d 1.2 x 1 0 " M 3 1 6
for A T P
(glucose), and 2.6 x 1 0 ~ M 3 1 6
for M g * * . M o r e recent m e a s u r e m e n t s give the K
2
M for glucose as
0.31 x 1 0 ~ M and for A T P 0.33 x 1 0 " M . T h e p H o p t i m u m of hexokinase (yeast) is 8 - 9
7 6 1 7 1 5 1 6
; for
dilute enzyme solutions in 50 m M tris buffer the o p t i m u m has been found to be p H 8 . 4 . 17
T h e equilibrium of the glucose-6-phosphate dehydrogenase reaction lies in favour of the gluconolactone.

With sufficient substrate, 1 mole of enzyme converts 12000 mole substrate/min. at 25 °C and p H 8**.
The values for the Michaelis constants* are 1.76 x 1 0 " M o r 0 . 6 9 x 1 0 4 2 0 - 4
M 2 1
for G - 6 - P a n d 2 . 6 x 1 0 " 5
M 2 0
or 3.3 x 1 0 " M 5 2 1
for N A D P ; the p H o p t i m u m is 8 . 5 ' . F o r further data, see
1 9 2 0 2 2
" 2 4
.
Equipment
3 3 4 , or 365 n m .
* In the hexokinase reaction (two substrate reaction) the value for K d e p e n d s o n the concentration of M
b o t h s u b s t r a t e s , a n d the different m e a s u r e m e n t s consequently c a n n o t be c o m p a r e d directly.

20
** T h e exergonic hydrolysis of the lactone gives virtually a quantitative yield of g l u c o n a t e . 18

Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 6. H e x o k i n a s e , H K
2. N i c o t i n a m i d e - a d e n i n e dinucleotide from yeast, crystalline suspension in 3.2 M
phosphate, N A D P ammonium sulphate solution; ^ 140 U / m g .
disodium salt, N A D P - N a H ; commercial p r e p
2 (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p . 473.
aration, see p . 546. 7. P e r c h l o r i c a c i d , A . R .
3. M a g n e s i u m c h l o r i d e , M g C l 2 6 H 0 , A . R.
2 sp. gr. 1.67; ca. 70% (w/w) o r sp.gr. 1.53; ca.
4. G l u c o s e , A . R., C H 6 1 2 0 6 H 0 2 60% (w/w).
5. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , 8. P o t a s s i u m c a r b o n a t e , K C 0 , A . R .
2 3
G6P-DH 9. M e t h y l o r a n g e ( i n d i c a t o r )
from yeast, crystalline suspension in 3.2 M a m
m o n i u m sulphate s o l u t i o n ; ^ 140 U / m g . (25°C).
C o m m e r c i a l p r e p a r a t i o n , see p. 458.
Hexokinase must contain less t h a n 0.02% of M K , A T P a s e , G 6 P - D H , 6 - P G D H , P G l u M , a n d G R (based

on the specific activity of H K ) a n d only traces of C K a n d P G I . These c o n d i t i o n s are largely satisfied by the
commercial p r e p a r a t i o n s p r o d u c e d by Boehringer M a n n h e i m .
Glucose-6-phosphate d e h y d r o g e n a s e : the c o n t a m i n a t i n g activities indicated for H K should n o t be
exceeded. T h e content of H K should be less t h a n 0 . 1 % . This condition is satisfied by the commercial
p r e p a r a t i o n of purity grade I.
Preparation of Solutions (for ca. 25 determinations)
P r e p a r e all s o l u t i o n s w i t h f r e s h , d o u b l y d i s t i l l e d w a t e r .
I. T r i e t h a n o l a m i n e buffer ( 5 0 m M ; p H 7 . 5 - 7 . 6 ) :
D i s s o l v e 4 . 6 5 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in c a . 2 0 0 m l . d i s t i l l e d w a t e r , a d d 11 m l .
1 N N a O H a n d after c o o l i n g d i l u t e t o 5 0 0 m l . w i t h d i s t i l l e d w a t e r .
II. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e (ca. 10 m M N A D P ) :
D i s s o l v e 2 7 m g . N A D P - N a H in 3 . 0 m l . d i s t i l l e d w a t e r .
2
III. M a g n e s i u m c h l o r i d e (0.1 M ) :
D i s s o l v e 2 . 0 3 g. M g C l - 6 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
2 2
IV. G l u c o s e (0.5 M ) :
D i s s o l v e 9.91 g. g l u c o s e ( C H 6 1 2 0 6 • H 0 ) in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
2
V . G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , G 6 P - D H (1 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n , if n e c e s s a r y , w i t h 3 . 2 M a m m o n i u m s u l p h a t e s o l u t i o n .
VI. H e x o k i n a s e , H K (2 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n , if n e c e s s a r y , w i t h 3 . 2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V I I . P e r c h l o r i c a c i d (ca. 6 % w / w ) :
D i l u t e 5.2 m l . H C 1 0 , s p . gr. 1.67, t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r ; o r d i l u t e 6.6 m l . H C 1 0 ,
4 4
s p . gr. 1.53, t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r .
VIII. P o t a s s i u m c a r b o n a t e (ca. 5 M K C0 ):
2 3
D i s s o l v e c a . 7 2 g. K C 0 2 3 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
IX. Methyl orange indicator:
D i s s o l v e c a . 5 0 m g . in 1 0 0 m l . d i s t i l l e d w a t e r .
Store all solutions, stoppered, in a refrigerator at 1 - 4 °C. Prepare fresh N A D P a n d glucose solutions each
week. Enzyme suspensions are stable for several m o n t h s .
Procedure
Collection o f b l o o d and separation o f the c o m p o n e n t s as described o n p. 1448. Plasma normally

c o n t a i n s n o A T P a n d t h e A T P o f b l o o d is l o c a t e d in t h e e r y t h r o c y t e s 4 , 2 6
. Hexose monophos
p h a t e s o c c u r in traces in w h o l e b l o o d ( r a t ) . F o r t h e d e t e r m i n a t i o n o f A T P in t i s s u e s it is
4
essential that t h e s a m p l e s a r e f r o z e n w i t h i n a f r a c t i o n o f a s e c o n d . Wollenberger 25

and Bucket 26
h a v e s u c c e s s f u l l y u s e d " t o n g s " m a d e o f a l u m i n i u m o r light m e t a l b l o c k s , w h i c h w e r e c o o l e d

w i t h l i q u i d air. It is w e l l k n o w n t h a t t h e m e t a b o l i t e c o n c e n t r a t i o n in a t i s s u e d e p e n d s o n t h e
s p e e d w i t h w h i c h t h e o r g a n is r e m o v e d a n d o n t h e m a n i p u l a t i o n s d u r i n g t h e k i l l i n g o f the a n i m a l
(ether n a r c o s i s , d e c a p i t a t i o n , " q u i c k - f r e e z i n g " , e t c . ) .
Deproteinization:
T h e s a m p l e is d e p r o t e i n i z e d w i t h p e r c h l o r i c a c i d s o l u t i o n . T h e r a t i o o f t o t a l fluid v o l u m e t o
o r i g i n a l w e i g h t o f o r g a n s h o u l d b e 4 : 1. T h e a m o u n t o f p e r c h l o r i c a c i d t h u s d e p e n d s o n t h e
water content o f the sample.
W h o l e b l o o d is d e p r o t e i n i z e d a c c o r d i n g t o t h e m e t h o d o f Bticher ( s e e p . 1 3 1 6 ) . V a r i a t i o n o f t h e
ratio o f p e r c h l o r i c a c i d t o b l o o d f r o m 4 : 1 results in i n c o r r e c t v a l u e s f o r t h e A T P c o n t e n t . F o r
e x a m p l e : W h o l e b l o o d w a s d e p r o t e i n i z e d w i t h p e r c h l o r i c a c i d in t h e r a t i o 1 : 1 ( v / v ) a n d
c e n t r i f u g e d for 10 m i n . at 3 0 0 0 r p m at 2 ° C , y i e l d i n g s u p e r n a t a n t I. T h e large, g e l a t i n o u s ,
residual p r e c i p i t a t e r e t a i n e d v a r y i n g a m o u n t s o f A T P ; w a s h i n g this r e s i d u e t h r e e t i m e s w i t h
4 ml. portions 6 % perchloric acid, with h o m o g e n i z a t i o n and centrifugation, yielded supernatants
II-IV:
A m o u n t o f A T P in supernatant I 8 . 3 5 mg./lOO m l .
supernatant II 6.75 mg./lOO ml.
s u p e r n a t a n t III 2.50 mg./lOO ml.
supernatant IV 0.79 m g . / l 0 0 ml.
Total 18.39 mg./lOO ml.
In c o m p a r i s o n , t h e A T P c o n c e n t r a t i o n in s u p e r n a t a n t I, c a l c u l a t e d f o r w h o l e b l o o d , w o u l d b e
1 6 . 7 m g . / 1 0 0 ml.
Yeast c e l l s : T h e s e are o n l y p a r t i a l l y l y s e d o n d e p r o t e i n i z a t i o n w i t h p e r c h l o r i c a c i d o r t r i c h l o r o
acetic a c i d . T h e f o l l o w i n g d e p r o t e i n i z a t i o n a n d d i s i n t e g r a t i o n m e t h o d h a s p r o v e d s u c c e s s f u l .
2 7 27
D i s i n t e g r a t e cells a c c o r d i n g t o Merkenschlager et a l . * w i t h a " m e c h a n i c a l cell h o m o g e n i z e r "

2 8
in 3 0 m l . D u r a n t u b e s * w i t h g l a s s b e a d s (size 3 1 / 8 ) * .
* B. Braun, Melsungen ( G e r m a n y ) .
P i p e t t e 2 0 m l . o f a 5 . 4 % y e a s t s u s p e n s i o n ( p r e s s e d , fresh y e a s t ) i n t o a m i x t u r e o f 2.0 m l . 6 0 %
p e r c h l o r i c a c i d a n d 2 0 m l . g l a s s b e a d s , w h i c h h a s b e e n p r e v i o u s l y c o o l e d in t h e h o m o g e n i z e r
t u b e s w i t h ice w a t e r . I m m e d i a t e l y s h a k e v i g o r o u s l y a n d h o m o g e n i z e for 3 0 s e c o n d s at a f r e q u e n
cy o f c a . 4 0 0 0 / m i n . w i t h o u t further c o o l i n g ( t e m p e r a t u r e o f t h e h o m o g e n a t e after 30 sec. is c a .
12 ° C ) ; t h e n c e n t r i f u g e in t h e c o l d at ca. 4 0 0 0 g.
O r g a n or t i s s u e s a m p l e s are q u i c k l y p r e s s e d b e t w e e n t h e " j a w s " o f t h e " q u i c k - f r e e z e " t o n g s ,
w h i c h h a v e b e e n c o o l e d w i t h l i q u i d air. C u t o r b r e a k off a n y p o r t i o n s o f t h e s a m p l e p r o j e c t i n g
o v e r t h e e d g e s o f t h e b l o c k s a n d o n c e a g a i n i m m e r s e in l i q u i d air. Q u i c k l y w e i g h t h e s a m p l e *
(g. t i s s u e = V ) a n d p o w d e r in a p o r c e l a i n m o r t a r w i t h r e p e a t e d a d d i t i o n s o f l i q u i d air ( c a .
x
10 m l . p o r t i o n s ) . T a k e c a r e t h a t t h e p i e c e o f t i s s u e d o e s n o t t h a w at a n y t i m e d u r i n g t h e m a n i
pulations. Slowly add the calculated a m o u n t of perchloric acid (6.5 ml. H C 1 0 4 t o 2 g. t i s s u e ;
V x + g. H C 1 0 4 = V ) and grind with the tissue to form a p o w d e r .
2
H o m o g e n i z a t i o n : e i t h e r a) a l l o w t i s s u e p o w d e r in m o r t a r t o w a r m u p in c o l d r o o m u n t i l
t e m p e r a t u r e r e a c h e s c a . 2 - 4 ° C , a n d t h e n h o m o g e n i z e for 30 seconds**, o r b) after e v a p o r a t i o n
o f t h e l i q u i d air, q u i c k l y transfer t h e still dry p o w d e r i n t o a h o m o g e n i z e r t u b e , r e m o v i n g the last
traces w i t h a s m a l l r u b b e r p o l i c e m a n o r p l a s t i c s p a t u l a ; w h e n t h e m i x t u r e is j u s t b e c o m i n g
fluid, h o m o g e n i z e for 3 0 s e c o n d s , w h i l e c o o l i n g in ice.
1
Take p o r t i o n s (2 t o 4 m l . ) ( = V ) o f t h e c e n t r i f u g e d p e r c h l o r i c a c i d e x t r a c t for d u p l i c a t e
3
d e t e r m i n a t i o n s . E i t h e r titrate w i t h 5 M K C 0 2 3 s o l u t i o n ( V I I I ) u s i n g a 0.2 m l . c a p i l l a r y p i p e t t e 2 6
a n d m e t h y l o r a n g e ( s o l u t i o n I X ) a s i n d i c a t o r , w h i l e stirring m a g n e t i c a l l y o r b u b b l i n g n i t r o g e n
t h r o u g h , o r a d j u s t t o p H 7.4 b y m e a n s o f t h e m o r e s e n s i t i v e e n d - p o i n t t i t r a t i o n u s i n g a n
"automatic Titrigraph" +
(V 3 + ml. K C 0 2 3 required = V ) . A total o f a b o u t 0 . 1 2 - 0 . 1 5 ml.
4
c a r b o n a t e s o l u t i o n is r e q u i r e d . A l l o w t h e n e u t r a l i z e d s o l u t i o n t o s t a n d for 10 m i n . in i c e
water, decant from the sediment o f K C 1 0 4 a n d i m m e d i a t e l y a n a l y s e 0.1 m l . o r 0.2 m l . ( = V ) . 5
Stability of sample: A T P is h y d r o l y s e d o n s t a n d i n g in s o l u t i o n . S t o r a g e at 0 ° C , freezing, o r

l y o p h i l i z a t i o n g e n e r a l l y l e a d s t o a d e c r e a s e in t h e A T P v a l u e s .
Assay System
T h e v o l u m e o f s a m p l e is s o c h o s e n t h a t t h e m a x i m u m e x t i n c t i o n c h a n g e at 365 n m is 0 . 1 5 0 a n d
t h e a s s a y is c o m p l e t e in 15 m i n .
W a v e l e n g t h : 3 4 0 , H g 3 3 4 o r H g 3 6 5 n m ; light p a t h : 1 c m . ; final v o l u m e : 3 . 0 0 m l . ; ca. 25 ° C ;
m e a s u r e a g a i n s t air. B e f o r e t h e start o f t h e e x p e r i m e n t , b r i n g t h e s o l u t i o n s to 25 ° C .
T h e s m a l l c h a n g e s in e x t i n c t i o n t h a t o c c u r o n a d d i t i o n o f t h e e n z y m e s u s p e n s i o n s m u s t b e
d e t e r m i n e d o n c e for e a c h b a t c h o f e n z y m e , a n d t h e A E v a l u e s are c o r r e c t e d b y this a m o u n t .
* D o not place the tissue sample o n metal weighing p a n s ; the use of small plastic dishes, glazed p a p e r
or strips of film as s u p p o r t s prevents the sample from freezing to the p a n . Weighing in a cold r o o m is
recommended.
** Ultra-Turrax, Type 18/2, J a n k e & K u n k e l , Stauffen i. B. ( G e r m a n y ) .
f
H o m o g e n i z e r for scientific purposes from Biihler, Tubingen ( G e r m a n y ) .
+
Radiometer, C o p e n h a g e n ( D e n m a r k ) .
T r i e t h a n o l a m i n e buffer (I) 2.27 ml. 38 m M

N A D P solution (II) 0.10 ml. 0.33 m M
MgCl 2 solution (III) 0.20 ml. 6.66 m M
Sample 0.10 ml. u p t o 0.1 m M A T P
M i x , w a i t 1 - 3 m i n . for c o n s t a n t v a l u e t o b e r e a c h e d ,
and read extinction E. 1
G 6 P - D H suspension (V) 0.01 m l . 3.3/ig./ml.^462 mU/ml.
M i x , read e x t i n c t i o n E 2 after a b o u t 5 m i n . , a n d c h e c k
for a further 1 - 2 m i n . t o e n s u r e t h a t v a l u e is c o n s t a n t .
E2~Ei = A E _ _ . G 6 P
Glucose solution (IV) 0.30 ml. 50 m M
M i x , r e a d e x t i n c t i o n E i m m e d i a t e l y , a n d start t h e A T P
3
r e a c t i o n w i t h H K after n o t m o r e t h a n 3 0 s e c *
Hexokinase suspension (VI) 0.02 ml. 1 3 . 3 jug./ml. = 1.8 U / m l .
O b s e r v e c h a n g e in e x t i n c t i o n u n t i l v a l u e becomes
c o n s t a n t ( E ) . T h e A T P r e a c t i o n is n o r m a l l y c o m p l e t e
4
after a b o u t 12 m i n . T h e c h a n g e in e x t i n c t i o n is f o l l o w e d
for 15 m i n . for safety. E - E 4 3 = A E A X P .
* T h o u g h the glucose-6-phosphate dehydrogenase p r e p a r a t i o n s contain less t h a n 0 . 1 % of hexokinase,

the addition of glucose initiates a slight but definite conversion of A T P (A E ca. 0.002 per min.). T h e
change in extinction can be observed after 3 - 5 m i n . ; the hexokinase should therefore be a d d e d soon
after the glucose, a n d in any case after n o t m o r e t h a n 1 min. This is also why the glucose is a d d e d after
G6P-DH
Calculations
U n d e r the conditions indicated, the reactions are stoichiometric, and to within the experimental error,
twice the quantity of deproteinized sample gives d o u b l e the values.
F o r m u l a (2) on p . 312 is valid, a n d the A T P concentration in the assay mixture is therefore:

c = 4.92 x ZIE A X P 4.82 x AEATP 8.70 x AE ATP [/miole/ml.]
In the case of biological samples the fluid content of the tissue and the dilution due to the pretreatment of the
sample must be taken into account.
If
V x = weight (g.) or volume (ml.) of tissue
V = V + g. (ml.) perchloric acid for deproteinization
2 t
V = volume of perchloric acid extract before neutralization

3
V = V + ml. K C 0
4 3 2 3 required
V = volume of deproteinized solution in the cuvette, the dilution factor is F

V 2 x V 4
5
V, x V 3
Adenosine-5'-triphosphate 2107
If the a m o u n t of A T P is to be calculated per g. tissue, then b o t h V\ a n d V are expressed in g. F o r 6% per 2
chloric acid: 1 ml. = 1.035 g. Therefore V [g.] = V [g.] + (ml. perchloric acid) x 1.035. 2 x
If the a m o u n t of A T P is to be calculated per ml. tissue, then V a n d V\ are expressed in ml. T h e weight of 2
tissue divided by the density of the tissue is V [ml.]. This value is then used to calculate V x 2 [ml.].
If the a m o u n t is to be given in pg. instead of pinole, then the result must be multiplied either by the molecular
weight of A T P (507.2) or by the molecular weight of G-6-P (260.2).
To obtain the A T P content of the cells of a tissue, the a m o u n t of A T P present in the occluded b l o o d of
the tissue m u s t be subtracted from the total A T P content. F o r this correction the following e q u a t i o n is
valid : 26
A T P content of the cells = [(total A T P content of the tissue) - (the fraction by weight of blood in the
tissue x A T P content of the b l o o d ) ] : [1 - fraction by weight of b l o o d in the
tissue]
This same formula is obviously valid for o t h e r metabolites.

T h e fraction by weight of b l o o d in the tissue is determined according to Bucher et a l . 2 6
from extinction
m e a s u r e m e n t s at 578, 560 a n d 540 n m . A s s u m i n g t h a t the p r o p o r t i o n of o x y h a e m o g l o b i n ( H b 0 ) in the 2
circulating b l o o d a n d in the tissue is approximately the same, it follows t h a t the fraction of blood x in
the tissue is
x =
AE
»*°*
X d i l u t i o n X d
' x 100 [wt.%]
zlEH o x dilution x d
b 2 2
where
d E H b o 2
=
extinction difference of the tissue extract
d EHI>O 2
=
extinction difference of the b l o o d dilution
dj a n d d 2 = light p a t h s of the cuvettes
AE H b 0 2 and A E H b Q 2 are c a l c u l a t e d 26
w i t h o u t the use of graphical m e t h o d s from the extinction m e a s u r e
ments at 578, 560 a n d 540 n m according to the formula
A E Q or A E
Hb 2 H b 0 2 = (E 5 7 8 - E 5 6 0 ) + [(E 5 4 0 - E 5 7 8 ) x 0.47]
Example
2.8035 g. liver from a normally fed rat were deproteinized with 9.10 ml. perchloric acid solution (VII). E a c h
4.0 ml. perchloric acid extract required 0.14 ml. K C 0 2 3 solution (VIII) for neutralization.
Vx = 2.8035 g.
V 2 = 2.8035 + (9.10 x 1.035) = 12.222 g.
V 3 = 4.0 ml.
V 4 = 4.14 ml.
V 5 = 0.1 a n d 0.2 ml.
Extinction values (365 n m )

for V 5 - 0.1 ml. for V 5 = 0.2 ml.
E x = 0.034 E 2 - E x = 0.026
C h a n g e due
E 2 = 0.060 to enzyme 0.002
AE H M P = 0.024 dE H M P = 0.046
E 3 = 0.060 E 4 - E 3 = 0.063
E 4 = 0.123 Change due
to enzyme 0.003
AE ATP = 0.060 AE ATP = 0.119
The following values are o b t a i n e d ( e 3 6 5 n m = 3.45 c m . / / i m o l e ) :2
2.354 nmole A T P / g . liver ( V = 0 . 1 5 ml.)

2.334 /miole A T P / g . liver ( V = 0 . 2 ml.) 5
0.841 /miole hexose m o n o p h o s p h a t e / g . liver ( V = 0 . 1 ml.) 5
0.806 /miole hexose m o n o p h o s p h a t e / g . liver ( V = 0 . 2 ml.) 5
Determination of ATP in whole blood of same rat: 6.4 ml. of perchloric acid were used for 2.0 g. of blood.
Since the quantity of b l o o d was 3.5485 g., a further 3.15 ml. of perchloric acid h a d t o be a d d e d . 4.0 ml. of t h e
centrifuged perchloric acid extract required 0.14 ml. 5 M K C 0 - s o l u t i o n for neutralization. V 2 3 5 = 0.1 ml.
and 0.2 ml.
Extinction differences o b t a i n e d :
for V 5 = 0.1 m l . : A E A T P = 0.015; A E H M P = 0 ; for V 5 = 0.2 m l . : A E A X P = 0.030; A E H M p = 0.
This is equivalent to 0.495 /miole A T P / g . b l o o d .
Amount of blood in liver of same animal:
Dilution of the b l o o d sample 1 : 125,

Dilution of the liver sample 1 : 12.5.
All m e a s u r e m e n t s in cuvettes with 1 cm. light p a t h .
The extinctions o b t a i n e d by m e a s u r e m e n t s at 578, 560, a n d 540 n m give
for b l o o d A E H b Q 2 = 0.237
0.168
The a m o u n t of blood in the liver is

0.168 x 12.5 x 1
x 100 = 7 . 1 %
0 . 2 3 7 x 125 x 1
Corrections for blood: Substitution in the formula gives the following v a l u e s :
1 A T D 2.354-(0.071 x0.495) _ . A X D /
1. A T P j QQ,^ — = 2 . 5 0 /miole A T P / g . liver

- A _ 2.334-(0.071 x0.495) 0 . A T 1 > / R
2. A T P — = 2 . 4 7 /miole A T P / g . liver
1-0.071 H I B
1. Hexose m o n o p h o s p h a t e ^ Q Q ^ | = 0 9 1 /miole hexose m o n o p h o s p h a t e / g . liver
2. Hexose m o n o p h o s p h a t e ^ Q Q ^ = 0 - 8 7 /miole hexose m o n o p h o s p h a t e / g . liver
1. Almost without exception, interference can be traced to c o n t a m i n a t i o n of the hexokinase or g l u c o s e s -

p h o s p h a t e dehydrogenase with o t h e r enzymes (especially with N A D P H oxidase, glutathione reductase or
t o o large an a m o u n t of hexokinase in the glucose-6-phosphate dehydrogenase).
2. In the presence of large a m o u n t of P O | ~ (e. g. deproteinized solutions from i n c u b a t i o n s carried out in

Krebs-Ringer p h o s p h a t e saline) a fine crystalline precipitate of m a g n e s i u m a m m o n i u m p h o s p h a t e often
appears in the cuvette d u r i n g the m e a s u r e m e n t s ; this leads to an a p p a r e n t increase in extinction.
Few systematic studies have been carried o u t on the specificity of hexokinase (yeast) t o w a r d s nucleoside
triphosphates. Inosine t r i p h o s p h a t e (ITP) reacts with yeast hexokinase, b u t at a m u c h slower rate t h a n
A T P . Bucher et a l .
3 0 2 6
have found by c h r o m a t o g r a p h y that the m a x i m u m n o n - A T P fraction of nucleoside
triphosphates in liver extracts is a b o u t 20%. E r r o r s d u e t o the d e t e r m i n a t i o n of o t h e r energy-rich nucleoside
triphosphates as well as A T P are possible in all assay m e t h o d s in which A T P acts as an agent for the transfer
of energy a n d of p h o s p h a t e g r o u p s . In the determination of A T P with p h o s p h o g l y c e r a t e kinase (p. 2097),
I T P , G T P , a n d U T P react at almost the same rate as A T P ; the reaction with C T P is slower. According to
Kaji ,31
if the conversion of A T P is t a k e n as 1, the conversions of o t h e r energy-rich nucleoside t r i p h o s p h a t e s
with yeast hexokinase a r e : d e o x y - A T P = 0.5, I T P = 0.03, G T P = 0.008, U T P = 0.004, C T P = 1.3 x 1 0 " , 3
d e o x y - C T P and d e o x y - G T P = 2.5 x 1 0 . - 6
T h e determination described here is therefore specific for A T P c o m p a r e d with the other nucleoside
triphosphates, and is superior to the other m e t h o d s for the determination of A T P in this respect. T h e
specificity is p o o r e r with hexokinase p r e p a r a t i o n s from animal tissues. A T P a n d also A D P can be replaced
in a similar m a n n e r by other nucleotides in the C K reaction (see p . 785 ) . 3 2
G 6 P - D H is strictly specific for glucose-6-phosphate, and N A D P c a n n o t be replaced by N A D .
O t h e r M e t h o d s for t h e D e t e r m i n a t i o n o f A T P
1. T h e enzymatic m e t h o d of Bucher et a l . 2 6
with phosphoglycerate kinase is described on p . 2097.
2. T h e fluorimetric enzymatic analysis with luciferase (McElroy et a l . , p . 2112) allows the detection of less
3 3
t h a n 1 pg. A T P / m l . sample a n d is extremely specific.
3. Using p o t a t o apyrase, adenylate kinase a n d 5 ' - A M P deaminase, A T P , A D P a n d A M P can be determined

in one operation according to the m e t h o d of Kalckar et a l . 3 7
by the s p e c t r o p h o t o m e t r i c m e a s u r e m e n t of the
extinction at 265 n m .
4. F o r differentiation between purine a n d pyrimidine nucleotides, see p . 2078.
References
1 O. Warburg & W. Christian, Biochem. Z . 242, 206 [1931]; 287, 440 [1936]; 291 [1936].
2 O. Warburg, W. Christian & A. Giese, Biochem. Z. 282, 157 [1935].
3 A. Kornberg, J. biol. C h e m . 182, 805 [1950].
4 W. Lamprecht & I. Trautschold, Hoppe-Seylers Z. physiol. C h e m . 311, 245 [1958]; I. Trautschold,
Diplom-Arbeit, Techn. H o c h s c h u l e M u n c h e n [1956]; W. Lamprecht, Habilitationsschrift, Techn.
Hochschule M u n c h e n [1957]; I. Trautschold, Dissertationsschrift, Techn. H o c h s c h u l e M u n c h e n [1958];
W. Lamprecht & Th. Hockerts, Die Medizinische 8, 289 [1957]; W. Lamprecht & Th. Hockerts: Struk-
t u r u. Stoffwechsel des Herzmuskels. G. Thieme Verlag, Stuttgart 1959.
5 O. Meyerhof, Biochem. Z . 183, 176 [1927].
6 O. Meyerhof 8L H. Green, J. biol. C h e m . 178, 655 [1949].
7 R. Robison: T h e Significance of P h o s p h o r i c Esters in M e t a b o l i s m . University Press, N e w Y o r k 1932.
8 C. F. Cori & F. Lipmann, J. biol. C h e m . 194, 417 [1952]; A. F. Brodie & F. Lipmann, ibid 212, 611
[1955].
9 F. Eisenberg & /. B. Field, J. biol. C h e m . 222, 293 [1956].
10 J.L. Gamble jr. & V.A.Najjar, Science (Washington) 120, 1023 [1954]; J. biol. C h e m . 217, 595 [1955].
11 L. Berger, M. W. Slein, S. P. Colowick & C. F. Cori, J. gen. Physiol. 29, 379 [1946].
12 M. W. Slein, G. T. Cori & C. F. Cori, J. biol. C h e m . 186, 763 [1950].
13 M. Dixon & D. M. Needham, N a t u r e [ L o n d o n ] 158,432 [1946].
14 / . Wajzer, C. R. h e b d . Seances A c a d . Sci. 236, 2116 [1953].
15 M. Kunitz & M. R. McDonald, J. gen. Physiol. 29, 393 [1946].
16 D. M. Greenberg, Chemical P a t h w a y s of M e t a b o l i s m . A c a d e m i c Press, N e w Y o r k 1954, p . 74.
17 W. Lamprecht & /. Sellmair, u n p u b l i s h e d ; J. Sellmair, Zulassungsarbeit z. wissenschaftl. Priifung f. d.

L e h r a m t , Universitat M u n c h e n 1957.
18 B. L. Horecker & P. Z . Smyrniotis, Biochim. biophys. A c t a 12, 98 [1953].
19 E. Negelein & W. Gerischer, Biochem. Z. 284, 289 [1936].
20 W. Lamprecht & G. Michal, u n p u b l i s h e d ; G. Michal, Diplom-Arbeit, Techn. H o c h s c h u l e M u n c h e n 1957.
21 L. Glaser & D. Brown, J. biol. C h e m . 216, 67 [1955].
22 R. Nordlie & H. Lardy in P. D. Boyer, H. Lardy & K. Myrbdck: T h e E n z y m e s 6,4 [1962]. R. K. Crane in
P. D. Boyer, H. Lardy & K. Myrbdck: T h e Enzymes 6, 47 [1962].
23 N. R. Lazarus c. s., Biochemistry 5, 4003, 4017 [1966]; 7, 2390 [1968].
24 E. A. Noltmann & S. A. Kuby in P. D. Boyer, H. Lardy & K Myrbdck: T h e Enzymes 7, 233 [1963].
25 A. Wollenberger, Naturwissenschaften 45, 294 [1958].
27 W. Lamprecht & D. Lommer, u n p u b l i s h e d ; D. Lommer, D i p l o m - A r b e i t , Techn. Hochschule M u n c h e n
1960.
28 M. Merkenschlager, K. Schlossmann & W. Kurz, Biochem. Z. 329, 332 [1957].
29 W. Lamprecht & P. Stein, unpublished.
30 N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c Press, N e w York 1957, Vol. I l l , p . 874.
31 A. Kaji, unpublished, see R. A. Darrow & S. P. Colowick i n : M e t h o d s in Enzymology, Vol. V, p . 226
[1962].
32 T. Nihei, L. Noda & M. F. Morales, J. biol. C h e m . 236, 3203 [1961].
33 B. L. Strehler & W. D. McElroy, M e t h o d s in Enzymology. A c a d e m i c Press, New York 1957, Vol. I l l ,
p . 871.
34 A. Munch-Petersen & H. M. Kalckar, M e t h o d s in Enzymology. A c a d e m i c Press, N e w York 1957, Vol.
I l l , p . 869.
Determination with Formyltetrahydrofolate Synthetase

Jesse C. R a b i n o w i t z
Principle
Is t h e s a m e a s for t h e d e t e r m i n a t i o n o f f o r m a t e , p . 1 5 4 6 .
Reagents, see p. 1547.
See p. 1547, N o . I - I H , V, VII a n d VIII. A d d i t i o n a l :

X . S o d i u m formate (0.4 M ; p H 8.0):
D i s s o l v e 2 . 7 2 g. H C O O N a in 5 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 8.0 w i t h 1 N KOH
X I . Reagent mixture (0.2 M triethanolamine; 80 m M formate; 20 m M M g C l ; 4 m M D L - T H F ; 2
0.4 M mercaptoethanol):
M i x 1 v o l u m e t r i e t h a n o l a m i n e buffer (I)
1 volume sodium formate solution (X)
1 volume M g C l 2 solution (V)
2 v o l u m e s T H F s o l u t i o n (III)
Procedure
Assay System
W a v e l e n g t h : 3 5 0 n m ; l i g h t p a t h : 1 c m . ; i n c u b a t i o n v o l u m e : 1.01 m l . ; i n c u b a t i o n t e m p e r a t u r e :
37 ° C ; final v o l u m e : 3.01 m l . ; r e a d a t r o o m t e m p e r a t u r e a g a i n s t a b l a n k ( m i n u s e n z y m e
suspension VIII).
P i p e t t e i n t o 2 test t u b e s (1.1 x 10 c m . ) : C o n c e n t r a t i o n in assay mixture
Reagent mixture (XI) 0.5 m l . 0.10 M triethanolamine;

4 0 m M f o r m a t e ; 10 m M M g C l ; 2
2mM DL-THF;
0.2 M 2-mercaptoethanol
Sample 0.5 m l . u p t o 100 fiM ATP
M i x b y s h a k i n g . P l a c e t h e t u b e s for 2 m i n . in a
3 7 ° C w a t e r b a t h a n d t h e n a d d 10 o n e o f t h e t u b e s
(No.l):
Enzyme suspension (VIII) 1 fil 0.005 mg. protein/ml.
M i x a n d after 10 m i n . ( o r l o n g e r ) at 37 ° C p i p e t t e
into both tubes:
Perchloric acid (VII) 2.0 ml. 0.133 M
C e n t r i f u g e off t h e p r o t e i n a n d 1 0 - 3 0 m i n . after a d
dition o f the acid read the extinctions o f both tubes.
E
tube I _ E
tube 2 = ^ E is u s e d for t h e c a l c u l a t i o n s .
A n a l y s e t h e s t a n d a r d s in t h e s a m e w a y .
Calculations
(2) o n p . 312 applies. T h e extinction coefficient of 5,10-methenyltetrahydrofolic acid at 350 n m is
24.9 c m . / / m i o l e . W i t h the m e t h o d described here the A T P concentration of the sample is given b y :
2
c = AE x 0.242 [^mole/ml.]
S o u r c e s o f Error and Specificity o f M e t h o d
As described for the d e t e r m i n a t i o n of formate o n p . 1549.

Adenosine-5 -triphosphate and Creatine Phosphate
Determination with Luciferase
B e r n a r d L. Strehler
Bioluminescence was first used as an indicator of metabolic activity by E. N. Harvey . 1

H e used luminous
bacteria (Achromobacter fischeri) to detect the photosynthetic oxygen p r o d u c t i o n of Elodea leaves by
immersing the leaves in a thick suspension of the bacteria. Harvey a n d his collaborators also used the
inhibition or stimulation of luminescence to study the effect of narcotics, respiratory inhibitors and pressure
on enzymes and enzyme s y s t e m s ' . T h e first application of bioluminescent reactions in vitro to determine
2 3
i m p o r t a n t metabolic intermediates was described by Strehler a n d Totter " in 1952. They used extracts of 4
the luminous organ of the firefly, Phontinus pyralis, to assay adenosine t r i p h o s p h a t e ( A T P ) and a n u m b e r of
metabolically related substrates a n d enzymes. T h e m e t h o d was based u p o n the earlier discovery by Mc-
Elroy 5
that a q u e o u s extracts of firefly lanterns which were n o longer l u m i n o u s emitted light once again on
the addition of A T P . McElroy a n d Strehler 6
then showed that in addition to the enzyme, A T P and oxygen,
two other c o m p o n e n t s were necessary for the emission of light: a divalent cation (e. g. M g , M n 2 + 2 +
,
Fe 2 +
, Co 2 +
, Zn 2 +
) and a fluorescent c o m p o u n d called luciferin. D u r i n g the intervening years, McElroy
et al. ' 1 9
have c o n t r i b u t e d m u c h to the u n d e r s t a n d i n g of the m e c h a n i s m of firefly bioluminescence.
Strehler a n d Totter 10
a n d Strehler a n d McElroy 11
have published reviews of this m e t h o d of analysis
and its application. T h e d e t e r m i n a t i o n of A T P with luciferase, because of its specificity and sensitivity, has
been used for studies on p h o t o s y n t h e s i s , neural f u n c t i o n , radiation effects , oxidative p h o s p h o r y l a t i o n
12 13 14 4
and muscle c h e m i s t r y 15,16

. Luciferase p r e p a r a t i o n s are available commercially. T h e quantitative determina
tion of A T P a n d creatine p h o s p h a t e is described here. Assay m e t h o d s for related substrates (e. g. mixtures
of A T P , A D P , A M P a n d creatine p h o s p h a t e ) a n d enzymes (myokinase, hexokinase, apyrase, creatine
kinase) can be found in the original p a p e r s of Strehler a n d Totter*' .
10
P r i n c i p l e and O p t i m u m C o n d i t i o n s for M e a s u r e m e n t s
(1) A T P + Luciferin , luciferase

» Adenyl-luciferin + P y r o p h o s p h a t e
Mg 2 +
(2) Adenyl-luciferin Adenyl-oxyluciferin + H 0 + Light2
Reaction (1) is reversible a n d the equilibrium lies far to the right. Reaction (2) is practically irreversible,
a n d its p r o d u c t , adenyl-oxyluciferin, is a p o t e n t inhibitor of the luminescent reaction.
T h e Michaelis constant for A T P is 50 uM at p H 7.4. At A T P concentrations which are a little below the
Michaelis half saturation value, the rate of the over-all reaction is p r o p o r t i o n a l to the A T P concentration.
In the presence of A T P the reaction reaches its m a x i m u m rate almost immediately and then decreases
essentially as a first order process with time as A T P is consumed or as inhibitors accumulate. T h e determi
nation of A T P therefore involves the m e a s u r e m e n t of the relative intensity of the light emitted by a luci
ferase solution within a few seconds after the addition of an A T P solution (in the presence of a d e q u a t e
a m o u n t s of M g 2 +
, oxygen a n d luciferin).
T h e proportionality between the light intensity and the A T P concentration is shown in Figure 1. The time
course of luminescence in the presence of various buffers is shown in Figure 2.
Since the intensity of the emitted light is p r o p o r t i o n a l to the A T P concentration u n d e r defined conditions
A T P a n d Creatine P h o s p h a t e , D e t e r m i n a t i o n with Luciferase 2113
3 30Y
Oh
B
Fig. 1. Relationship between light intensity and A T P c o n c e n t r a t i o n

Light intensity measured with a q u a n t u m counter. 10\
Abscissa:
A T P c o n c e n t r a t i o n [fig. ~ P/5 ml.]. 1 fig. ~ P c o r r e s p o n d s to
8.15 fig. A T P 1
with the m e t h o d described here it is also possible to determine any substance which affects the A T P concen
tration. By addition of the a p p r o p r i a t e coupling enzymes or enzyme systems the following substances can be
d e t e r m i n e d : A D P , A M P , creatine p h o s p h a t e a n d glucose. In the same way the activity of the following
enzymes or enzyme systems can be assayed: hexokinase, myokinase, various A T P a s e s or apyrases and A T P
synthesizing systems such as p h o s p h o r y l a t i n g m i t o c h o n d r i a or p h o s p h o r y l a t i n g c h l o r o p l a s t s .
4 17
Fig. 2. Time course of luminescence in presence

of different buffers ( p H 7.4)
Curve A: Succinate + 20 m M p h o s p h a t e
Curve B: Succinate + 12.5 m M arsenate
Curve C: Succinate + 10 m M p h o s p h a t e
Curve D: Succinate -f 5 m M arsenate
Curve E: Succinate buffer
Abscissa:
T i m e after mixing luciferase a n d A T P solution
[min.]
Equipment
T h e sensitivity of the m e t h o d depends on the sensitivity of the instrument used for the m e a s u r e m e n t of
the light. These light measuring devices consist of the following essential p a r t s : a lightproof housing for the
p h o t o m u l t i p l i e r ; a lightproof sample c h a m b e r ; a shutter between the t w o ; a p h o t o m u l t i p l i e r ; a stabilized
high voltage supply a n d an instrument for m e a s u r i n g the a m o u n t of c u r r e n t flowing t h r o u g h the p h o t o
multiplier. F o r routine assays of A T P c o n c e n t r a t i o n s of the o r d e r of 1 fig.lmX. the Farrand fluorimeter* or
a modified A m i n c o p h o t o m e t e r * * is suitable. T h e use of a q u a n t u m counter, which c o u n t s individual
photo-electrons increases the sensitivity by a factor of 100 to 1000. T h e essential c o m p o n e n t of this c o u n t e r
is a photomultiplier which is immersed in a D e w a r flask containing liquid nitrogen (see Fig. 3).
T h e light reaches the photosensitive surface of the photomultiplier t h r o u g h an unsilvered w i n d o w in the
D e w a r flask. O p e r a t i o n at the t e m p e r a t u r e of liquid nitrogen has the a d v a n t a g e of a m u c h lower rate of
* M a n u f a c t u r e r : F a r r a n d Optical C o . , Inc., N e w York 70, N . Y., U S A

* M a n u f a c t u r e r : A m e r i c a n I n s t r u m e n t C o . , Silver Spring, M d . , U S A .
emission of thermal electrons from the p h o t o c a t h o d e and d y n o d e s t h a n occurs at r o o m t e m p e r a t u r e .

T h u s the b a c k g r o u n d c o u n t i n g rate is decreased. T h e single photo-electron pulses, after amplification, con
sist of a b o u t a million electrons which arrive practically simultaneously at the a n o d e . These pulses last
for less t h a n a micro-second. Each impulse is then amplified by a linear amplifier, for example, an Al
preamplifier-Al amplifier c o m b i n a t i o n . Such amplifiers, which were primarily developed for scintillation
counting, are available commercially from the R a d i a t i o n C o u n t e r L a b o r a t o r i e s , Chicago, U S A . They can
be used without major changes for the c o u n t i n g of individual light q u a n t a .
Fig. 3. Q u a n t u m counter (for details, see Text).

A : Lightproof housing for the p h o t o m u l t i p l i e r
B : D e w a r flask containing liquid nitrogen
C : Photomultiplier
D : Unsilvered w i n d o w in the D e w a r flask
E : Shutter
F : Test t u b e
G : Sample
H : L i g h t p r o o f housing for the sample
I : High voltage source
K : Lightproof filling device for liquid nitrogen
L : To the c o u n t e r
After amplification the impulse passes t h r o u g h a discriminator which discards all pulses which have t o o
low an amplitude and therefore did n o t originate from the p h o t o c a t h o d e of the photomultiplier. T h e pulses
passed by the discriminator are given a uniform size and are directed to the o u t p u t terminal of the Al ampli
fier. They are then counted with a scaler which gives c o u n t s per unit time, or an integrator m a y be used
which gives a DC voltage p r o p o r t i o n a l t o the counting rate. This voltage m a y then be employed to drive a
recorder.
H J K L
A B C 0 E F 6
11 o >
1
0 o
Fig. 4. A p p a r a t u s for the continual m e a s u r e m e n t of the A T P c o n c e n t r a t i o n in illuminated chloroplasts.

A Tungsten filament l a m p H : Sample cuvette (light p a t h : 2 m m . )
B : C o n d e n s i n g lens J : C o r n i n g filter N o . 9782
C Water (heat filter) K : Wratten filter N o . 65
D : Wratten filter N o . 25 L : Photomultiplier
E : Wratten filter N o . 26 M : Aminco photometer
F : Wratten filter N o . 29 N : High voltage source (Baird A t o m i c M o d e l 312)
G : C o r n i n g filter N o . 2408 O : Recorder
A T P a n d C r e a t i n e P h o s p h a t e , D e t e r m i n a t i o n with Luciferase 2115
A recorder is particularly useful when time courses of very low levels of luminescence have to be
measured (e. g. A T P formation as a result of the action of physical or chemical factors on biological
material, e. g. illumination of luciferase-chloroplast mixtures). A q u a n t u m c o u n t i n g p h o t o m e t e r with a
simple recorder costs a b o u t $ 2500 .Of particular i m p o r t a n c e in the design of such a n instrument is the
complete protection of the photomultiplier from external light, either d u r i n g the m e a s u r e m e n t s o r d u r i n g
the changing of the sample it m a y yield some low level p h o s p h o r e s c e n c e which can give false results o r
m a y even d a m a g e the photomultiplier. W i t h a properly constructed h o u s i n g the b a c k g r o u n d c o u n t i n g
rate with a cooled 1P21 or 1P22 p h o t o m u l t i p l i e r is three o r four impulses/min. T h e use of the m o r e expensive
1P21 photomultiplier h a s n o appreciable a d v a n t a g e over the m u c h less expensive 1P22, since b o t h tubes
have practically n o d a r k c u r r e n t at the t e m p e r a t u r e of liquid nitrogen. F o r further details of the construction
and applications of a q u a n t u m counter, s e e 1 8 , 1 9
.
A relatively simple a p p a r a t u s 1 7
for the c o n t i n u o u s m e a s u r e m e n t of the A T P c o n c e n t r a t i o n in chloroplasts
is illustrated in Figure 4. A n easily constructed a d a p t a t i o n of the A m i n c o p h o t o m e t e r suitable for the
routine assay of m i c r o g r a m quantities of A T P is shown in F i g u r e 5.
Fig. 5. A simple housing for p h o t o m u l t i p l i e r a n d sample.

A : Lightproof wooden housing
B : Photomultiplier (inserted into a hole in the t o p of the housing
in such a way t h a t light is excluded)
C : Tube m o u n t e d rigidly in the t o p of the h o u s i n g
D : R o t a t a b l e , b u t lightproof t u b e inserted in t u b e C ( C o n t a i n e r for
the test t u b e with sample))
E : O p e n i n g in t u b e C
F : O p e n i n g in t u b e D
G : H a n d l e for r o t a t a b l e t u b e D
H : Lightproof c a p for t u b e D
T h e tubes C a n d D with their openings E a n d F form the equivalent
of a shutter, which is opened a n d closed by r o t a t i o n of t u b e D . 0 k 8 121620cm
Determination of Adenosine-5'-triphosphate
Reagents
1. A d e n o s i n e t r i p h o s p h a t e , A T P 7. M a g n e s i u m s u l p h a t e , M g S 0 - 7 H 0 4 2
crystalline s o d i u m salt, A T P - N a H • 3 H 0 ; 2 2 2 8. S o d i u m a c e t a t e , 1 M .
commercial p r e p a r a t i o n , see p. 527. 9. D i p o t a s s i u m h y d r o g e n p h o s p h a t e ,
2. Luciferase K HP0
2 4
In most cases it is n o t necessary to use a highly 10. S o d i u m c h l o r i d e , N a C l

purified p r e p a r a t i o n . F o r m e t h o d s of p r e p a r i n g 1 1 . T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris
the enzyme, see A p p e n d i x , p . 2125. 12. P h e n a z i n e m e t h o s u l p h a t e , P M S
3. S o d i u m a r s e n a t e , Na As0 12H 0
3 4 2 13. A d e n o s i n e - 5 ' - m o n o p h o s p h a t e , AMP
4. M a g n e s i u m chloride, M g C l - 6 H 0 2 2 disodium salt, A M P - N a ; commercial prepara
2
5. H y d r o c h l o r i c a c i d , 1 N tion, see p. 526.

6. Glycylglycine
F o r other reagents, see the sections "Collection, Treatment and Stability o f S a m p l e " (p. 2116),
"Assay System" (p. 2117) and " A p p e n d i x " (p. 2123).
U s e distilled w a t e r free f r o m h e a v y m e t a l i o n s .
I. A T P s t a n d a r d s o l u t i o n
a) S t o c k s o l u t i o n ( 0 . 1 6 m M ) :
D i s s o l v e 25 m g . A T P - N a H - 3 H 0 in 2 5 0 m l . d i s t i l l e d w a t e r . S t o r e t h e s o l u t i o n
2 2 2
f r o z e n at - 2 0 ° C .
b) Dilute solution (1.6 /xM):
D i l u t e 5 m l . s o l u t i o n a) t o 5 0 0 m l . w i t h d i s t i l l e d w a t e r . S t o r e t h e s o l u t i o n b e t w e e n 0
a n d 4 ° C a n d p r e p a r e freshly e a c h d a y . F o r t h e d e t e r m i n a t i o n o f p u r i t y , see " A p p e n
d i x " , p. 2 1 2 3 .
II. Luciferase
S o l u t i o n s , see " A p p e n d i x " , p . 2 1 2 5 .
III. A r s e n a t e - m a g n e s i u m buffer (0.1 M a r s e n a t e ; 5 0 m M M g 2 +
; p H 7.4):
D i s s o l v e 4 2 . 5 g. N a A s 0 - 1 2 H 0 a n d 10 g. M g C l - 6 H 0 in 5 0 0 m l . distilled w a t e r ,
3 4 2 2 2
a d j u s t t o p H 7.4 w i t h 1 N H C 1 a n d d i l u t e t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r .
I V . G l y c y l g l y c i n e - m a g n e s i u m buffer ( 2 5 m M g l y c y l g l y c i n e ; 0.1 M M g 2 +
; p H 7.5):
D i s s o l v e 1.65 g. g l y c y l g l y c i n e a n d 2.3 g. M g S 0 - 7 H 0 in 4 0 0 m l . d i s t i l l e d w a t e r , adjust
4 2
t o p H 7.5 w i t h 1 N N a O H a n d d i l u t e t o 5 0 0 m l . w i t h d i s t i l l e d w a t e r .
V. Suspension m e d i u m :
D i s s o l v e 0 . 1 3 9 g. K H P 0 , 0 . 4 9 5 g. N a C l , a n d 0.341 g. M g C l - 6 H 0 in d i s t i l l e d w a t e r ,
2 4 2 2
a d d 9 5 m l . 0.1 M tris buffer ( p H 8.0) a n d m a k e u p t o 5 0 0 m l . w i t h d i s t i l l e d w a t e r .

V I . Tris buffer (0.1 M ; p H 8 . 0 ) :
D i s s o l v e 6 . 0 5 g. tris in 4 0 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 8.0 w i t h 3 0 m l . 1.0 N H C 1
VII. Phenazine methosulphate, P M S (0.5 m M ) :
D i s s o l v e 0 . 1 4 5 g. P M S in 1 0 0 0 m l . d i s t i l l e d w a t e r .
VIII. Adenosine m o n o p h o s p h a t e , A M P (20 m M ) :
D i s s o l v e 1.03 g. A M P - N a 2 in 125 m l . distilled w a t e r .
F o r o t h e r s o l u t i o n s , see t h e s e c t i o n s " A s s a y s y s t e m " ( p . 2 1 1 7 ) a n d " A p p e n d i x " ( p . 2 1 2 3 ) .
T h e stability of dilute A T P solutions is variable, even at — 20 °C. Therefore p r e p a r e the standard A T P

solutions freshly each day. T h e luciferase p r e p a r a t i o n s prepared according to p . 2125 keep for a year at
- 2 0 °C.
Procedure
B e c a u s e o f its i n h i b i t o r y a n d d e n a t u r i n g a c t i o n o n e n z y m e s t r i c h l o r o a c e t i c a c i d is n o t s u i t a b l e
for t h e e x t r a c t i o n o f A T P . In m a n y c a s e s , e s p e c i a l l y w i t h h i g h l y d i s p e r s e d s u s p e n s i o n s ( t i s s u e
c u l t u r e s , b l o o d , b a c t e r i a , a l g a e , m i t o c h o n d r i a , e t c . ) it is sufficient t o h e a t t h e s a m p l e s at 100 ° C
for b e t w e e n 5 a n d 15 m i n . ( b o i l i n g w a t e r b a t h ) . H o w e v e r , it is i m p o r t a n t t h a t t h e s a m p l e s are
b r o u g h t a s q u i c k l y a s p o s s i b l e t o 100 ° C s o t h a t e n z y m a t i c d e g r a d a t i o n o f s u b s t r a t e s in t h e
A T P and Creatine Phosphate, Determination with Luciferase 2117
s a m p l e d u r i n g t h e h e a t i n a c t i v a t i o n o f t h e p r o t e i n s is a v o i d e d . It is p r e f e r a b l e t o inject t h e s a m p l e
i n t o t w o o r t h r e e v o l u m e s o f b o i l i n g w a t e r . I m m e d i a t e l y after h e a t i n g , transfer t h e s a m p l e
t o a n ice b a t h o r d e e p - f r e e z e a n d s t o r e t h e r e u n t i l t h e a s s a y .
B e f o r e u s i n g this m e t h o d for t h e e x t r a c t i o n o f A T P it s h o u l d b e c o m p a r e d w i t h o t h e r p r o c e d u r e s
t o s e e if s i m i l a r results are o b t a i n e d . In s t u d i e s o n t h e effect o f l i g h t o n p h o t o s y n t h e t i c p h o s
p h o r y l a t i o n in g r e e n a l g a e , h e a t i n g t h e s a m p l e at 1 0 0 ° C for 1 0 - 1 5 m i n . w a s f o u n d t o b e
sufficient t o e x t r a c t all t h e A T P . F o r larger, a n d e s p e c i a l l y for s o l i d s a m p l e s this s i m p l e e x
t r a c t i o n b y h e a t i n g is o b v i o u s l y n o t sufficient.
T i s s u e s w h i c h c o n t a i n h i g h c o n c e n t r a t i o n s o f e n z y m e s c a p a b l e o f r e a c t i n g w i t h A T P require
s p e c i a l t r e a t m e n t . Carlson 20
h a s d e v e l o p e d a p r o c e d u r e for t h e a n a l y s i s o f A T P in m u s c l e :
freeze t h e t i s s u e s a m p l e in dry i c e - p e t r o l e u m e t h e r , g r i n d in a c o l d m o r t a r w i t h f r o z e n 8%
p e r c h l o r i c a c i d (1 m l . p e r c h l o r i c a c i d / 1 0 0 m g . m u s c l e ) . T h a w t h e m i x t u r e a n d t h e n a l l o w t o
s t a n d for 10 t o 15 m i n . at 0 ° C . F i l t e r , n e u t r a l i z e t h e filtrate w i t h 1 N N a O H o r K O H ( r e m o v e
K C I O J a n d d i l u t e t o 2 5 m l . w i t h d i s t i l l e d w a t e r . T h e s o l u t i o n c a n b e s t o r e d at — 2 0 ° C .
In c e r t a i n c a s e s , for e x a m p l e , in t h e a s s a y o f A T P in c h l o r o p l a s t s , e x t r a c t i o n is n o t n e c e s s a r y .
U s u a l l y t h e s a m p l e s n e e d n o t b e d e p r o t e i n i z e d . T h e l u c i f e r a s e p r e p a r a t i o n is c o n t a m i n a t e d
w i t h a p p r e c i a b l e q u a n t i t i e s o f m y o k i n a s e , p y r o p h o s p h a t a s e a n d a p y r a s e ' . If t h e s a m p l e
8 9
contains light-scattering or absorbing particles (chloroplasts, m i t o c h o n d r i a , suspended lipids),

a k n o w n a m o u n t o f A T P s h o u l d b e a d d e d t o t h e r e a c t i o n m i x t u r e after t h e d e t e r m i n a t i o n o f
the A T P c o n t a i n e d in t h e s a m p l e . T h e e x p e r i m e n t a l results are t h e n r e l a t e d t o this i n t e r n a l
standard.
Assay System
Preliminary remarks: F o u r m e t h o d s for t h e d e t e r m i n a t i o n o f A T P are d e s c r i b e d b e l o w . T h e y

are b a s e d o n t w o p r i n c i p l e s : 1. t h e s a m p l e c o n t a i n i n g A T P is r a p i d l y i n j e c t e d i n t o t h e l u c i f e r a s e
s o l u t i o n . T h e m a x i m u m l i g h t i n t e n s i t y o b s e r v e d is a m e a s u r e o f t h e A T P c o n c e n t r a t i o n . H o w
ever, t h e m a x i m u m i n t e n s i t y d e p e n d s o n t h e rate at w h i c h t h e s a m p l e a n d e n z y m e are m i x e d a n d
a l s o o n t h e p r e s e n c e o f A D P . 2. T h e r a p i d d e c a y o f l u m i n e s c e n c e is r e t a r d e d b y u s e o f a h i g h
c o n c e n t r a t i o n o f a r s e n a t e ( o r p h o s p h a t e ) a n d m a g n e s i u m in t h e a s s a y m i x t u r e . T h e A T P
4
c o n c e n t r a t i o n is o b t a i n e d b y e x t r a p o l a t i o n o f t h e l i g h t i n t e n s i t y t o z e r o t i m e . A l s o w i t h t h i s
m e t h o d , t h e s a m p l e a n d e n z y m e m u s t b e m i x e d r a p i d l y a n d in a r e p r o d u c i b l e m a n n e r .
Method:
a) If a F a r r a n d f l u o r i m e t e r is a v a i l a b l e , p r o c e e d a s f o l l o w s : d i l u t e
0.2 m l . l u c i f e r a s e s o l u t i o n ( p r e p a r a t i o n a, p . 2 1 2 5 )
t o 0.6 m l . w i t h d i s t i l l e d w a t e r . I m m e d i a t e l y b e f o r e t h e m e a s u r e m e n t s a d d
0.2 ml. o f sample,
m i x r a p i d l y a n d m e a s u r e t h e light i n t e n s i t y at 5, 10, 15, 2 0 , 25 a n d 3 0 sec. after m i x i n g in t h e
sample.
b) If a q u a n t u m c o u n t e r is a v a i l a b l e , p r o c e e d a s f o l l o w s :
dilute the
luciferase solution (preparation b, p. 2125)
five-fold w i t h a r s e n a t e - m a g n e s i u m buffer ( s o l u t i o n III).

Add
V volume of sample
5
a n d proceed as described under a).
c) A c c o r d i n g to McElroy 21
mix
0.1 m l . l u c i f e r a s e s o l t u i o n ( p r e p a r a t i o n c, p . 2 1 2 5 )
with
2.1 m l . g l y c y l g l y c i n e - m a g n e s i u m buffer ( s o l u t i o n I V ) .
P l a c e t h e s o l u t i o n in t h e i n s t r u m e n t in f r o n t o f t h e p h o t o m u l t i p l i e r .
W i t h a 0 . 2 5 m l . s y r i n g e r a p i d l y inject
0.2 m l . s a m p l e
and proceed as described under a).
d) F o r c o n t i n u o u s m e a s u r e m e n t o f the A T P concentration in chloroplast preparations 1 7
proceed as follows :
P r e p a r e t h e luciferase s o l u t i o n a c c o r d i n g t o p r o c e d u r e a) ( p . 2 1 2 5 ) , b u t u s e p h o s p h a t e buffer
without adding M g S 0 : 4
M i x the follow solutions:
0.2 ml. luciferase solution

1.4 m l . s u s p e n s i o n m e d i u m ( s o l u t i o n V )
0.1 m l . P M S s o l u t i o n ( V I I )
0.1 m l . A M P s o l u t i o n ( V I I I )
A d d t o this m i x t u r e in s u b d u e d light
0.2 m l . c h l o r o p l a s t s u s p e n s i o n ( a b o u t 7 0 0 /ig. c h l o r o p l a s t s / m l . s u s p e n s i o n ) .
P o u r t h e m i x t u r e i n t o a c u v e t t e ( 2 m m . light p a t h ) a n d p l a c e in t h e a p p a r a t u s s h o w n in F i g . 4.
T h e result o f a t y p i c a l e x p e r i m e n t is i l l u s t r a t e d in F i g . 6.
Fig. 6. T i m e course of the A T P concen

tration in chloroplasts illuminated at
intervals (measured with the a p p a r a t u s
shown in Fig. 4). T h e figure shows the
fall in A T P concentration in the d a r k
d u e t o the action of myokinase con
tained in b o t h the chloroplasts a n d the
luciferase solution. It also illustrates the
gradual rise in the base line luminescence
due to the accumulation of A T P and
ADP.
1 1 1 1 1 1
L = Start of illumination
Time [min.] D = E n d of illumination
A T P a n d C r e a t i n e P h o s p h a t e , D e t e r m i n a t i o n with Luciferase 2119
Calculations
Since the luminescence o b t a i n e d with luciferase is directly p r o p o r t i o n a l t o A T P c o n c e n t r a t i o n (in the physio

logical c o n c e n t r a t i o n range), the light intensity p r o d u c e d by the sample is c o m p a r e d t o t h a t p r o d u c e d by
a s t a n d a r d . It is advisable t o c o n s t r u c t a s t a n d a r d curve t o check w h e t h e r t h e relationship between the lumi
nescence a n d the A T P c o n c e n t r a t i o n is linear.
Example
A T P synthesis in Chlorella pyrenoidosa . 22

T h e reaction m i x t u r e p r e p a r e d a c c o r d i n g t o m e t h o d d) contained
2 m g . algae (wet weight). T h e following values were m e a s u r e d :
D u r a t i o n of illumination [ s e c ] Luminescence [counts/15 s e c ]

0 1052.
10 1362 d 2 0 = 1410 c o u n t s / 1 5 s e c
20 2 4 6 2 ^
30 2311
60 2091
d n 1410
di = 70 c o u n t s / 1 5 s e c / s e c illumination
2
d u r a t i o n of illumination 20
0.152 pg. ~ P* gave A 1 = 5000 counts/15 sec. T h e r e f o r e : 7 0

* Q
Q Q
1 5 2
= 0.002 pg. ~ P * / s e c illumi
n a t i o n was synthesized in the reaction mixture. This c o n t a i n e d 2 m g . ( = 2 0 0 0 pg.) algae (wet weight).
Therefore t h e algae synthesized 1 0 ~ times** their weight of A T P .
6
Adenosine diphosphate (ADP): First d e t e r m i n e the A T P c o n t e n t if the s a m p l e as described above. T h e n

convert the A T P t o A D P with A T P a s e p r e p a r e d from crayfish muscle a c c o r d i n g t o Lohmann . 23
Boil the
C
120 r
03
Fig. 7. T h e time course of the

luminescence in the A D P assay.
T h e reaction m i x t u r e (0.8 ml.)
c o n t a i n e d : 0.2 ml. luciferase solu
tion ( p r e p a r a t i o n b), p . 2125;
0.2 ml. arsenate buffer (0.1 M ;
p H 7.4; 0.05 M M g S 0 ) a n d A D P :
4 o °
curve A : 0.650 pmole; curve B : £
0.260 pmole; curve C : 0.130 B 60 120
S3
pmole; curve D : 0.065 pmole. H-l T i m e after starting t h e reaction [ s e c ]
1 pg. ~ P is equivalent t o 8.15 pg. A T P

0.002 _ 2 x 1 0 " 3
- = 10" 6
2000 2 10x 3
reaction mixture for 5 to 10 min., a n d determine the total A D P ( A T P + A D P ) as described for A T P . T h e

luciferase p r e p a r a t i o n contains m y o k i n a s e which converts A D P to A T P . O b t a i n the A D P content by
c o m p a r i s o n with a s t a n d a r d curve. T h e time course of the luminescence with A D P is illustrated in Fig. 7.
Specificity
T h e reaction is specific for A T P , a l t h o u g h substances which can alter the c o n c e n t r a t i o n of A T P available

to the luciferase affect the luminescence. A D P in the absence of myokinase, creatine p h o s p h a t e in the absen
ce of creatine kinase, adenosine t e t r a p h o s p h a t e , inosine t r i p h o s p h a t e , cytidine t r i p h o s p h a t e , uridine tri
p h o s p h a t e , acetyl p h o s p h a t e and other p h o s p h o r y l a t e d intermediates d o n o t react.
Possible sources of error a r e :

1. Precipitation of buffer, enzyme or activator.
2. Depletion of oxygen d u e to respiratory activity of the sample or the luciferase.
3. Quenching of luminescence by m o n o v a l e n t anions, turbidity, a b s o r p t i o n or n a t u r a l inhibitors (see Fig. 8).
Fig. 8. Effect of chloride ions on the intensity

of the luminescence. T h e reaction mixture
(0.8 ml.) contained 0.2 ml. luciferase (prepar
ation b, p.2125);0.2 ml. A T P solution contain
ing 250 fig. ~ P / m l . * ) ; 2 mg. M g S 0 and the
4
5 M N a C l solution in reaction a m o u n t s of 5 M N a C l solution stated on the

mixture [ml.] abscissa.
4. Utilization of A T P by side reactions or binding of A T P at inactive sites (high concentrations of arsenate

9
buffer decrease this error).

5. Luminescent o r phosphorescent i m p u r i t i e s . 24
6. Presence of a m m o n i u m ions in p h o t o s y n t h e t i c p h o s p h o r y l a t i o n test systems.

7. Hydrolytic enzymes in the sample. This source of error can be obviated if the samples are deproteinized
with perchloric acid.
* 1 fig. ~ P c o r r e s p o n d s to 1 6 / i m o l e A T P .
8. Injection of the sample into the reaction mixture w i t h o u t p r o p e r protection from light; d a m a g e to the
photomultiplier by over-exposure to light; faulty performance of the i n s t r u m e n t , leading to non-linear
response.
Nearly all such sources of error can be eliminated or corrected by use of internal s t a n d a r d or s t a n d a r d
curves.
Other m e t h o d s for the estimation of A T P can be found o n pages 2097, 2101 a n d 2110. T h e ion exchange
m e t h o d of Cohn a n d Carter 25
which is described in the A p p e n d i x (p. 2123) is also useful. However, the most
sensitive a p p e a r s to be the d e t e r m i n a t i o n of A T P with luciferase, especially if a q u a n t u m c o u n t e r is available.
It has the advantages of being rapid a n d simple a n d it permits the continual m e a s u r e m e n t of A T P concen
tration in certain systems.
Determination of Creatine Phosphate

Reagents
1. A T P , s e e p . 2 1 1 5 . 5. S o l i u m a r s e n a t e , N a A s 0 - 1 2 H 0
3 4 2
2. L u c i f e r a s e , s e e p . 2 1 1 5 . 6. C r e a t i n e k i n a s e , C K
3. C r e a t i n e p h o s p h a t e lyophilized p o w d e r from m u s c l e ; commercial
26
sodium salt; c o m m e r c i a l p r e p a r a t i o n , see p.529. p r e p a r a t i o n , see p . 444.

4. A d e n o s i n e m o n o p h o s p h a t e , A M P
sodium salt, A M P - N a ; commercial p r e p a r a
2
tion, see p . 526.
S o l u t i o n s I & II f r o m p . 2 1 1 6 . In a d d i t i o n :
III. C r e a t i n e p h o s p h a t e s t a n d a r d s o l u t i o n (0.1 m M ) :
D i s s o l v e 1 m g . c r e a t i n e p h o s p h a t e ( N a salt) in 5 m l . d i s t i l l e d w a t e r . P r e p a r e freshly
e a c h d a y . F o r t h e d e t e r m i n a t i o n o f t h e c o n t e n t o f t h e s o l u t i o n , see " A p p e n d i x " , p . 2 1 2 4 .
IV. A d e n o s i n e m o n o p h o s p h a t e (20 m M A M P ) :
Dissolve 40 mg. A M P - N a 2 in 5 m l . distilled w a t e r
V . S o d i u m a r s e n a t e (0.1 M ; p H 7 . 4 ) :
D i s s o l v e 4 2 . 5 g. N a A s 0 1 2 H 0 in 5 0 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7.4 w i t h 1 N H C 1
3 4 2

VI. Creatine kinase, C K :
D i s s o l v e t h e C K p r e p a r a t i o n in sufficient d i s t i l l e d w a t e r s o t h a t t h e i n t e n s i t y o f t h e l u m i
n e s c e n c e is r e a d i l y m e a s u r a b l e d u r i n g t h e 1 0 - 3 0 sec. r e a c t i o n t i m e . P r e p a r e t h e s o l u t i o n
freshly e a c h d a y .
Procedure
Assay System
The following should be prepared: experimental tube, control tube without creatine kinase and
A M P ( e s p e c i a l l y if t h e s a m p l e c o n t a i n s m u c h A T P a n d A D P * ) , a n d a s t a n d a r d t u b e c o n t a i n i n g
c r e a t i n e p h o s p h a t e s t a n d a r d s o l u t i o n (III) i n s t e a d o f t h e s a m p l e . A s t a n d a r d c u r v e s h o u l d
a l s o b e p r e p a r e d ( s e e F i g . 10).
P i p e t t e i n t o a test t u b e :
0 . 2 0 m l . luciferase s o l u t i o n ( p r e p a r a t i o n a, p . 2 1 2 5 )
0.20 ml. s o d i u m arsenate solution (V)
0.12 ml. A M P solution (IV)
0.01 m l . c r e a t i n e k i n a s e ( V I )
0 . 0 2 t o 0.2 m l . s a m p l e
distilled w a t e r t o 1.0 m l .
M i x t h o r o u g h l y a n d r e a d t h e l u m i n e s c e n c e e v e r y 19 sec. T h e t i m e c o u r s e o f t h e l u m i n e s c e n c e
is s h o w in F i g . 9.
Fig. 9. Time course of the

luminescence in the creatine
p h o s p h a t e assay. T h e reaction
mixture (1 ml.) c o n t a i n e d :
0.01 ml. of a crude creatine
kinase solution; 0.2 ml. 0.1 M
N a arsenate solution ( p H 7.4);
10 mg. M g S 0 ; 1 mg. A M P ;
4
0.2 ml. luciferase solution and

the following a m o u n t s of crea
tine p h o s p h a t e :
C u r v e A : 0.664 //mole;
C u r v e B : 0.332 umole;
C u r v e C : 0.199 /rniole;
C u r v e D : 0.132 umole;
C u r v e E : 0.0664 umole.
Calculations
T h e creatine p h o s p h a t e content of the sample is read off from a s t a n d a r d curve (see Fig. 10).
See p . 2 1 2 0 .
* Alternatively, the A T P a n d A D P contained in the sample can be converted to A M P with A T P a s e (from

crayfish ) a n d myokinase, a n d then the A T P a s e is inactivated by boiling.
23
40 r
Fig. 10. R e l a t i o n s h i p between lumines

cence (30 sec. after mixing) a n d creatine
p h o s p h a t e content. F o r constituents of
the assay mixture, see Legend to Fig. 9.
Creatine p h o s p h a t e content of the assay mixture [fig. ~ P]
Creatine p h o s p h a t e can also be determined by the m e t h o d described in the " A p p e n d i x " (p. 2124). A n o t h e r
m e t h o d can be found o n p . 1777.
Appendix
Assay of the Purity of the A T P Standard Solutions
Reagents and Solutions
1. D o w e x 1 ( N H ^ form), 2 0 0 - 4 0 0 m e s h 4. S o d i u m c h l o r i d e - H C l (20 m M N a C l ;
2. H y d r o c h l o r i c acid 0.003 N ; 0.01 N ; 1 N 0.01 N HC1):
3. A m m o n i u m chloride (10 m M ) : Dissolve 1.169 g. N a C l in 100 ml. 0.01 N HC1.
Dissolve 0.053 g. N H C 1 in 100 ml. distilled
4 5. S o d i u m c h l o r i d e - H C l (0.2 M N a C l ;
water 0.01 N H C 1 ) :
Dissolve 11.69 g. N a C l in 100 ml. 0.01 N HC1.
Procedure
Suspend D o w e x 1 in distilled water, p r e p a r e a 1.5 cm. high c o l u m n (ca. 1 c m . diameter) a n d convert the resin
to the chloride form with ca. 50 ml. 1 N HC1. Wash the c o l u m n with water until t h e washings are neutral.
Adjust the p H of the A T P solution to 8 to 9 a n d p o u r the solution o n the c o l u m n * . Wash the c o l u m n with ca.
50 ml. distilled water a n d then elute successively with 100 ml. of each of the following solutions; 10 m M
N H C 1 (elutes adenosine a n d a d e n i n e ) ; 0.003 N HC1 (elutes A M P a n d inorganic p h o s p h a t e ) ; 20 m M N a C l
4
in 0.01 N HC1 (elutes A D P ) ; 0.2 M N a C l in 0.01 N HC1 (elutes A T P ) * * .

D e t e r m i n e the a d e n i n e nucleotide c o n t e n t of the eluate by m e a s u r e m e n t of the extinction at 260 n m ( p H 2).
U n d e r these conditions the m o l a r extinction coefficient of adenosine a n d its p h o s p h o r y l a t e d derivatives is
a b o u t 14.2 x 10 c m / m o l e .
6 2
* To ensure t h a t all the nucleotides are a d s o r b e d o n the resin the a n i o n c o n c e n t r a t i o n of the solution should
n o t exceed 0.01 N .
** According to Cohn (personal c o m m u n i c a t i o n ) the adenosine p h o s p h a t e s can also be separated by
elution with 0.1 M N a S 0 in 0.01 N H S 0 , with 1.5 M a m m o n i u m f o r m a t e or with 0.2 M a m m o n i u m
2 4 2 4
formate in 4 M formic acid. These reagents are approximately as effective as 0.1 M N a C l in 0.01 N HC1
in eluting A T P from t h e c o l u m n .
Assay of the Purity of the Creatine Phosphate Solutions 27
Reagents and Solutions
1. A m m o n i u m m o l y b d a t e : 3. Hydrochloric acid, 0.4 N .

Dissolve 25 g. ( N H ) M o 0 - 4 H 0 in 500
4 6 7 2 4 2 4. Sodium hydroxide, 0.4 N .
ml. 10 N H S 0 2 4 and dilute to 1000 ml. with 5. Sodium chloride, 0.4 M .
distilled water. 6. a - N a p h t h o l , recrystallized, 1 % (w/v).
2. F e r r o u s s u l p h a t e : 7. Diacetyl (butane-2.3-dione).
Dissolve 5 g. F e S 0 - 7 H 0 in 50 ml. distilled
4 2
water containing 1 ml. 1 N H S 0 . Prepare 2 4
the solution freshly each day.
Procedure
a) Fiske Subbarowphosphate determination:

Pipette into 10 ml. test t u b e s :
Tube A Tube B
2.0 ml. creatine p h o s p h a t e s t a n d a r d solution (III) 2.0 ml. distilled water
1.0 ml. 0.4 N HC1 2.0 ml. 0.4 M N a C l solution
H e a t t u b e A in a water b a t h at 65 °C for 10 min., cool a n d a d d
1.0 ml. 0.4 N N a O H .
Pipette into b o t h tubes
1.0 ml. m o l y b d a t e solution (1)
0.1 ml. F e S 0 solution (2).
4
Mix, allow to stand for 5 min. a n d read the extinction at 600 n m or a suitable adjacent wavelength (A
against B). O b t a i n the p h o s p h a t e content of t u b e A from a s t a n d a r d curve.
b) Determination of creatine:
Pipette into 10 ml. test tubes (graduated to 10 m l . ) :
TubeC Tube D Tube E
creatine p h o s p h a t e s t a n d a r d solution (III) 2.0 ml. 2.0 ml. —
distilled water 1.0 ml. 1.0 ml. 3.0 ml.
0.4 M N a C l solution — 2.0 ml. 2.0 ml.
Warm tube C to 65 °C in a water bath, a d d
1.0 ml. 0 . 4 N H C 1
and keep at 65 °C for 9 min. Then a d d
1.0 ml. 0.4 N N a O H
a n d quickly cool to r o o m t e m p e r a t u r e in an ice b a t h .
To all three tubes in the d a r k , a d d
2.0 ml. 1 % a-naphthol solution*)
1.0 ml. diacetyl (butane-2,3-dione)
distilled water to 10 ml.,
* If an u n k n o w n sample is analysed instead of the creatine p h o s p h a t e s t a n d a r d solution a n d it contains

proteins or other S H groups, a d d 1 ml. 0.5 M N a - p - c h l o r o m e r c u r i b e n z o a t e solution.
and keep in the d a r k for 20 min. at 20 °C. M e a s u r e the extinction at 520 n m (C a n d D against E ) . With the
difference E C - E D 2 0 2 0
o b t a i n the creatine content from a s t a n d a r d curve. If the s t a n d a r d curve has been
prepared with a pure solution of creatine, then the a m o u n t of creatine c o r r e s p o n d i n g to E C - E D 2 0 2 0
m u s t be
multiplied by 1.122, because the creatine p h o s p h a t e forms some creatinine on hydrolysis. Naturally, this
correction is n o t necessary if the s t a n d a r d curve is p r e p a r e d with creatine p h o s p h a t e .
Preparation of Luciferase
Method a
This simple m e t h o d was described by Strehler a n d Totter*, w h o used p r e p a r a t i o n s o b t a i n e d in this way to

determine A T P with the F a r r a n d fluorimeter.
G r i n d 50 mg. of vacuum-dried firefly lanterns* (10 or 12 lanterns) with 5 ml. ice-cold 0.1 M N a arsenate
buffer (solution V from p . 2121) or N a p h o s p h a t e buffer** ( p H 7.4) for 5 to 10 min. Filter the mixture into
a test tube standing in an ice b a t h . Dissolve 50 mg. M g S 0 • 7 H 0 in the filtrate. T h e p r e p a r a t i o n keeps
4 2
for several days at 4 °C.
Method b
A p u r e r p r e p a r a t i o n can be obtained according t o 6 1 0

. This m e t h o d involves fractional precipitation with
a m m o n i u m sulphate, a n d the final p r e p a r a t i o n contains relatively little apyrase. However, it is n o t suitable
for the c o n t i n u o u s m e a s u r e m e n t of the A T P concentration in chloroplasts, since N H 4 ions inhibit the
photosynthetic p h o s p h o r y l a t i o n . 28
G r i n d t h o r o u g h l y 4 g. vacuum-dried firefly lanterns with sand a n d extract with t w o 50 ml. p o r t i o n s of

distilled water. Adjust the extract to p H 6 with 1 N HC1, centrifuge a n d discard the precipitate. To the super
n a t a n t add 10 g. ( N H ) S 0 , allow to stand for 15 min. at 0 °C, centrifuge a n d discard the precipitate.
4 2 4
Adjust the p H of the s u p e r n a t a n t to 7.5 with 1 N N a O H , again a d d 10 g. ( N H ) S 0 , allow to stand for

4 2 4
15 min. at 0 °C, centrifuge a n d discard the precipitate. Adjust the p H of the s u p e r n a t a n t to 4.5 with 1 N HC1,
a d d 30 g. ( N H ) S 0 , allow to stand for 10 min. at 0 °C, filter a n d discard the filtrate. Dissolve the precipitate
4 2 4
in 50 ml. distilled water, store the clear amber-yellow coloured solution in small p o r t i o n s at —20 °C (keeps
for several years) or lyophilize a n d store the residue in a deep-freeze.
Method c
McElroy 21
r e c o m m e n d s the following modification.
G r i n d thoroughly 5 g. dried firefly lanterns in a m o r t a r a n d extract with 25 ml. cold distilled water. Adjust
the p H of the suspension to ca. 7.7 with 1 N N a O H , centrifuge for 10 min. in the cold at 3 000 g a n d save the
supernatant. Extract the precipitate once again with 25 ml. cold distilled water a n d centrifuge as already
described. C o m b i n e the s u p e r n a t a n t s a n d use this c r u d e enzyme p r e p a r a t i o n for the d e t e r m i n a t i o n s . Store
frozen. O n thawing, centrifuge off any precipitate which a p p e a r s a n d adjust the p H of the s u p e r n a t a n t to ca.
7.5.
* Obtainable from Sigma Chemical C o . St. Louis, M o . ; Schwarz Bio-Research Inc., O r a n g e b u r g , N e w

Y o r k ; Worthington Biochemical C o r p . , Freehold, N e w Jersey, U S A .
** N a p h o s p h a t e buffer (0.1 M ; p H 7.4):
Dissolve 17.8 g. N a H P 0 • 2 H 0 a n d 13.8 g. N a H P 0 • H 0 in 1 000 ml. distilled water.
2 4 2 2 4 2
References
1 E. N. Harvey, Plant Physiol. 3, 85 [1928].

2 E. N. Harvey, Biol. Bull. W o o d s ' H o l e 29, 308 [1915].
3 F. H. Johnson, H. Eyring & E. W. Williams, J. cellular c o m p a r a t . Physiol. 20, 247 [1942].
4 B. L. Strehler & J. R. Totter, Arch. Biochem. Biophysics 40, 28 [1952].
5 W. D. McElroy, P r o c . N a t . A c a d . Sci. U S A 33, 342 [1947].
6 W. D. McElroy & B. L. Strehler, Arch. Biochem. Biophys. 22, 420 [1949].
7 W. D. McElroy & J. Coulombre, J. cellular c o m p a r a t . Physiol. 39, 475 [1951].
8 J. W. Hastings & W. D. McElroy in F. H. Johnson: T h e Luminescence of Biological Systems. Amer.
Assoc. A d v . Sci., Washington, D . C. 1955, p . 257.
9 W. D. McElroy & A. Green in O. H. Gaebler: Enzymes, Units of Biological Structure and F u n c t i o n .
Academic Press, N e w York 1956, p . 369.
10 B.L. Strehler & J. R. Totter in D. Glick: M e t h o d s of Biochemical Analysis. Interscience Publishers,
N e w York 1954, Vol. I, p . 341.
11 B.L. Strehler & W. D. McElroy in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. Academic
Press, N e w York 1957, Vol. I l l , p . 871.
12 B.L. Strehler in W. D. McElroy & H. B. Glass: P h o s p h o r u s S y m p o s i u m . T h e J o h n s H o p k i n s Press,
Baltimore, M d . 1952, Vol. II, p. 4 9 1 .
13 R. Grenell in H. E. Himwich: Tranquilizing D r u g s . A m e r . Assoc. Sci., Washington, D . C. 1957, Publ.
N o . 46, p . 6 1 .
14 D. Billen, B. L. Strehler, G. E. Stapleton & E. Brigham, Arch. Biochem. Biophys. 43, 1 [1953].
15 F. D. Carlson & A. Siger, J. gen. Physiol. 43, 301 [1959].
16 L. B. Nanninga & W. F. H. M. Mommaerts, P r o c . N a t . Acad. Sci. U S A 46,1155 [I960].
17 B.L. Strehler & D. D. Hendley in W. D. McElroy & H. B. Glass: S y m p o s i u m on Light and Life. T h e
J o h n s H o p k i n s Press, Baltimore, M d . 1961, p . 601.
18 B. L. Strehler, Arch. Biochem. Biophysics 34, 239 [1951].
19 W. E. Arthur & B. L. Strehler, Arch. Biophys. 70, 507 [1957].
20 F. Carlson, Private c o m m u n i c a t i o n .
21 W. D. McElroy, Private c o m m u n i c a t i o n .
22 B. L. Strehler, Arch. Biochem. Biophys. 43, 67 [1953].
23 K.Lohmann, Biochem. Z. 282, 109 [1935].
24 B. L. Strehler & W. A. Arnold, J. gen. Physiol. 34, 809 [1951].
25 W. E. Cohn & C. E. Carter, J. A m e r . chem. Soc. 72, 4 2 7 3 [1950].
26 K. Lohmann, Biochem. Z. 277, 264 [1934].
27 A. H. Ennor in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c Press, N e w
York 1957, Vol. I l l , p . 850.
28 A. T. Jagendorf m C. Fuller et a l . : T h e Photochemical A p p a r a t u s , Its Structure a n d F u n c t i o n . B r o o k -
haven N a t i o n a l L a b o r a t o r y , U p t o n , N . Y. 1958, p . 236.
Adenosine-5 -diphosphate and Adenosine-5 -monophosphate
Dieter Jaworek, Wolfgang Gruber and Hans Ulrich Bergmeyer
T h e nucleoside p h o s p h a t e s , A D P a n d A M P , together with a d e n o s i n e - 5 ' - t r i p h o s p h a t e , are the m o s t widely

distributed p h o s p h a t e c o m p o u n d s in N a t u r e . T h e y occur in all cells including erythrocytes a n d t h r o m
bocytes, b u t are n o t n o r m a l l y present in serum.
T h e energy-rich p h o s p h a t e s represent the "energ y p o o l " from which the cells o b t a i n their energy require
ments. In pathological situations, where m e t a b o l i s m is altered, the specific d e t e r m i n a t i o n of nucleotides is
of diagnostic value a n d is increasingly used in heart, liver a n d kidney diseases.
T h e enzymatic d e t e r m i n a t i o n with m y o k i n a s e , M K ( A T P : A M P p h o s p h o t r a n s f e r a s e , E C 2.7.4.3),
pyruvate kinase, P K ( A T P : 2-O-pyruvate phosphotransferase, E C 2.7.1.40) a n d lactate dehydrogenase, L D H
(L-Lactate: N A D oxidoreductase, E C 1.1.1.27) estimates A D P a n d A M P in a single assay system. This
m e t h o d is preferable to the existing c h r o m a t o g r a p h i c m e t h o d s , especially with serial m e a s u r e m e n t s ,
because of its simplicity a n d speed.
Principle
(1) AMP + ATP 2 ADP
(2) 2 ADP + 2 PEP 2 A T P + 2 Pyruvate
(3) 2 Pyruvate + 2 N A D H + 2 H +
- ^ " ^ 2 Lactate + 2 N A D +
T h e decrease of N A D H , as m e a s u r e d by the change in extinction at 340 (334, 365) n m is p r o p o r t i o n a l t o the

a m o u n t of A M P a n d A D P present.
T h e equilibrium of reaction (3), K « 1 0 l./mole, is sufficiently far to the r i g h t to r e m o v e all the pyruvate.
4 1
T h e equilibrium of reaction (2), K = 2 x 1 0 at 30 ° C , is so far t o the right t h a t it can equilibrate with the
3 2
A D P d i s m u t a t i o n (1), which lies ca. 6 6 % to the left, a n d still remove all the A M P .
Equipment
334 or 365 n m ; b e n c h centrifuge.
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 4 . P o t a s s i u m c h l o r i d e , A . R.
2. P o t a s s i u m c a r b o n a t e , A . R. 5. P h o s p h o e n o l p y r u v a t e , PEP
3. M a g n e s i u m s u l p h a t e , MgS0 -7H 0,4 2 t r i c y c l o h e x y l a m m o n i u m salt; commercial
A . R. p r e p a r a t i o n , see p . 548.
6. S o d i u m h y d r o g e n c a r b o n a t e , NaHC0 , 3 9. P y r u v a t e k i n a s e , P K
A . R. from rabbit skeletal muscle, crystalline suspens
7. R e d u c e d n i c o t i n a m i d e - a d e n i n e di ion in 3.2 M a m m o n i u m sulphate solution;
nucleotide. N A D H ^ 200 U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n ,
disodium s a l t , N A D H - N a ; commercial
2 see p. 509.
preparation, see p . 545. 10. L a c t a t e d e h y d r o g e n a s e , L D H
8. M y o k i n a s e , M K from rabbit muscle, suspension in 3.2 M
from rabbit skeletal muscle, crystalline suspens a m m o n i u m sulphate s o l u t i o n ; ^550 U/mg.
ion in 3.2 M a m m o n i u m sulphate s o l u t i o n ; (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 481.
^ 360 U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , 1 1 . P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , s p . gr.
see p. 486. 1.67
12. T r i c h l o r o a c e t i c a c i d , A . R.
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y d i s t i l l e d w a t e r . To a v o i d t h e g r o w t h o f m i c r o - o r g a n i s m s
I. T r i e t h a n o l a m i n e h y d r o c h l o r i d e / K C 0
2 3 (0.43 M t r i e t h a n o l a m i n e ; 0.55 M K C 0 ) : 2 3
D i s s o l v e 1.6 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e a n d 1.5 g. K C 0 2 3 in distilled w a t e r

and m a k e u p to 20 ml.
II. P h o s p h o e n o l p y r u v a t e / M g S O J K C l ( 1 4 m M P E P ; 0.5 M M g S 0 ; 1.8 M K C 1 ) : 4
D i s s o l v e 2 0 m g . P E P , 3 7 0 m g . M g S 0 - 7 H 0 a n d 4 0 0 m g . KC1 in 3 m l . distilled w a t e r .
4 2
III. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 1 4 m M / ? - N A D H ) :
D i s s o l v e 12 m g . N A D H - N a 2 in 1 ml. 5 % N a H C 0 3 solution.
I V . M y o k i n a s e , M K (5 m g . p r o t e i n / m l . ) :
Dilute the stock suspension accordingly with 3.2 M a m m o n i u m sulphate solution
and mix.
V. P y r u v a t e k i n a s e , P K ( 1 0 m g . p r o t e i n / m l . ) :
D i l u t e s t o c k s u s p e n s i o n a c c o r d i n g l y w i t h 3.2 M a m m o n i u m s u l p h a t e s o l u t i o n and
mix.
VII. Perchloric acid:

a) 0.2 M : d i l u t e 1.7 m l . p e r c h l o r i c a c i d t o 100 m l . w i t h d i s t i l l e d w a t e r .
b) 0.9 M : d i l u t e 7.7 m l . p e r c h l o r i c a c i d t o 100 m l . w i t h d i s t i l l e d w a t e r .
V I I I . T r i c h l o r o a c e t i c a c i d (1 M ) :
D i s s o l v e 1 6 . 3 g. t r i c h l o r o a c e t i c a c i d in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
Store all solutions, stoppered, in a refrigerator at 0 - 4 °C. Solutions I, VII a n d VIII are stable indefinitely,
the undiluted enzyme suspensions are stable for ca. one year. P r e p a r e the P E P a n d N A D H solutions
freshly each week.
Adenosine-5'-diphosphate and Adenosine-5'-monophosphate 2129
Procedure
D r o p b l o o d d i r e c t l y f r o m t h e v e i n i n t o a c i d a n d stir i m m e d i a t e l y . C o l l e c t t i s s u e w i t h f r e e z e -
s t o p t o n g s ( s e e " C e l l a n d T i s s u e D i s i n t e g r a t i o n " p. 4 0 0 ) .
Deproteinization :
F o r t h e d e t e r m i n a t i o n o f A D P a n d A M P in b l o o d a n d t i s s u e e x t r a c t s refer t o d e t e r m i n a t i o n
of A T P (p. 2099).
B l o o d : M i x 5 m l . b l o o d a n d 5 m l . 1 M t r i c h l o r o a c e t i c a c i d a n d i m m e d i a t e l y c e n t r i f u g e for
10 m i n . at c a . 3 0 0 0 r p m . P i p e t t e 5 m l . s u p e r n a t a n t fluid i n t o a g r a d u a t e d test t u b e , n e u t r a l i z e
w i t h 2.2 m l . s o l u t i o n I a n d d i l u t e t o 10 m l . w i t h distilled w a t e r . T a k e 2 m l . o f this s o l u t i o n for t h e
assay.
T i s s u e : S e e d e t e r m i n a t i o n o f A T P , p. 2 0 9 9 .
A n y c h a n g e in t h e p h y s i o l o g i c a l state c a u s e s i m m e d i a t e c h a n g e s in t h e c o n t e n t o f the e n e r g y -
rich n u c l e o t i d e s . T h e r e f o r e a l l o w b l o o d t o d r o p directly i n t o a c i d , freeze o r g a n s in situ w i t h
d e e p - c o o l e d m e t a l b l o c k s a n d s t o r e at — 30 ° C until r e a d y for w o r k u p . F o r i n f o r m a t i o n o n
stability, s e e T a b l e 4 o n p . 1 6 6 .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : for A D P 2 . 2 4 m l . , for

A M P 2 . 2 6 m l . ; r o o m t e m p e r a t u r e ; R e a d a g a i n s t air. F o r e a c h s e r i e s o f m e a s u r e m e n t s p r e p a r e
a b l a n k c o n t a i n i n g distilled w a t e r i n s t e a d o f s a m p l e .
n e u t r a l i z e d , buffered) 2.00 ml. 10-80 pM
P E P / M g S 0 / K C l solution
4 (II) 0.15 ml. 0.94 m M P E P ; 33.8 m M M g S 0 ; 4
0.12 M KC1
L D H suspension (VI) 0.02 ml. 4 4 . 3 /xg./ml. = 2 4 U / m l .
M i x a n d after 5 m i n . r e a d e x t i n c t i o n E.
t
P K suspension (V) 0.02 ml. 8 8 . 5 / x g . / m l . = 18 U / m l .
M i x a n d w a i t for the c o m p l e t i o n o f t h e r e a c t i o n (ca.

5 m i n . ) , o t h e r w i s e t a k e a further 3 - 5 r e a d i n g s at 2 m i n .
intervals a n d e x t r a p o l a t e E t o t h e t i m e o f a d d i t i o n o f
2
suspension V.
M K suspension %
(IV) 0.02 ml. 4 4 . 3 / i g . / m l . = 16 U / m l .
M i x a n d w a i t for the c o m p l e t i o n o f t h e r e a c t i o n (ca.

15 m i n . ) , o t h e r w i s e t a k e a further 3 - 5 r e a d i n g s at
2 min. intervals and extrapolate E 3 to the time of
addition of suspension IV.
^ E A D P = (E x — E )2 s a m p l e — (E t — E ) 2 b l a n k
AE A M P = (E 2 - E )
3 s a m p l e - (E 2 - E ) i3 b a n k
Calculations
U n d e r the above conditions the reactions proceed stoichiometrically a n d therefore the calculation formula
(2) on p . 312 applies. T h e results are obtained as /rniole A D P or A M P per ml. sample. This value must be
multiplied by a factor to correct for the dilution on deproteinization. Blood c o n t a i n s ca. 80 % of its weight as
water and 1 ml. blood weighs 1.06 g. T h e addition of 5 ml. blood = 5.3 g. gives on deproteinization 5.3 x
8 0 / 1 0 0 + 5 = 9 . 2 4 ml. acid extract; this extract is diluted on neutralization 5 + 5. T h e dilution factor is
therefore
F _(5xl.06x0.8) + 5 ; c 10 3 6 %
T h e A D P or A M P concentration of b l o o d is calculated as follows:

ADP c = AE x 0.679 AE x 0.666 AE x 1.218 [/xmole/ml.]
c = AE x 290 AE x 284 AE x 520 [/*g./ml.]
AMP c = AE x 0.342 AE x 0.336 AE x 0.614 [/imole/ml.]
c = AE x 119 AE x 117 AE x 213 [/ig./ml.]
Adenosine-5'-diphosphate and Adenosine-5'-monophosphate 2131
With a m e a n value of 35 pg. A D P / m l . b l o o d a s t a n d a r d deviation of 3.6 pg. A D P was f o u n d ; coefficient

of variation is 5 . 1 % . W i t h a m e a n value of 8 pg. A M P / m l . blood a s t a n d a r d deviation of 2.2 fig. A M P
was f o u n d ; coefficient of variation is 13.5%.
N o r m a l Values
T h e n o r m a l values for b l o o d are 2 . 1 - 4 . 8 mg. A D P / 1 0 0 ml. a n d 0.5-1.2 mg. A M P / 1 0 0 m l . .

1
Interference in the assay technique: T h e N A D H p r e p a r a t i o n m a y contain A M P ; this will be corrected for

by the blank.
I D P , G D P , U D P a n d C D P also react, but at different rates. Neither I D P , G D P nor U D P can be distin

guished from A D P by the rate of the reaction, but a correction by extrapolation is possible in the case
of C D P . 2
References
1 G. Laudahn, Klin. Wschr. 37, 850 [1959].

2 H. Adam in H. U. Bergmeyer: M e t h o d e n der enzymatischen Analyse, Verlag Chemie, Weinheim/Berg-
straBe, 1st. edn., 1962, p . 577.
Adenosine Phosphates
Hans Mollering and Hans Ulrich Bergmeyer
The m e t h o d described here determines A - 2 - M P , A - 3 - M P , A - 5 - M P , A - 2 , 5 - d i p h o s p h a t e , A - 3 , 5 - d i p h o s p h a t e ,

A D P and A T P . A - 5 - M P , A D P or A T P can be determined separately. Kalckar 1
in 1947 was first to publish
a suitable m e t h o d for adenosine p h o s p h a t e s with adenosine d e a m i n a s e (Adenosine a m i n o h y d r o l a s e ,
EC 3.5.4.4) a n d "alkaline" phosphatase (Orthophosphoric-monoester phosphohydrolase, alkaline
o p t i m u m , E C 3.1.3.1). Alkaline p h o s p h a t a s e hydrolases adenosine p h o s p h a t e s to adenosine and inorganic
p h o s p h a t e . Adenosine d e a m i n a s e catalyses the d e a m i n a t i o n of adenosine to inosine (see " A d e n o s i n e " ,
p. 1919).
Application of Method: In biochemistry and in clinical biochemistry.
Principle
(1) Adenosine P h o s p h a t e s p h o s p h a t a s e
> A d e n o s i n e + Pi
(2) Adenosine Inosine + N H 3
T h e decrease of extinction at 265 n m due to the removal of adenosine is m e a s u r e d .
The equilibria of reactions (1) and (2) are completely to the right. T h e p H o p t i m u m of alkaline p h o s p h a t a s e
is between p H 9 - 1 0 and that of the deaminase a b o u t p H 7.4. A suitable p H for coupling the two reactions
is p H 8.0; values above p H 8 are to be avoided (see " A d e n o s i n e " , p . 1919).
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for p r e c i s e m e a s u r e m e n t s at 2 6 5 n m ; b e n c h c e n t r i f u g e .
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 4. Alkaline p h o s p h a t a s e
2. S o d i u m h y d r o x i d e , A . R . , 0.1 N for analytical purposes, from calf intestine,
3. A d e n o s i n e d e a m i n a s e suspension in 3.2 M ammonium sulphate
from calf intestinal mucosa, suspension in solution; ^ 500 U / m g . (p-nitrophenylphos-
3.2 M a m m o n i u m sulphate solution: ^ 200 p h a t e as substrate, 25 °C). C o m m e r c i a l prep
U / m g . (25 ° C ) ; commercial p r e p a r a t i o n , see aration, see p . 496.
p . 426. 5. P e r c h l o r i c a c i d , A . R . 7 0 % ( w / w ) , s p .
gr. 1.67.
Purity of Reagents
Adenosine deaminase must be free from A - 5 ' - M P deaminase a n d phosphodiesterase, similarly it should
contain not m o r e t h a n 0 . 0 1 % of alkaline p h o s p h a t a s e , A T P a s e , nucleoside p h o s p h o r y l a s e a n d xanthine
oxidase (relative to the specific activity of the deaminase). Alkaline p h o s p h a t a s e m a y be c o n t a m i n a t e d
Adenosine Phosphates 2133
with deaminase, b u t should n o t c o n t a i n m o r e t h a n 0 . 5 % p y r o p h o s p h a t a s e a n d 0 . 0 1 % nucleoside p h o s

phorylase or 0.02% A - 5 ' - M P d e a m i n a s e if the sample contains p y r o p h o s p h a t e - c o n t a i n i n g nucleotides
(NAD, N A D P , CoA).
P r e p a r e all s o l u t i o n s w i t h freshly p r e p a r e d , d o u b l y d i s t i l l e d w a t e r . T o a v o i d g r o w t h o f m i c r o
I. T r i e t h a n o l a m i n e buffer ( 5 0 m M ; p H 8 . 0 ) :
D i s s o l v e 9 3 0 m g . t r i e t h a n o l a m i n e - H C 1 in 6 0 m l . distilled w a t e r , a d j u s t t o p H 8.0 w i t h
0.1 N N a O H a n d d i l u t e t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r .
II. A d e n o s i n e d e a m i n a s e (0.1 m g . p r o t e i n / m l . ) :
III. A l k a l i n e p h o s p h a t a s e (5 m g . p r o t e i n / m l . ) :
I V . P e r c h l o r i c a c i d (1 M ) :
D i l u t e 9 m l . 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h d i s t i l l e d w a t e r .
Store all solutions, stoppered, in a refrigerator at 0 - 4 °C. Adenosine d e a m i n a s e a n d alkaline p h o s p h a t a s e

are stable for ca. 1 year, t h e buffer a n d perchloric acid solutions are stable indefinitely.
Procedure
Deproteinization :
Mixtures o f nucleotides, nucleic acid hydrolysates and similar experimental material d o n o t

require t o b e d e p r o t e i n i z e d . P r o t e i n - c o n t a i n i n g s a m p l e s ( e . g . w h o l e b l o o d , t i s s u e s ) m u s t b e
d e p r o t e i n i z e d w i t h p e r c h l o r i c a c i d b e c a u s e o f t h e h i g h e x t i n c t i o n at 2 6 5 n m d u e t o p r o t e i n
(see p . 1 9 2 0 ) . T r i c h l o r o a c e t i c a c i d is n o t s u i t a b l e a s a d e p r o t e i n i z i n g a g e n t b e c a u s e it a b s o r b s
at 2 6 5 n m .
Stability of sample
T h e perchloric acid extract s h o u l d be neutralized immediately and used for the m e a s u r e m e n t s .

A d e n o s i n e p h o s p h a t e s are u n s t a b l e in n e u t r a l i z e d e x t r a c t s , w h e r e a s a d e n o s i n e is s t a b l e f o r
at least 2 4 hr. at 2 5 ° C . I n a l k a l i n e h y d r o l y s a t e s ( c a . p H 1 0 - 1 1 ) o f n u c l e i c a c i d s t h e a d e n o s i n e
p h o s p h a t e s are s t a b l e f o r 2 4 hr. at 7 0 ° C .
2134 M e t a b o l i t e s : Nucleic Acids, Purines, Pyrimidines,"Nucleosides, C o e n z y m e s
Assay System
A s t h e s a m p l e s u s u a l l y c o n t a i n o t h e r n u c l e o t i d e s a n d n u c l e o s i d e s , t h e test is r e a d a g a i n s t a
b l a n k w h i c h c o n t a i n s sufficient s a m p l e s o t h a t at least 7 5 % o f t h e e x t i n c t i o n at 2 6 5 n m w h i c h is
n o t d u e t o a d e n o s i n e p h o s p h a t e s is c o m p e n s a t e d .
W a v e l e n g t h : 2 6 5 n m ; silica c u v e t t e s , light p a t h : 1 c m . ; final v o l u m e : 3 . 0 4 m l . ; r o o m t e m p e r
a t u r e . R e a d a g a i n s t buffer s o l u t i o n (I) o r buffer s o l u t i o n -f sample.
Buffer s o l u t i o n (I) 2.50 ml. ca. 4 2 m M

Sample (deproteinized, neutralized) 0.50 ml. up to 40 pg.
adenosine phosphates
M i x with a plastic spatula and read extinction E t
( s h o u l d n o t b e greater t h a n 0 . 5 0 0 ) .
Adenosine deaminase (II) 0.02 ml. 0.66 /ig./ml. = 132 m U / m l .
M i x a n d after c a . 5 m i n . r e a d t h e final e x t i n c t i o n E . 2
E — E =
{ AE 2 {
Alkaline phosphatase (III) 0.02 ml. 33 /ig./ml. = 11.6 U / m l .
M i x a n d after c a . 5 - 2 0 m i n . r e a d final e x t i n c t i o n E . 3
E 2
—
E 3 = A E .
2
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o a d d i t i o n o f t h e e n z y m e s u s p e n s i o n s b y t h e further
a d d i t i o n o f 0 . 0 2 m l . o f t h e s u s p e n s i o n s at t h e e n d o f t h e o v e r a l l r e a c t i o n . A d d t h e a p p r o p r i a t e
extinction change to the corresponding A E.
Calculations
AE Y = Ei — E 2 is propo'rtional t o the a m o u n t of adenosine a n d AE 2 = E 2 —E to the a m o u n t of

3
adenosine p h o s p h a t e s . W i t h absolutely p u r e adenosine we found an extinction difference A E of 0.303 for

10 pg. adenosine/ml. cuvette contents (37.4 pM) instead of the published value of A E 0 . 2 6 3 . T h e extinction 1
coefficient for 265 n m is therefore 8.1 c m . / ^ m o l e . T h e calculation formula (2) on p . 312 applies and the
2
results are obtained in /miole adenosine or jrniole adenosine p h o s p h a t e per ml. sample. This value must
be multiplied by a factor if the sample has been deproteinized, neutralized o r diluted in any way.
F o r this m e t h o d the concentrations of samples which have n o t been deproteinized are obtained as follows:
Adenosine c = AE l x 0.750 [^mole/ml.]

Adenosine c = AE X x 200 [/zg./ml.]
Adenosine p h o s p h a t e s c = AE 2 x 0.750 [/^mole/ml.]
If the sample has been deproteinized according to p . 1920 it is necessary t o multiply by the dilution factor
(2 + 0.05).
If a range of the p h o t o m e t e r scale is chosen so t h a t a A E of 0.010 can be read with sufficient accuracy,
it is possible to determine as little as 2 pg. adenosine p h o s p h a t e / m l . sample.
Adenosine Phosphates 2135
After alkaline hydrolysis of yeast ribonucleic acids we found in the hydrolysate 20.03 g. adenosine p h o s p h a t e
per 100 g. nucleic acid (2 s = 2.32). T h e coefficient of variation is 5.8%.
According to Kalckar 1
dinucleotides (e.g. N A D , N A D P , C o A ) or cyclic A - 3 : 5 - M P in the sample can
be determined in a third reaction step by addition of a d e n y l p y r o p h o s p h a t a s e or p h o s p h o d i e s t e r a s e
respectively.
See Adenosine p . 1922 a n d u n d e r " P u r i t y of R e a g e n t s " .
See Adenosine. T h e cyclic adenosine p h o s p h a t e s d o n o t react, b u t various analogues of adenosine p h o s

phates d o (see p . 1922).
References

Adenosine-3': 5-monophosphate, cyclic
Gerhard Michal and Peter Wunderwald
Cyclic adenosine 3 ' : 5 ' - m o n o p h o s p h a t e ( A - 3 : 5 - M P ) plays an i m p o r t a n t role in m e t a b o l i s m as t r a n s

mitter of n u m e r o u s h o r m o n a l effects ("second messenger", for reviews, see Robison, Butcher and Suther
land ).
1,2
A m o n g t h e m are the effects of glucagon, a d r e n o c o r t i c o t r o p h i c h o r m o n e , catecholamines, thyroid
stimulating h o r m o n e , luteotrophic h o r m o n e , vasopressin, prostaglandins, histamine, some of the releasing
h o r m o n e s in the anterior pituitary, etc. A - 3 : 5 - M P occurs in almost all cells of animals a n d also in micro
o r g a n i s m s . Some evidence suggests it occurs in higher p l a n t s . A - 3 : 5 - M P is formed by adenylate cyclase
3 4
(EC 4.6.1.1) from A T P and it is degraded by 3':5'-cyclic nucleotide p h o s p h o d i e s t e r a s e ( P D E ; E C 3.1.4.17)

to A - 5 - M P . A p a r t from a very slight side reaction, purified phosphodiesterase from Crotalus terrificus
terrificus (EC 3.1.4.1), crude v e n o m from Naja naja and ribonuclease (EC 3.1.4.22) d o not split A - 3 : 5 - M P . 5
The biological concentration of A - 3 : 5 - M P is very low in all tissues studied so far. Generally it is in the
range of 1 0 " - 1 0 "8 1 1
mole/g. t i s s u e . L o w concentrations of A - 3 : 5 - M P have also been found in u r i n e .
2,6 7
The only other cyclic nucleotide found so far in N a t u r e is cyclic g u a n o s i n e - 3 ' : 5 ' - m o n o p h o s p h a t e ( G - 3 : 5 -
M P ) . T h e biological concentration of this c o m p o u n d is usually a b o u t 1/30 of that of A - 3 : 5 - M P , but in
8 9
urine it is a b o u t 1/3 . O t h e r cyclic nucleotides could n o t be d e t e c t e d

8 8,10
, a l t h o u g h the occurence of a
u r i d i n e - 3 : 5 - M P splitting phosphodiesterase in heart has been d e s c r i b e d . 11
While m e a s u r e m e n t s of m i c r o m o l a r quantities of A - 3 : 5 - M P can be easily a n d specifically p e r f o r m e d 12
with s p e c t r o p h o t o m e t r i c assays, the low concentrations of A - 3 : 5 - M P present in biological samples

require m o r e sensitive techniques. A multitude of m e t h o d s have been described so far. They can be classified
as follows:
a) Activation of enzyme activities (e. g. of the whole phosphorylase s y s t e m 7 , 1 3

~ 1 7
o r of protein k i n a s e 18
" ) 2 0
by A - 3 : 5 - M P .
b) Isolation of A - 3 : 5 - M P by ion exchange c h r o m a t o g r a p h y w i t h o u t 21

or with use of high p r e s s u r e 2 2 , 2 3
a n d m e a s u r e m e n t of the ultraviolet a b s o r p t i o n .
c) Microbiological determination by the A - 3 : 5 - M P d e p e n d e n t m o v e m e n t of myxamoebae . 24
d) Conversion of A - 3 : 5 - M P to A - 5 - M P a n d then to A T P , which is d e t e r m i n e d by enzymatic c y c l i n g 25,26

,
by an exchange m e t h o d with labelled A T P 2 7
or by the luciferase luminescence r e a c t i o n . Similar 28
m e t h o d s can be employed for determination of G - 3 : 5 - M P 9 , 1 0 , 2 9 , 3 0

.
e) Isotope derivative m e t h o d s : Conversion of A - 3 : 5 - M P to A - 5 - M P , reaction of this c o m p o u n d with

32
P - l a b e l l e d A T P , c h r o m a t o g r a p h i c isolation of the 3 2
P - A D P formed a n d m e a s u r e m e n t of its radio
activity . Alternative: F o r m a t i o n of 2' ( [ H ] - a c e t y l ) - A - 3 : 5 - M P by reaction with [ H]-acetic anhydride,
6 3 3
isolation of the derivative and m e a s u r e m e n t of its r a d i o a c t i v i t y . 31
f) Enzymatic isotope dilution analysis: Decrease of the rate of [ H ] - A - 3 : 5 - M P splitting by p h o s p h o 3
diesterase in the p r e s e n c e 32
of unlabelled A - 3 : 5 - M P . F o r theoretical considerations, s e e 3 3 - 3 5
.
g) R a d i o i m m u n o a s s a y : Competitive binding of a labelled A - 3 : 5 - M P derivative a n d of an unlabelled

A - 3 : 5 - M P to a specially prepared antibody, precipitation of this complex with a second a n t i b o d y a n d
measurement of its r a d i o a c t i v i t y . 36
h) Protein binding assay: Competitive binding of labelled a n d unlabelled A - 3 : 5 - M P to the " b i n d i n g

p r o t e i n " of various tissues, isolation of the complex a n d m e a s u r e m e n t of the radioactivity present either
in the complex or in the u n b o u n d c o m p o u n d . Using a " b i n d i n g p r o t e i n " from lobster m u s c l e , a
3 7 - 3 9 40
similar test was developed for G - 3 : 5 - M P . These binding activities are most likely identical with the
4 1
cyclophosphate binding p r o p e r t y of protein k i n a s e s . 3 7 , 4 2 , 4 3

A d e n o s i n e - 3 ' : 5 ' - m o n o p h o s p h a t e (cyclic) 2137
While most of these m e t h o d s yield s o m e w h a t similar results, m a n y of t h e m a r e difficult t o h a n d l e , a n d , d u e

to the multitude of steps required, they are sensitive t o interference. Therefore, the r a d i o i m m u n o - a n d
the protein b i n d i n g techniques a r e preferable, since they require only few o p e r a t i o n s a n d have a high
accuracy a n d reproducibility. T h e widely used m e t h o d of Gilman , 31
f u r t h e r m o r e , gives a favorable
calibration curve ( c f . ) . It is outlined here.
44
Application of Method: In biochemistry, p h a r m a c o l o g y a n d medical research.
Principle
(1) [ H ] - A - 3 : 5 - M P + Binding p r o t e i n —• C o m p l e x (radioactive)

3
(2) A - 3 : 5 - M P + Binding p r o t e i n —• C o m p l e x
The following m e a s u r e m e n t s are carried o u t (Fig. 1):

(1) A given q u a n t i t y of binding protein (P mg.) binds a certain fraction of A moles of isotope-labelled
A - 3 : 5 - M P (shaded area). This complex is isolated by a d s o r p t i o n on Millipore® filters, a n d the r a d i o
activity c present in it is determined.
G
(2) If in addition to A moles of labelled A - 3 : 5 - M P there are also X moles of unlabelled A - 3 : 5 - M P present
(white area, sample or s t a n d a r d ) , i n c u b a t i o n with the same q u a n t i t y of b i n d i n g p r o t e i n m a y result in the
binding of m o r e substance to the protein (depending on the s a t u r a t i o n of the protein), b u t the fraction
of radioactive substance b o u n d , c , is smaller t h a n before. M e a s u r e m e n t s with k n o w n quantities of
x
unlabelled A - 3 : 5 - M P p e r m i t the establishment of a calibration curve, which is used for d e t e r m i n a t i o n

of u n k n o w n quantities of A - 3 : 5 - M P . Preferably, for the calibration curve, a plot of c / c versus the q u a n t i t y
0 x
X of the unlabelled material is used. This results (except with large a m o u n t s of p r o t e i n , see below) in a
practically straight line with an o r d i n a t e intercept of 1.0. T h e plot of c / c h a s the further a d v a n t a g e t h a t
Q x
the calibration curve is (within certain limits) insensitive t o changes in t h e b i n d i n g capacity of t h e p r o t e i n ,

and thus shows a " s e l f - c o m p e n s a t i n g " effect. A detailed theory of the b i n d i n g assay a n d its c o r r e l a t i o n
with experimental d a t a is given in'
(1)
(2)
Fig. 1. Principle of the protein
binding test. The areas represent X [mole]+ CxCCpm]
the quantities of the c o m p o n e n t s A [mole]
in the test. C [cpm]
A p H value of 4.0 is o p t i m u m for binding of low quantities of A - 3 : 5 - M P ; t h e K 3 7

m is lower t h a n at m o r e
physiological p H values. A b o u t 100 min. are required to reach equilibrium at 0 °C. At higher t e m p e r a t u r e s ,
the equilibrium is obtained faster, but thereafter a decline of binding occurs, possibly by d e n a t u r a t i o n of
the p r o t e i n . It is an a d v a n t a g e not to use such small quantities of binding protein that full saturation
37
is obtained, since then the b o u n d radioactivity is s m a l l 44

and the useful range of m e a s u r e m e n t s is limited.
O n the o t h e r h a n d , large quantities of binding protein result in a small slope of the calibration curve a n d
thus in decreased sensitivity . F u r t h e r m o r e , noticeable deviations of the curve from linearity occur in this
44
s i t u a t i o n . Likewise, large quantities of labelled A - 3 : 5 - M P in the assay mixture decrease the sensitivity,
44
small quantities d o n o t p e r m i t accurate radioactivity m e a s u r e m e n t s . Therefore, intermediate, m a t c h e d

44
concentrations b o t h of [ H ] - A - 3 : 5 - M P a n d binding protein are required. If 0.8 p m o l e of [ H ] - A - 3 : 5 - M P

3 3
(specific activity n o t less t h a n 20 C i / m m o l e ) are used in the assay mixture, we found a quantity of binding
protein, giving a saturation of 3 0 - 4 0 % most suitable ( c f . ) . 37
In order to avoid interference (see below), the extraction p r o c e d u r e should n o t introduce noticeable
quantities of salts. T h e recovery of A - 3 : 5 - M P d u r i n g the p r e p a r a t i o n of tissue extracts should be checked.
Equipment
Ice-bath, filtration apparatus with detachable top, scintillation counting apparatus with a
c o u n t i n g efficiency for [ H ] o f m o r e t h a n 2 0 % .
3
Reagents*
1. G l a c i a l a c e t i c a c i d , A . R., s p . gr. 1.05, 6. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , A . R.,

100% KH P0 2 4
2. S o d i u m h y d r o x i d e , A . R . , 1 N 7. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , A . R.,
3. A d e n o s i n e - 3 ' : 5 ' - m o n o p h o s p h o r i c acid, K HP02 4
c y c l i c , as c r y s t a l l i z e d free a c i d , 8. 2 - ( M e t h o x y ) - e t h a n o l ( e t h y l e n e g l y c o l
A-3:5-MPH 0; 2
m o n o m e t h y l e t h e r ) , A . R.
commercial p r e p a r a t i o n s : see p . 526. 9. T o l u e n e , for s c i n t i l l a t i o n measurements
4. [ H ] - A d e n o s i n e - 3 ' :5'-monophosphoric
3
10. 2 , 5 - D i p h e n y l o x a z o l e ( P P O )
a c i d , c y c l i c (specific a c t i v i t y n o t less t h a n 11. M i l l i p o r e ® filters H A W P 2 5 ,
20 C i / m m o l e ) as a m m o n i u m salt; p r o d u c e d by Millipore C o r p . , Bedford, Mass.,
commercial p r e p a r a t i o n s available. USA.
5. A - 3 : 5 - M P b i n d i n g p r o t e i n , p r e p a r e d a c
c o r d i n g t o Gilman 31
a n d t o Miyamoto et a l . ,
45
see A p p e n d i x , p. 2 1 4 3 .
Purity of Reagents
If the content of the a d e n o s i n e - 3 ' : 5 ' - m o n o p h o s p h o r i c acid p r e p a r a t i o n differs from the formula A - 3 : 5 -
M P H 0 (mol. wt. 347.2), correct the a m o u n t used for p r e p a r a t i o n of solution II correspondingly.
2
P r e p a r e all s o l u t i o n s w i t h d o u b l y distilled w a t e r .
I. A c e t a t e buffer ( 0 . 2 M a c e t a t e ; p H 4 . 0 ) :
D i l u t e 0 . 5 7 m l . g l a c i a l a c e t i c a c i d t o 4 5 m l . A d j u s t t o p H 4 . 0 w i t h 1 N N a O H (ca. 1.3 m l . )
* Complete reagent kits are available commercially (Boehringer M a n n h e i m ) .

II. A - 3 : 5 - M P s t a n d a r d s o l u t i o n ( 0 . 2 / i M ) :
D i s s o l v e 1 3 . 8 9 m g . A - 3 : 5 - M P H 0 (exactly2 4 0 / / m o l e ) in d i s t i l l e d w a t e r w i t h t h o r o u g h
stirring a n d m a k e u p t o 1 0 0 m l . D i l u t e 0 . 0 5 m l . o f this s o l u t i o n t o 1 0 0 m l . a g a i n w i t h
t h o r o u g h stirring.
III. [ H ] - A - 3 : 5 - M P (1 ^ C i / m l . ; c a . 4 0 n M ) :
3
D i s s o l v e 1.5 pCi [ H ] - A - 3 : 5 - M P (specific a c t i v i t y n o t less t h a n 2 0 C i / m m o l e ) in 1.5 m l .

3
water.
IV. A - 3 : 5 - M P binding p r o t e i n :
U s e t h e s t a n d a r d i z e d s o l u t i o n o f A - 3 : 5 - M P b i n d i n g p r o t e i n d e s c r i b e d in t h e A p p e n d i x .
V . P o t a s s i u m p h o s p h a t e buffer ( 2 0 m M , p H 6 . 0 ) :
D i s s o l v e 2 . 3 8 g. K H P 02 4 a n d 0 . 4 3 g. K H P 02 4 in w a t e r , m a k e u p t o 1 0 0 0 m l .
VI. Scintillation cocktail:
M i x 2 0 0 m l . 2 - ( m e t h o x y ) - e t h a n o l w i t h 8 0 0 m l . o f t h e s o l u t i o n o f 8 g. 2 , 5 - d i p h e n y l o x a z o l e
( P P O ) in 1 0 0 0 m l . t o l u e n e .
Store solutions I - I V frozen below —15 °C. In this state they are stable for m o r e t h a n 1 m o n t h . A - 3 : 5 - M P
binding protein may lose some binding capacity when repeatedly frozen a n d thawed. This may be c o n
trolled by determining the c value. Store solution V at 4 °C, it is stable for at least 1 m o n t h . Store the
Q
scintillation fluid in a b r o w n bottle at r o o m t e m p e r a t u r e . T h e solution is stable for m o r e t h a n 1 m o n t h .

Note: Solutions I - V are kept d u r i n g use in a n ice b a t h .
Procedure
F o r d e t e r m i n a t i o n o f t h e A - 3 : 5 - M P c o n c e n t r a t i o n in t i s s u e s * , u s e t h e f r e e z e - s t o p t e c h n i q u e
(p. 4 0 0 ) . H o m o g e n i z e f r o z e n p i e c e s o f t i s s u e o f a b o u t 5 0 m g . w i t h 1.0 m l . 6 % ( w / v ) t r i c h l o r o a c e t i c
a c i d in a n i c e - c o o l e d g l a s s h o m o g e n i z e r . C e n t r i f u g e in t h e c o l d a n d p i p e t t e off t h e s u p e r n a t a n t
fluid. A c i d i f y t h e s u p e r n a t a n t w i t h 0.1 m l . 0.1 N H C 1 . E x t r a c t 4 t i m e s w i t h a f o u r - f o l d v o l u m e
o f w a t e r - s a t u r a t e d e t h e r . E v a p o r a t e t h e s a m p l e in vacuo t o d r y n e s s . T a k e u p t h e r e s i d u e in a n
a p p r o p r i a t e a m o u n t o f d o u b l y d i s t i l l e d w a t e r for a n a l y s i s ( f o r m o s t t i s s u e s , 0.5 m l . are a d e q u a t e
w h e n 0.05 m l . are u s e d for a n a l y s i s ) .
To c h e c k r e c o v e r y , a d d t o a p a r a l l e l s a m p l e 2 - 4 n C i [ H ] - A - 3 : 5 - M P , c a r r y t h r o u g h t h e s a m e
3
procedure and determine the yield of radioactivity.

T h e p r e s e n c e o f i n t e r f e r i n g f a c t o r s m a y b e t e s t e d for b y a d d i n g a d e f i n i t e a m o u n t o f A - 3 : 5 - M P
( e . g . , 0.01 ml. o f s t a n d a r d s o l u t i o n II) t o t h e s a m p l e a n d b y c h e c k i n g f o r q u a n t i t a t i v e r e c o v e r y
u s i n g the c a l i b r a t i o n c u r v e . In c a s e o f d e v i a t i o n s o f t h e r e s u l t s , e s t a b l i s h a r e v i s e d c a l i b r a t i o n
c u r v e as f o l l o w s : a d d t o h a l f o f t h e n e u t r a l i z e d s a m p l e sufficient 3 ' : 5 ' - c y c l i c nucleotide
p h o s p h o d i e s t e r a s e * * ( 1 5 0 m U h y d r o l y s e 2 5 p m o l e A - 3 : 5 - M P in 3 0 m i n . ) , i n c u b a t e at 37 ° C ,
s t o p t h e r e a c t i o n b y i m m e r s i n g in a b o i l i n g w a t e r b a t h , c e n t r i f u g e a n d e v a p o r a t e t h e s u p e r n a t a n t
to dryness. Take u p the residue with d o u b l y distilled water (half the original v o l u m e o f sample).
* T h e m e t h o d was checked with liver. F o r o t h e r materials, modifications m a y b e c o m e necessary.

!
* Enzyme from beef heart, commercial p r e p a r a t i o n s , see p . 497.
A d d , e. g., 0 . 0 5 m l . o f t h i s s o l u t i o n t o t h e s t a n d a r d s w h e n e s t a b l i s h i n g t h e c a l i b r a t i o n c u r v e a n d
c o m p e n s a t e for this v o l u m e c h a n g e b y t h e a m o u n t o f w a t e r a d d e d . U s e t h e s a m e a m o u n t o f t h e
untreated sample for determining the a m o u n t o f A - 3 : 5 - M P .
W h e n large a m o u n t s o f i n h i b i t o r s are p r e s e n t p u r i f i c a t i o n b y c h r o m a t o g r a p h y o n a n i o n
exchangers ' 2 1 3 1
or by a c o m b i n a t i o n of electrophoresis and paper c h r o m a t o g r a p h y 4 6
is t o
b e preferred, i n s t e a d o f t a k i n g a c c o u n t o f the i n t e r f e r e n c e f a c t o r s in the c a l i b r a t i o n c u r v e .
F o r p l a s m a a n a l y s i s ( c f . ) let 5 m l . b l o o d flow freely i n t o a c e n t r i f u g e t u b e w h i c h c o n t a i n s 10 m g .
4 7
t h e o p h y l l i n e a n d 1 m g . h e p a r i n . M i x a n d c e n t r i f u g e ( 2 5 0 0 g, 15 m i n . ) . A d d t o 2 m l . p l a s m a
8 ml. ethanol, mix v i g o r o u s l y a n d centrifuge ( 6 0 0 0 g, 15 m i n . ) . S u s p e n d t h e p r e c i p i t a t e in
1 - 2 ml. ethanol/water mixture ( 4 + 1 , v/v). Centrifuge again and c o m b i n e the supernatants.
E v a p o r a t e t h e m in vacuo t o d r y n e s s a n d d i s s o l v e t h e r e s i d u e in 0 . 5 0 m l . w a t e r . U s e 0.05 m l .
for a n a l y s i s . T h e d e t e r m i n a t i o n o f t h e r e c o v e r y c a n b e p e r f o r m e d a s d e s c r i b e d for tissue
extracts.
U r i n e a n a l y s i s c a n b e p e r f o r m e d d i r e c t l y after d i l u t i o n o f t h e s a m p l e w i t h w a t e r ( 1 + 4 9 o r
1+99).
Assay System
U s e at least 3 s t a n d a r d s , e . g . w i t h 0 . 0 1 , 0 . 0 5 a n d 0 . 1 0 m l . o f s o l u t i o n II ( c o n t a i n i n g 2, 10 a n d
20 p m o l e of A - 3 : 5 - M P , respectively).
Pipette into
ice-cold Zero C o n c e n t r a t i o n in
Blank Standards Sample
microreagent standard assay mixture
tubes
A c e t a t e buffer (I) 0.05 ml. 0.05 ml. 0.05 ml. 0.05 ml. 0.05 M
S t a n d a r d (II) +
water — — 0.11 m l . u p to 100 n M
Sample + water — — —' 0.11 ml. u p t o 100 n M
Water 0.13 ml. 0.11 m l . — —

[ H]-A-3:5-MP
3
(III) 0.02 ml. 0.02 ml. 0.02 ml. 0.02 ml. 4nM
M i x w e l l . P i p e t t e i n t o t h e s o l u t i o n a n d rinse t h e t i p o f t h e p i p e t t e w i t h t h e
test m i x t u r e a f t e r w a r d s :
A-3:5-MP
binding protein — 0.02 ml. 0.02 ml. 0.02 ml. A-3:5-MP
(IV) binding protein,
sufficient f o r
binding o f ca.
4 0 % o f the
[ H]-A-3:5-MP
3
M i x by turning tubes carefully (avoid a n y shaking!). A l l o w to stand at

0 ° C for 1 0 0 m i n . A d d 1 m l . o f ice c o l d (!) p h o s p h a t e b u f f e r ( V ) . M o i s t e n
Millipore® filters with p h o s p h a t e buffer (V) a n d place t h e m o n the
filtration a p p a r a t u s . A p p l y t h e t e s t m i x t u r e t o t h e filters u n d e r s u c t i o n .
R i n s e e a c h w i t h 10 m l . o f i c e c o l d p h o s p h a t e b u f f e r ( V ) . D r y t h e filters
b y s u c t i o n . P l a c e t h e d r y filters i n t o s c i n t i l l a t i o n v i a l s a n d d i s s o l v e t h e m
in 1 m l . o f 2 - ( m e t h o x y ) - e t h a n o l w i t h g e n t l e a g i t a t i o n . A d d 10 m l . o f
s c i n t i l l a t i o n s o l u t i o n ( V I ) a n d s h a k e t h e c l o s e d v i a l s v i g o r o u s l y for 2 m i n .
A l l o w t o s t a n d for 2 hr. w i t h o c c a s i o n a l s h a k i n g u n t i l all m a t e r i a l h a s
b e e n d i s s o l v e d . D e t e r m i n e t h e c o u n t s / m i n . ( c p m ) for s t a n d a r d s a n d
s a m p l e s b y c o u n t i n g 1 0 0 0 0 c o u n t s . C o u n t t h e b l a n k f o r 10 m i n .
Calculations
Subtract the b l a n k from each m e a s u r e d v a l u e :
C
o = C
P z e r o standard ~~
m C
P blank
m
c = cpm
x s t a n d a r d - cpm b l a n k or
c = c p m ^ e - cpm
x b l a n k , respectively.
Calibration curve: Calculate the c / c values found from m e a s u r e m e n t s with different a m o u n t s of s t a n d a r d ,

0 x
considering that 0.01 ml. of solution II contains 2 pmole. Plot these values against the pmole used. A
straight line should result, which intersects the c / c axis at 1. Q x
Calculation: Take the c o r r e s p o n d i n g c / c value m e a s u r e d for the sample a n d read the n u m b e r of p m o l e

0 x
present from the calibration curve.
With the conditions described, 0.5 p m o l e m a y be measured with high accuracy. T h e coefficient of variation
u p to 20 pmole in the test is between 5 a n d 1 0 % . F o r m e a s u r e m e n t s on different days it is unnecessary t o
establish a new calibration curve every time. Small variations in the binding ability of the binding protein
are c o m p e n s a t e d for by the calculation of c / c . If c changes by m o r e t h a n 2 0 % , a new calibration curve
c x D
must be set u p .
N o r m a l Values
D u e to the present line of research, m o s t d a t a were m e a s u r e d on animals. T h e following d a t a were o b

tained by the different types of analytical systems, as indicated by letters at the beginning of this chapter.
W i t h the exception of fluids, the d a t a relate to g r a m wet weight of the organ. Liver ( r a t ) : 0 . 4 0 - 1 . 4 1 nmole/g
( a , d , d , e , g , h , h , ) ; skeletal muscle ( r a t ) : 0 . 2 7 - 0 . 6 6 nmole/g ( a , d , f , h , a ) ; skeletal
20 2 6 2 7 3 1 3 6 3 8 3 9 1 5 2 6 32 3 8 4 7
muscle (frog): 0.60 nmole/g. ( a ) ; brain ( m o u s e ) : 0 . 8 - 1 . 2 nmole/g. (anaesthetized, d ) , 1.5-1.8 nmole/g.

1 5 2 5
(decapitated, d ) ; brain ( r a t ) : 1.15-5.13 nmole/g. ( d , e , g , h ) ; kidney ( r a t ) : 0 . 5 - 1 . 5 2 nmole/g.

2 5 2 7 3 1 3 6 3 8
( e , g ) ; pituitary gland ( r a t ) : 0 . 8 - 1 . 4 nmole/g. ( g ) ; a d r e n a l gland ( r a t ) : 1-2.6 nmole/g. ( a , h ) ;

3 1 3 6 3 6 48 3 8
plasma ( m a n ) : 2 - 2 . 4 x 1 0 ~ M ( ) ; urine ( m a n ) : 2 - 7 ^ m o l e / d a y ( a , f , h ) or 0 . 5 6 - 5 uM, respectively

8 4 9 7 50 5 1
( c , d , d ) ; urine ( r a t ) : 0 . 2 4 - 0 . 8 9 uM ( d ) .
24 2 7 2 8 2 7
Sources of Error
Various c o m p o u n d s m a y inhibit the binding of labelled A - 3 : 5 - M P to the protein and thus simulate the
presence of an extra a m o u n t of A - 3 : 5 - M P in the sample. Besides structural analogues of A - 3 : 5 - M P
(see "Specificity of M e t h o d " ) , salts interfere with the test. T h e presence of 15 m M a m m o n i u m sulphate
decreases the c -value to less t h a n 50% of the original value. To avoid this source of error for absolute
0
measurements, the calibration curve has to be m e a s u r e d with the interfering substances of the sample
present. This m a y be d o n e by destroying A - 3 : 5 - M P with the specific phosphodiesterase as described
u n d e r "Collection, T r e a t m e n t a n d Stability of S a m p l e " .
According to Gilman , 31
the binding of A - 3 : 5 - M P at a 40 n M c o n c e n t r a t i o n is inhibited 5 0 % by the follow
ing concentrations of structurally related c o m p o u n d s , thus simulating the presence of extra A - 3 : 5 - M P :
300 n M i n o s i n e - 3 ' ^ ' - m o n o p h o s p h a t e , 5000 n M G - 3 : 5 - M P , 700 /iM G T P o r 1000 pM A T P . Still higher
concentrations of U T P , C T P , A - 5 - M P or A D P are required to cause this inhibition. Adenosine or theo
phylline d o not inhibit at a concentration of 1000 / / M . Synthetic analogues of A - 3 : 5 - M P such as N - 6
m o n o b u t y r y l - A - 3 : 5 - M P , b u t n o t N - 0 - 2 ' - d i b u t y r y l - A - 3 : 5 - M P or 0 - 2 ' - m o n o b u t y r y l - A - 3 : 5 - M P ,
6 52
may
also bind a n d interfere in this way. In samples of biological material, the interference by G - 3 : 5 - M P can
be neglected, while the influence of A T P m a y become a p p a r e n t only in tissues with very small concentrations
of A - 3 : 5 - M P a n d , therefore, a very u n f a v o u r a b l e ratio of the c o n c e n t r a t i o n s of b o t h c o m p o u n d s . In these

cases, a dilution of the sample is r e c o m m e n d e d . N o interference is to be expected from the physiological
37
concentrations of the other nucleosides a n d nucleotides m e n t i o n e d a b o v e .
Appendix
Preparation of A-3 :5 - M P Binding Protein
A - 3 : 5 - M P binding protein is p r e p a r e d from bovine muscle according to the p r o c e d u r e published by

Gilman 31
and Miyamoto et a l . . T h e following steps are carried out at 4 ° C : h o m o g e n i z e beef muscle with
4 5
3 volumes of neutral 4 m M E D T A . Centrifuge at 27000 g for 30 min. Precipitate by addition of 1 N acetic

acid to p H 4.8. Centrifuge at 27000 g for 30 min. Re-adjust the s u p e r n a t a n t to p H 6.5 with 1 M p o t a s s i u m
p h o s p h a t e buffer, p H 7.2. F r a c t i o n a t e by addition of 32.5 g. solid ( N H ) S 0 to each 100 ml. s u p e r n a t a n t .
4 2 4
Centrifuge at 16000 g for 20 min. Dissolve the precipitate in a v o l u m e of 5 m M p o t a s s i u m p h o s p h a t e

buffer, p H 7.0, which c o r r e s p o n d s to 6% of the initial crude extract. Dialyse against 20 volumes 5 m M
potassium p h o s p h a t e buffer p H 7.0. Centrifuge at 27000 g for 30 min. A p p l y the protein solution o n a
column containing 56 g./g. protein of D E A E cellulose (equilibrated with 5 m M p o t a s s i u m p h o s p h a t e
buffer, p H 7.0). Wash with 2 bed volumes 0.1 M p o t a s s i u m p h o s p h a t e buffer, p H 7.0, elute with 0.3 M
potassium p h o s p h a t e buffer, p H 7.0. Dialyse against 5 m M p o t a s s i u m p h o s p h a t e buffer, p H 7.0. P e r f o r m
the test procedure. T h e n dilute the dialysed solution in such a way, t h a t the c value o b t a i n e d with 0.02 ml.
D
of the solution in the test p r o c e d u r e c o r r e s p o n d s to 3 0 - 4 0 % of the total radioactivity applied. K e e p this

solution frozen in small p o r t i o n s below —15 °C.
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50 A. E. Broadus, N. I. Kaminski, R. C. Northcutt, J. G. Hardman, E. W. Sutherland8i G. W. Liddle, J. Clin.
Inv. 49, 2237 [1970].
51 M. I. Paul, B. R. Ditzion, G. L. Pauk & D. S. Janowski, A m . J. Psychiatry 126, 137 [1970].
52 B. L. Brown, J. G. Salway, D. M. Albano, R. P. Hullin & R. P. Ekins, Brit. J. Psychiatry 120,405 [1972].
53 E. Kaukel, K. Mundhenk & H. Hilz, Eur. J. Biochem. 27, 197 [1972].
Appendix
New Principle: Measurement by AMP-inhibition of Fructose-1,6-diphosphatase
Eric A . N e w s h o l m e
T h e principle briefly outlined here m a y p r o v e t o be a simple experimental m e t h o d for the determination of

small a m o u n t s of A - 3 : 5 - M P . A detailed m e t h o d based on this principle has not yet been worked out.
Fructose-1,6-diphosphatase, F D P a s e (D-Fructose-1,6-bisphosphate 1 - p h o s p h o h y d r o l a s e , E C 3.1.3.11) from
skeletal muscle is specifically inhibited by very small a m o u n t s of A - 5 - M P ( K j » 5 x 1 0 " M ) . Hydrolysis
7
of A - 3 : 5 - M P to A - 5 - M P would allow the d e t e r m i n a t i o n of the former in this concentration range.

T h e following steps would be required for the assay:
1. Collection of sample by the quick-freeze m e t h o d (see p . 400) a n d deproteinization.
2. Separation of A - 3 : 5 - M P from o t h e r c o m p o u n d s (e. g. A - 5 - M P ) by thin-layer c h r o m a t o g r a p h y , possibly
with the a d d i t i o n of radioactively labelled A-3 : 5 - M P to check recovery.
3. Elution of the c h r o m a t o g r a m , hydrolysis of A-3 : 5 - M P to A - 5 - M P by the specific phosphodiesterase
(see p . 2139) a n d inactivation of the enzyme by boiling.
4. A d d i t i o n of this pretreated sample t o the F D P a s e assay. A c o n t r o l consisting of the eluate from the
c h r o m a t o g r a m treated with inactive phosphodiesterase is also tested. F r o m the inhibition of F D P a s e
activity (relative to the c o n t r o l value) the a m o u n t of A - 5 - M P a n d t h u s of A - 3 : 5 - M P can be determined
by reference to a s t a n d a r d curve p r e p a r e d with A - 5 - M P .
Cytidine-5-triphosphate
Marianne Grassl
Cytidine-5'-triphosphate ( C T P ) has so far been detected in various animal o r g a n s a n d tissues, e. g. liver

a n d b r a i n , by c h r o m a t o g r a p h i c m e t h o d s which allow its separation from other nucleotides. To determine
1
the purity of C T P p r e p a r a t i o n s an enzymatic m e t h o d would seem preferable. T h e m e t h o d described

here is based on the reaction catalysed by fructose-6-phosphate kinase, F - 6 - P K ( A T P : D-fructose-6-
p h o s p h a t e 1-phosphotransferase, E C 2.7.1.11) in which fructose-1,6-diphosphate ( F - l , 6 - P ) a n d cyti 2
dine-5'-diphosphate ( C D P ) are formed from C T P a n d fructose-6-phosphate (F-6-P). In the presence of

aldolase ( D - F r u c t o s e - l , 6 - b i s p h o s p h a t e D-glyceraldehyde-3-phosphate-lyase, E C 4.1.2.13) F - l , 6 - P 2 is
converted to dihydroxyacetone p h o s p h a t e ( D A P ) a n d glyceraldehyde p h o s p h a t e ( G A P ) ; the latter is in
equilibrium with D A P via triose p h o s p h a t e isomerase, T I M (D-Glyceraldehyde-3-phosphate ketol-
isomerase, E C 5.3.1.1). Finally the D A P is converted to glycerol-3-phosphate with reduced nicotinamide-
adenine dinucleotide ( N A D H ) a n d glycerol-3-phosphate dehydrogenase, G D H ( M - G y c e r o l - 3 - p h o s p h a t e :
N A D 2-oxidoreductase, E C 1.1.1.8).
Principle
(1) C T P + F-6-P , ~ ~ * F - l , 6 - P
F 6 P K
2 + CDP
i 1
(2) F-l,6-P 2 (
a l d o l a s e
- GAP + DAP
(3) GAP t
T I M
- DAP
(4) 2DAP + 2NADH + 2 H +

t
G D H
- 2 Glycerol-3-P + 2 N A D +
T h e decrease of N A D H , as m e a s u r e d by the extinction change at 340 (334, 365) n m , is p r o p o r t i o n a l to

the a m o u n t of C T P present a n d t w o mole N A D H are oxidized per mole C T P .
The equilibrium of the indicator reaction is p H - d e p e n d e n t and in the neutral range lies on the side of
glycerol-3-phosphate.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r suitable for precise m e a s u r e m e n t s at 340, 334

o r 365 n m .
Reagents
1. H y d r o c h l o r i c a c i d , A . R . 3. Fructose-6-phosphate
2. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , t r i s disodium salt; c o m m e r c i a l preparation, see
p . 535.
4. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo 7. Aldolase
tide, N A D H from rabbit muscle, crystalline suspension in
2 3.2 M a m m o n i u m sulphate s o l u t i o n ; ^ 9 U / m g .
aration, see p . 545. (25 °C); commercial p r e p a r a t i o n , see p . 430.
5. M a g n e s i u m c h l o r i d e , M g C l 2 • 6 H 0 , A . R.
2 8. F r u c t o s e - 6 - p h o s p h a t e k i n a s e , F - 6 - P K
6. G l y c e r o l - 3 - p h o s p h a t e d e h y d r o g e n a s e / from yeast, crystalline suspension in 3.2 M
triose p h o s p h a t e i s o m e r a s e G D H / T I M ammonium sulphate s o l u t i o n ; ^ 70 U/mg.
from rabbit muscle, crystalline suspension in (25 °C).
3.2 M a m m o n i u m sulphate s o l u t i o n ; ^ 3 6 U
GDH/mg. (25 °C) and ^450 U TIM/mg.
(25 °C); commercial p r e p a r a t i o n , see p. 468.
Purity of Reagents
The enzymes must be as free as possible from p h o s p h a t a s e s ( 0 . 0 1 % relative to the activity of the individual
enzymes). W h e n the m e t h o d is applied to solutions which contain other metabolites the content of other
NAD-specific dehydrogenases should not exceed 0.01 % (relative to the activity of the individual enzymes).
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y distilled w a t e r . T o p r e v e n t t h e g r o w t h o f m i c r o

I. H y d r o c h l o r i c a c i d ( I N ) :
D i l u t e 2 0 m l . c o n e . HC1 (ca. 12 M ) w i t h 2 0 0 m l . distilled w a t e r .
II. Tris buffer (0.1 M ; p H 8 . 0 ) :
D i s s o l v e 1.2 g. tris in ca. 8 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 8.0 w i t h 1 N H C 1 (I) a n d d i l u t e
t o 100 m l . w i t h distilled w a t e r .
III. F r u c t o s e - 6 - p h o s p h a t e ( 1 4 m M ) :
D i s s o l v e 5 4 m g . F - 6 - P , d i s o d i u m salt, in 1 m l . distilled w a t e r .
IV. R e d u c e d nicotinamide-adenine dinucleotide (12 m M ) :
V . M a g n e s i u m c h l o r i d e (0.1 M ) :
D i s s o l v e 2 . 0 3 g. M g C l 2 • 6 H 0 in 100 m l . distilled w a t e r .
2
V I . G l y c e r o l - 3 - p h o s p h a t e d e h y d r o g e n a s e / t r i o s e p h o s p h a t e i s o m e r a s e , G D H / T I M (1.8 m g .
G D H / m l . ; 0.2 m g . T I M / m l . ) :
If n e c e s s a r y , d i l u t e t h e s t o c k s u s p e n s i o n s w i t h 3 . 2 M a m m o n i u m s u l p h a t e s o l u t i o n .
V I I . A l d o l a s e (10 m g . p r o t e i n / m l . ) :
V I I I . F r u c t o s e - 6 - p h o s p h a t e k i n a s e , F - 6 - P K (5 m g . p r o t e i n / m l . ) :
U s e the s t o c k s u s p e n s i o n u n d i l u t e d .
Solution I is stable indefinitely at r o o m t e m p e r a t u r e . Store solutions II, III, IV a n d V, stoppered, in a

refrigerator to avoid the growth of micro-organisms. U n d e r these conditions they are usable for ca.
2 weeks. T h e enzyme suspensions are stable for 6 to 12 m o n t h s at 4 °C.
Cytidine-5 '-triphosphate 2147
Procedure
T h e a s s a y h a s s o far o n l y b e e n carried o u t o n p r o t e i n - f r e e a q u e o u s s o l u t i o n s .
Stability of sample: In a p p r o x i m a t e l y n e u t r a l s o l u t i o n C T P is s t a b l e f o r s e v e r a l h o u r s ( 4 ° C ) .
T h e rate o f d e c o m p o s i t i o n is g r e a t l y a f f e c t e d b y s m a l l a m o u n t s o f c o n t a m i n a n t s (e. g. h e a v y
metals).
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e 2 . 9 4 m l . ; r e a d a g a i n s t air.
Tris buffer (II) 2.50 ml. 85 m M

F-6-P solution (III) 0.10 ml. 0.41 j n M
N A D H solution (IV) 0.05 ml. 0.2 m M
M g C l solution
2 (V) 0.10 ml. 3.4 m M
Sample 0.10 ml. 0.02-0.1 m M CTP
G D H / T I M suspension (VI) 0.02 ml. 0.43 U G D H / m l .
6.3 U T I M / m l .
Aldolase (VII) 0.02 ml. 0.6 U a l d o l a s e / m l .
M i x , f o l l o w e x t i n c t i o n c h a n g e until c o n s t a n t ( c a . 2 0
m i n . ) , t h e n read e x t i n c t i o n E .x
F-6-PK suspension (VIII) 0.05 m l . 5.9 U F - 6 - P K / m l .
Mix. After ca. 2 0 min. read the extinction every 2 m i n . ;

by extrapolation o f these values to the time o f F - 6 - P K
a d d i t i o n (see p . 3 0 8 ) d e t e r m i n e e x t i n c t i o n E 2 E!-E 2 =
A E is u s e d f o r t h e c a l c u l a t i o n s .
T h e i n c r e a s e in e x t i n c t i o n d u e t o a d d i t i o n o f F - 6 - P K a l o n e is d e t e r m i n e d b y t h e further a d d i t i o n
o f 0.05 m l . s u s p e n s i o n V I I I at t h e e n d o f t h e r e a c t i o n . T h e c h a n g e in e x t i n c t i o n m u s t b e u s e d t o
correct E . 2
Calculations
U n d e r the above conditions t h e reaction proceeds stoichiometrically with t h e oxidation of 2 mole N A D H

per mole C T P . Therefore in the calculation formula (2) o n p . 312 a n additional factor of 2 must a p p e a r
in the d e n o m i n a t o r . T h e results are obtained as /miole C T P / m l . sample. C h a n g e s in the v o l u m e of sample
must be taken into account. T h e following relationships hold :

c = AE x 1.16 AE x 1.14 AE x 2.09 [mg./ml.]
Interference in the assay technique: Insufficient purity of the enzymes (see p . 2146) can result in false
values. Side reactions which oxidize N A D H are a particular source of interference; it is then n o longer
possible to measure the end-point of the reaction with sufficient accuracy.
Fructose-6-phosphate kinase can react with practically all naturally occurring nucleoside triphosphates
as p h o s p h a t e d o n o r s . T h e rate of reaction with C T P is s o m e w h a t slower t h a n with purine nucleosides, but
a b o u t equal to the rates with other pyrimidine nucleotides . 2
U n d e r the conditions described here it is not possible to differentiate the individual triphosphates (refer
also to p . 2081).
C T P can also be determined with the aid of reactions catalysed by hexokinase (with glucose-6-phosphate
dehydrogenase as indicator enzyme) a n d by phosphoglycerate kinase (glyceraldehyde p h o s p h a t e d e h y d r o
genase as indicator enzyme). However, the rate of reaction in this assay system is very low a n d the assay
can last for at least 2 to 5 hr. and often does n o t give a quantitative result.
A n o t h e r possibility is the use of the reaction catalysed by gluconate kinase, coupled with the 6 - P G D H
reaction. In this system C T P can be quantitatively determined in ca. 30 m i n . . 3
References
1 H. Schmitz, R. B. Hurlbert & V. R. Potter, J. biol. C h e m . 209, 41 [1954].

2 W. Gruber, H. Mollering & H. U. Bergmeyer, Enzym. biol. clin. 7, 115 [1966].
3 H. Mollering, unpublished results.
Cytidine-5 -diphosphate, Guanosine-5-diphosphate
and Uridine-5'-diphosphate
Marianne Grassl
T h e nucleoside d i p h o s p h a t e s ( X D P ) are found in m a n y a n i m a l tissues a n d until n o w have been isolated by

c h r o m a t o g r a p h i c m e t h o d s a n d determined by m e a n s of their specific a b s o r p t i o n in the U V 1 , 2
. T h e con
centration of these c o m p o u n d s can be determined enzymatically with the aid of the reactions catalysed
by pyruvate kinase ( A T P : pyruvate 2-O-phosphotransferase, EC 2.7.1.40) a n d lactate dehydrogenase (L-
L a c t a t e : N A D oxidoreductase, E C 1.1.1.27). T h e m e t h o d described here is based on that for adenosine-
5'-diphosphate.
Principle
(1) X D P + PEP t
p y r u v a t e
- X T P + Pyruvate
kinase
I
D T H ^ M , • L-Lactate + NAD +
T h e decrease of N A D H , as m e a s u r e d by the extinction change at 340 (334, 365) n m , is p r o p o r t i o n a l t o

the a m o u n t of nucleoside d i p h o s p h a t e present.
T h e equilibrium of reaction (2) is strongly d e p e n d e n t o n the p H . In the n e u t r a l range it is practically

quantitatively on the side of N A D . T h e equilibrium position of reaction (2) displaces the equilibrium of
reaction (1) further in favour of p y r u v a t e formation.
Equipment
3 3 4 o r 365 n m , b e n c h c e n t r i f u g e .
Reagents
1. P e r c h l o r i c a c i d , A . R., 7 0 % ( w / w ) , s p . gr. 5. M a g n e s i u m s u l p h a t e , M g S 0 - 7 H 0 , A . R .
4 2
1.67 6. P o t a s s i u m c h l o r i d e , A . R.
2. P o t a s s i u m h y d r o x i d e , A . R. 7. P h o s p h o e n o l p y r u v a t e , P E P
3. T r i e t h a n o l a m i n e h y d r o c h l o r i d e t r i c y c l o h e x y l a m m o n i u m salt; commercial
4. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleo p r e p a r a t i o n , see p . 548.
tide, N A D H
2

8. L a c t a t e d e h y d r o g e n a s e , LDH 9. P y r u v a t e k i n a s e , P K
from rabbit skeletal muscle, crystalline suspens from rabbit muscle, crystalline suspension in
ion in 3.2 M a m m o n i u m sulphate solution; 3.2 M a m m o n i u m sulphate solution; ^ 200
^ 550 U / m g . (25 ° C ) ; commercial p r e p a r a t i o n , U / m g . (25 °C); c o m m e r c i a l p r e p a r a t i o n , see
see p . 4 8 1 . p . 509.
Purity of Reagents
L D H and P K must be as free as possible from p h o s p h a t a s e (less t h a n 0.01 %, relative to the activity of L D H
or P K ) . P E P should contain only traces of pyruvate.
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y distilled w a t e r . T o p r e v e n t t h e g r o w t h o f m i c r o

D i l u t e 5.2 m l . 7 0 % p e r c h l o r i c a c i d t o 100 m l . w i t h distilled w a t e r .
II. P o t a s s i u m h y d r o x i d e (2 N ) :
D i s s o l v e 11.2 g. K O H in c o l d w a t e r a n d m a k e u p t o 100 m l .
III. T r i e t h a n o l a m i n e buffer (0.1 M ; p H 7 . 6 ) :
D i s s o l v e 1.86 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in ca. 80 m l . d i s t i l l e d w a t e r , adjust t o
p H 7.6 w i t h 2 N K O H a n d d i l u t e t o 100 m l . w i t h distilled w a t e r .
IV. R e d u c e d nicotinamide-adenine dinucleotide (12 m M ) :
V . P h o s p h o e n o l p y r u v a t e / m a g n e s i u m s u l p h a t e / p o t a s s i u m c h l o r i d e ( 3 2 m M P E P ; 83 m M
M g S 0 ; 0.135 M K C 1 ) :
4
D i s s o l v e 15 m g . P E P - ( C H A ) 3 in 1 m l . a q u e o u s M g S 0 / K C l s o l u t i o n , w h i c h c o n t a i n s
4
5 g. M g S 0 - 7 H 0 a n d 5 g. KC1 in 5 0 0 m l . distilled w a t e r .
4 2
Store solutions I I - V and suspensions VI a n d VII, stoppered, in a refrigerator at 0 - 4 °C. Solutions I and II
are stable indefinitely at r o o m t e m p e r a t u r e (do n o t allow the K O H to remain u n s t o p p e r e d ) . Solutions I I - V
can be used for ca. one week (buffer solution III can only be kept for short periods because of the possibility
of bacterial growth). T h e enzyme suspensions are stable for ca. 12 m o n t h s .
Procedure
We h a v e s o far n o t carried o u t a n y d e t e r m i n a t i o n s o f C D P , G D P a n d U D P in b i o l o g i c a l
material. D u e to the instability o f nucleoside d i p h o s p h a t e s the samples s h o u l d be collected
as for the d e t e r m i n a t i o n o f A D P (see p . 2 1 2 9 ) .
Cytidine-, G u a n o s i n e - a n d U r i d i n e - 5 ' - d i p h o s p h a t e 2151
Deproteinization :
S e e u n d e r A D P , p . 2 1 2 9 . If t h e d e p r o t e i n i z e d a n d n e u t r a l i z e d s a m p l e c o n t a i n s less t h a n
0.3 / m i o l e n u c l e o s i d e d i p h o s p h a t e / m l . , i n c r e a s e t h e v o l u m e o f s a m p l e t a k e n for t h e a s s a y
a n d t a k e c o r r e s p o n d i n g l y l e s s buffer s o l u t i o n ( I I I ) .
T h e n u c l e o s i d e d i p h o s p h a t e s are o n l y s t a b l e for a f e w h o u r s in n e a r - n e u t r a l s o l u t i o n at 4 ° C .
Assay System
Pipette into cuvettes :
assay mixture
T R A buffer (Ill) 2.60 ml. 87 m M T R A

N A D H solution (IV) 0.05 ml. 0.2 m M NADH
PEP solution (V) 0.15 ml. 1.63 m M P E P ;
4.2 mM MgS0 ; 4
6.8 m M KC1
L D H suspension (VI) 0.02 ml. 33 pg. L D H / m l . = 1 8 . 2 U / m l .
Sample 0.10 ml. u p t o ca. 0.15 m M X D P
P K suspension (VII) 0.02 ml. 6 7 pg. P K / m l . = 1 3 . 4 U / m l .
Mix. O n c o m p l e t i o n of the reaction read extinction

E . Ei — E
2 2 = A E is u s e d for t h e c a l c u l a t i o n s .
T h e i n c r e a s e in e x t i n c t i o n d u e t o t h e a d d i t i o n o f P K ( V I I ) a l o n e is d e t e r m i n e d b y the further
a d d i t i o n o f 0.01 m l . s u s p e n s i o n ( V I I ) at t h e e n d o f t h e r e a c t i o n . E 2 m u s t b e c o r r e c t e d for t h i s
extinction change.
Calculations
(2) on p . 312 applies. T h e results are obtained in /miole nucleoside d i p h o s p h a t e / m l . sample. T h e value
must, however, be multiplied by the a p p r o p r i a t e factor if the sample has been deproteinized, neutralized or
diluted in any way. In the case of whole b l o o d the specific gravity (ca. 1.06) a n d the fluid content (ca. 80%)
must also be t a k e n into account. T h e following relationships hold for the present m e t h o d :

CDP c = AE x 1.94 AE x 1.90 AE x 3.49 [mg./ml.]
GDP c = AE x 2.14 AE x 2.09 AE x 3.83 [mg./ml.]
UDP c = AE x 1.95 AE x 1.91 AE x 3.50 [mg./ml.]
Interference in the assay technique: Insufficient purity of the reagents (see p. 2150), in particular the enzymes,
can lead to false results.
Pyruvate kinase has wide specificity with regard to naturally-occurring nucleoside d i p h o s p h a t e s , so that
apart from C D P , G D P and U D P , A D P , I D P a n d d - T D P a n d the o t h e r c o r r e s p o n d i n g deoxynucleoside
diphosphates are determined in the assay s y s t e m 3 - 5
. F o r a specific enzymatic d e t e r m i n a t i o n of A D P , see
p. 2127.
References
1 H. Schmitz, Naturwiss. 41, 120 [1954].

3 J. L. Strominger, Biochim. Biophys. Acta 16, 616 [1955].
4 A. Tietz & S. Ochoa, Arch. Biophys. Biochem. 78, 477 [1958].
5 M. Grass I, unpublished.
Cytidine-5'-monophosphate and Uridine-5'-monophosphate
Marianne Grassl
C y t i d i n e - 5 ' - m o n o p h o s p h a t e ( C - 5 - M P ) a n d u r i d i n e - 5 ' - m o n o p h o s p h a t e ( U - 5 - M P ) occur in m a n y different

tissues , o r g a n s a n d m i c r o - o r g a n i s m s . U - 5 - M P h a s also been found in the culture m e d i u m of, for example,
1 2 3
Lactobacillus arabinosus . 4
T h e m e t h o d s used at present, such as m e a s u r e m e n t of the extinction after c h r o
m a t o g r a p h y , are time-consuming a n d require considerable p r e t r e a t m e n t of the sample.
The enzymatic d e t e r m i n a t i o n employs nucleoside m o n o p h o s p h a t e kinase ( A T P : n u c l e o s i d e m o n o p h o s -
p h a t e phosphotransferase, E C 2.7.4.4), which in the presence of a d e n o s i n e - 5 ' - t r i p h o s p h a t e forms the cor
responding nucleoside d i p h o s p h a t e . These can be quantitatively d e t e r m i n e d in the reaction catalysed by
pyruvate kinase ( A T P : p y r u v a t e 2 - 0 - p h o s p h o t r a n s f e r a s e , E C 2.7.1.40) a n d lactate dehydrogenase (L-
L a c t a t e : N A D oxidoreductase, E C 1.1.1.27).
Application of Method: In biochemistry a n d possibly in clinical chemistry a n d foodstuff chemistry.
Principle
(1) C-5-MP (U-5-MP) + A T P t

n u c l e o s i d e m o n
° - . CDP (UDP) + ADP
v 7
phosphate kinase
(2) CDP (UDP) + A D P + 2 PEP t

p y
™ v a t e
- C T P ( U T P ) + A T P + 2 Pyruvate
(3) 2 Pyruvate + 2 N A D H + 2 H + l a c t a t e
- 2 L-Lactate + 2 N A D +
dehydrogenase
The a m o u n t of C - 5 - M P or U - 5 - M P is p r o p o r t i o n a l to the decrease of N A D H , as measured at 340 (334,

365) nm, a n d 2 mole N A D H are equivalent to 1 mole nucleotide.
T h e e q u i l i b r i u m o f r e a c t i o n (3) is v e r y d e p e n d e n t o n t h e p H a n d in t h e n e u t r a l r a n g e is p r a c t i c a l l y
q u a n t i t a t i v e l y o n t h e s i d e o f N A D . T h e e q u i l i b r i u m p o s i t i o n o f r e a c t i o n (3) a l s o d i s p l a c e s t h e
e q u i l i b r i a o f r e a c t i o n s ( 1 ) a n d (2) in f a v o u r o f n u c l e o s i d e d i p h o s p h a t e o r p y r u v a t e f o r m a t i o n .
Equipment
3 3 4 o r 365 n m . ; b e n c h c e n t r i f u g e ; s t o p w a t c h .
Reagents
1. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , s p . gr. 4. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucle
1.67 otide, N A D H
2. P o t a s s i u m h y d r o x i d e , A . R . d i s o d i u m salt, N A D H - N a . C o m m e r c i a l p r e p
2
3. T r i e t h a n o l a m i n e h y d r o c h l o r i d e a r a t i o n , see p . 545.
5. A d e n o s i n e - 5 ' - t r i p h o s p h a t e , A T P 11 . P y r u v a t e k i n a s e , P K
disodium salt, A T P - N a H - 3 H 0 . Commercial
2 2 2
from rabbit skeletal muscle, crystalline, suspen
preparation, see p . 527. sion in 3.2 M a m m o n i u m sulphate solution;
6. M a g n e s i u m s u l p h a t e , M g S 0 - 7 H 0 ,4 2
^ 2 0 0 U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n ,
A . R. p r e p a r a t i o n , see p . 509.
7. P o t a s s i u m c h l o r i d e , A . R . 12 . M y o k i n a s e , M K
8. P h o s p h o e n o l p y r u v a t e , P E P from r a b b i t or h o g muscle, suspension in 3.2 M
tricyclohexylammonium salt. Commercial prep a m m o n i u m sulphate solution; ^360 U/mg.
aration, see p . 548. (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p . 486.
9. R e d u c e d g l u t a t h i o n e , G S H 13 . N u c l e o s i d e m o n o p h o s p h a t e k i n a s e ,
Commercial preparation, see p. 538. NMPK
10. L a c t a t e d e h y d r o g e n a s e , L D H from beef liver, lyophilized; ^ 0 . 5 U / m g . enzyme
from rabbit skeletal muscle, crystalline sus protein (25 °C). C o m m e r c i a l preparations, see
pension in 3.2 M ammonium sulphate solution; p. 489.
^ 550 U / m g . (25 °C). Commercial preparation,
see p . 481.
Purity of Reagents
L D H , M K a n d P K must be as free as possible from N M P K and p h o s p h a t a s e s . N M P K should not contain

m o r e than 0.001% p h o s p h a t a s e s a n d G - 5 - M P kinase (relative to the activity of N M P K ) . P E P should contain
only traces of pyruvate, a n d A T P should c o n t a i n less t h a n 1% A - 5 - M P a n d A D P .
P r e p a r e all s o l u t i o n s w i t h fresh d o u b l y d i s t i l l e d w a t e r . Sterilize c o n t a i n e r s t o a v o i d bacterial

contamination.
II. P o t a s s i u m h y d r o x i d e ( 2 N ) :
D i s s o l v e 1 1 . 2 g. K O H in c o l d d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
III. T r i e t h a n o l a m i n e buffer (0.1 M ; p H 7 . 6 ) :
D i s s o l v e 1.86 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in c a . 8 0 m l . d i s t i l l e d w a t e r , adjust t o
p H 7.6 w i t h 2 N K O H a n d d i l u t e t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r .
IV. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleotide (12 m M ) :
V. Adenosine-5'-triphosphate (16 m M A T P ) :
D i s s o l v e 10 m g . A T P - N a H - 3 H 0 in 1 m l . distilled w a t e r .
2 2 2
V I . P h o s p h o e n o l p y r u v a t e / m a g n e s i u m s u l p h a t e / p o t a s s i u m c h l o r i d e ( 3 2 m M P E P ; 83 m M
M g S 0 ; 0.135 M K C 1 ) :
4
D i s s o l v e 15 m g . P E P - ( C H A ) 3 i n 1 m l . a q u e o u s M g S 0 / K C l s o l u t i o n ( c o n t a i n i n g 5 g.
4
M g S 0 - 7 H 0 a n d 5 g. K C 1 in 5 0 0 m l . d i s t i l l e d w a t e r ) .
4 2
VII. Glutathione (33 m M ) :

D i s s o l v e 10 m g . G S H in 1 m l . d i s t i l l e d w a t e r .
V I I I . L a c t a t e d e h y d r o g e n a s e / p y r u v a t e k i n a s e , L D H / P K ( e a c h 2.5 m g . p r o t e i n / m l . ) :
Dilute the stock suspensions accordingly with 3.2 M a m m o n i u m sulphate solution and
mix.
Cytidine-5'-monophosphate and Uridine-5'-monophosphate 2155
I X . M y o k i n a s e , M K (5 m g . p r o t e i n / m l . ) :
If n e c e s s a r y , d i l u t e t h e s t o c k s u s p e n s i o n w i t h 3 . 2 M a m m o n i u m s u l p h a t e s o l u t i o n .
X. Nucleoside m o n o p h o s p h a t e kinase, N M P K :
D i s s o l v e 6 0 m g . l y o p h i l i z e d p o w d e r ( c o n t a i n i n g 10 m g . N M P K ) in 1 m l . d i s t i l l e d w a t e r .
Store solutions III to VII a n d solution X, a n d all suspensions, stoppered, in a refrigerator at 0 to 4 °C.
Solutions III to VII are stable for ca. 1 week (buffer solution (III) should be used as soon as possible because
of the danger of bacterial c o n t a m i n a t i o n ) . P r e p a r e the glutathione solution freshly each day. T h e L D H / P K
and the M K suspensions are stable for ca. 12 m o n t h s ; the N M P K solution keeps for a b o u t 2 weeks.
Perchloric acid (I) a n d K O H (II) are stable indefinitely (do n o t leave K O H u n s t o p p e r e d ) .
Procedure
S o far C - 5 - M P a n d U - 5 - M P h a v e n o t b e e n d e t e r m i n e d in b i o l o g i c a l m a t e r i a l .
Deproteinization:
A d d 5 v o l . p e r c h l o r i c a c i d (I) t o b i o l o g i c a l m a t e r i a l , r e m o v e t h e p r e c i p i t a t e b y c e n t r i f u g a t i o n a n d
n e u t r a l i z e t h e s u p e r n a t a n t fluid w i t h K O H (II). A l l o w t h e m i x t u r e t o s t a n d f o r c a . 3 0 m i n . in a n
ice b a t h a n d t h e n filter off p o t a s s i u m p e r c h l o r a t e p r e c i p i t a t e . U s e t h e filtrate for t h e d e t e r m i n
a t i o n ; if the c o n c e n t r a t i o n o f C - 5 - M P o r U - 5 - M P is < 0.5 ^ m o l e / m l . , t a k e a larger v o l u m e o f
s a m p l e a n d c o r r e s p o n d i n g l y less buffer s o l u t i o n (III).
C - 5 - M P a n d U - 5 - M P are s t a b l e in w e a k l y a c i d t o n e u t r a l s o l u t i o n s , p r o v i d i n g t h a t n o b a c t e r i a l
contamination occurs.
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m . ; light p a t h : 1 c m . ; final v o l u m e : 3 . 1 0 m l . ; r o o m
Pipette into t w o cuvettes: Blank Sample
assay mixture
T R A buffer (III) 2.60 ml. 2.60 ml. 84 m M TRA

N A D H solution (IV) 0.07 ml. 0.07 ml. 0.27 m M NADH
A T P solution (V) 0.05 ml. 0.05 ml. 0.27 m M A T P
PEP solution (VI) 0.15 ml. 0.15 ml. 1.55 m M P E P
4.0 mM MgS0 4
6.5 mMKCl
G S H solution (VII) 0.10 ml. 0.10 ml. 1.7 mMGSH
L D H / P K suspension (VIII) 0.04 ml. 0.04 ml. 18 U L D H / m l .
6 U PK/ml.
M K suspension (IX) 0.01 m l . 0.01 m l . 6 U MK/ml.
N M P K solution (X) 0.05 ml. 0.05 ml. 0.25 U N M P K / m l .
M i x a n d f o l l o w e x t i n c t i o n c h a n g e s in b l a n k a n d s a m p l e c u v e t t e s
until c o n s t a n t (ca. 10 m i n . ) . R e a d e x t i n c t i o n E.1

Sample 0.03 ml. u p t o c a . 0.1 m M
M i x a n d after ca. 10 m i n . r e a d t h e e x t i n c t i o n at 2 m i n . i n t e r v a l s .
Determine extinction E 2 by extrapolation o f these values to the
t i m e o f s a m p l e o r w a t e r a d d i t i o n (refer t o p . 3 0 8 ) . (E t — E )2
sample - (î ~ E )
2 b l a n k = A E is u s e d for t h e c a l c u l a t i o n s .
Calculations
U n d e r the above conditions the reaction proceeds stoichiometrically a n d t h e conversion of 1 mole C - 5 - M P

or U - 5 - M P corresponds to the oxidation of 2 mole N A D H . T h e calculation formula (2) on p . 312 must there
fore also include the factor 2 in the d e n o m i n a t o r . T h e results are o b t a i n e d in //mole C - 5 - M P or U - 5 - M P
per ml. sample. This value must be multiplied by the a p p r o p r i a t e factor if the sample has been deproteinized
or diluted in any way. Alterations in the v o l u m e of sample taken for assay m u s t be allowed for. T h e follo
wing relationships hold.

c = AE x 8.47 AE x 8.30 AE x 15.20 [//mole/ml.]
C-5-MP c = AE x 2.74 AE x 2.68 AE x 4.91 [mg./ml.]
U-5-MP c = AE x 2.75 AE x 2.69 AE x 4.93 [mg./ml.]
Interference in the assay: T h e commercially available N M P K p r e p a r a t i o n s contain myokinase so that

it is necessary to add p u r e m y o k i n a s e t o the assay mixture before the sample so t h a t the A - 5 - M P present
(from A T P ) is rapidly removed. Otherwise this reaction takes 20 to 30 min. to complete. If t o o m u c h
Cytidine-5'-monophosphate and Uridine-5'-monophosphate 2157
N A D H is used u p in the preliminary reaction d u e to the c o n t a m i n a t i o n of the reagents with pyruvate a n d

A - 5 - M P , m o r e must be a d d e d . T h e reaction is strongly inhibited if the solutions are several days old a n d
have n o t been stored in a refrigerator. T h e reaction catalysed by N M P K is inhibited if the m a g n e s i u m con
centration is t o o high. T h e presence of p h o s p h a t a s e s (ATPase) causes an interfering side reaction which
can be corrected for by m e a n s of the b l a n k cuvette, b u t which prevents an a c c u r a t e assay.
Specificity o f M e t h o d 5
N M P K is specific for C - 5 M P a n d U - 5 - M P , but the p r e p a r a t i o n c o n t a i n s m y o k i n a s e a n d possibly G - 5 - M P

kinase, so that u n d e r the c o n d i t i o n s described here it is n o t possible to distinguish between the four nucleo
tides. T h e assay can be carried out in the presence of G - 5 - M P , if the reaction is started by addition of N M P K
(and not with sample) a n d G - 5 - M P is removed with G - 5 - M P kinase. In this case myokinase must n o t be
added to the assay mixture, because this enzyme m a y still be c o n t a m i n a t e d with a G - 5 - M P kinase, that also
reacts with U - 5 - M P to a slight extent. I-5-MP a n d d e o x y - T - 5 - M P are virtually inactive. D e o x y - C - 5 - M P
and d e o x y - U - 5 - M P can also be determined in this way, but the reactions t a k e a long time to complete. C T P ,
G T P , I T P or U T P can serve as p h o s p h a t e d o n o r s .
References
1 R. B. Hurlbert, H. Schmitz, A. F. Brumm & V. R. Potter, J. biol. C h e m . 209, 23 [1954].

3 K. Okunuki, K. Iwasa, F. Imamoto & T. Higashiyama, J. Biochem. (Tokyo) 45, 795 [1958].
4 / . Baddiley & A. P. Mathias, C h e m . a n d Industr. 1954, 277.
5 M. Grassl, U. Haid&H. Schebesch, unpublished experiments.
Guanosine-5'-triphosphate and Inosine-5'-triphosphate
Marianne Grassl
Whereas guanosine-5'-triphosphate ( G T P ) a p p e a r s to be of widespread occurrence in n a t u r e 1 - 3

, little is
k n o w n a b o u t the occurrence of inosine-5'-triphosphate (ITP). These nucleoside triphosphates have so
far been determined, after purification by c o l u m n a n d thin layer c h r o m a t o g r a p h y , by m e a n s of the charac
4 5
teristic U V absorption or enzymatically with the aid of various enzyme s y s t e m s . T h e m e t h o d described here
6
is based on the m e t h o d for the d e t e r m i n a t i o n of adenosine-5'-triphosphate (see p. 2097). Phosphoglycerate

kinase, P G K ( A T P : 3-phospho-D-glycerate 1-phosphotransferase, E C 2.7.2.3) transfers a p h o s p h a t e
residue from the nucleoside t r i p h o s p h a t e ( X T P ) to 3-phosphoglycerate with formation of 1,3-diphospho-
glycerate, which is reduced by the N A D - d e p e n d e n t glyceraldehyde-3-phosphate dehydrogenase, G A P D H
( D - G l y c e r a l d e h y d e - 3 - p h o s p h a t e : N A D oxidoreductase, p h o s p h o r y l a t i n g , E C 1.2.1.12) to glyceraldehyde
phosphate (GAP).
P ri nc iple
(1) X T P + 3-Phosphoglycerate ^J=^=± X D P + 1,3-Diphosphoglycerate
i
(2) 1,3-Diphosphoglycerate + N A D H + H + G A P D H
> GAP + N A D +
+ P ;
The decrease in the concentration of N A D H , which is measured by the change in extinction at 340 (334,
365) n m , is p r o p o r t i o n a l to the q u a n t i t y of nucleoside t r i p h o s p h a t e present.
The equilibrium of the indicator reaction (2) is p H - d e p e n d e n t , a n d lies on the side of G A P in the neutral
range.
Equipment
3 3 4 , o r 365 n m ; l a b o r a t o r y c e n t r i f u g e .
Reagents
1. P e r c h l o r i c a c i d , A . R., 7 0 % ( w / w ) , s p . gr. 5. M a g n e s i u m s u l p h a t e , M g S 0 - 7 H 0 , A . R. 4 2
1.67 6. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA
2. P o t a s s i u m h y d r o x i d e , A . R . disodium salt, E D T A - N a H - 2 H 0 2 2 2
3. Triethanolamine hydrochloride 7. R e d u c e d n i c o t i n a m i d e - a d e n i n e
4. 3-Phosphoglycerate dinucleotide, NADH
disodium salt. C o m m e r c i a l p r e p a r a t i o n s , see disodium salt, N A D H - N a . Commercial prep 2
p. 540. aration, see p. 545.

G u a n o s i n e - 5 ' - t r i p h o s p h a t e a n d Inosine-5 ' - t r i p h o s p h a t e 2159
8. G l y c e r a l d e h y d e p h o s p h a t e 9. P h o s p h o g l y c e r a t e k i n a s e , P G K
dehydrogenase, G A P D H from yeast, crystalline suspension in 3.2 M
from rabbit muscle, crystalline suspension in ammonium sulphate solution; ^400 U/mg.
3.2 M a m m o n i u m sulphate s o l u t i o n ; ^ 3 6 U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 502.
(25 °C). C o m m e r c i a l p r e p a r a t i o n , see p. 466.
Purity of Reagents
G A P D H a n d P G K m u s t be as free as possible from p h o s p h a t a s e s a n d m y o k i n a s e (less t h a n 0.001 %,

relative to the activity of G A P D H or P G K ) . T h e content of o t h e r NAD-specific dehydrogenases when the
m e t h o d is applied to biological material should be less t h a n 0.001 %, relative to the activity of G A P D H or
PGK.
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y d i s t i l l e d w a t e r . Sterilize c o n t a i n e r s t o p r e v e n t the g r o w t h

of micro-organisms.
II. P o t a s s i u m h y d r o x i d e s o l u t i o n (2 N ) :
D i s s o l v e 11.2 g. K O H in c o l d d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l .
III. T r i e t h a n o l a m i n e / 3 - p h o s p h o g l y c e r a t e s o l u t i o n (0.1 M t r i e t h a n o l a m i n e , 7 m M 3 - p h o s p h o -
g l y c e r a t e , 3 m M M g S 0 , 1.4 m M E D T A ; p H 7 . 6 ) :
4
D i s s o l v e 1.86 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e , 2 1 0 m g . 3 - p h o s p h o g l y c e r a t e disodium
salt, 125 m g . M g S 0 4 • 7 H 0 , and 50 mg. E D T A - N a H
2 2 2 2 H 0 in c a . 8 0 ml. distilled w a t e r ,
2
adjust t o p H 7.6 w i t h 2 N K O H (II), a n d m a k e u p t o 100 m l . w i t h d i s t i l l e d w a t e r .

IV. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 1 2 m M ) :
D i s s o l v e 10 m g . N A D H - N a 2 in 1 ml. d i s t i l l e d w a t e r .
V. G l y c e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase/phosphoglycerate kinase, GAPDH/PGK
(5 m g . p r o t e i n / m l . e a c h ) :
M i x s t o c k s u s p e n s i o n s , e a c h c o n t a i n i n g 10 m g . e n z y m e p r o t e i n , in a r a t i o o f 1 : 1.
Solution I and II keep indefinitely at r o o m t e m p e r a t u r e (do not leave K O H solution open). Keep solutions
III and IV a n d enzyme suspensions in closed vessels in a refrigerator at 4 °C. Solutions III and IV are
fit for use for a b o u t a week, a n d the enzyme suspensions for a b o u t 6 t o 12 months^
Procedure
T h e s e n u c l e o s i d e t r i p h o s p h a t e s h a v e s o far b e e n d e t e r m i n e d o n l y in p r o t e i n - f r e e s o l u t i o n s .
H o w e v e r , the m e t h o d s h o u l d a l s o b e s u i t a b l e for a p p l i c a t i o n t o o r g a n e x t r a c t s a n d t h e l i k e . 6
Take b l o o d w i t h o u t v e n e s t a s i s . A d d i t i o n o f o x a l a t e , citrate, or E D T A (1 m g . / m l . in e a c h c a s e )
d o e s n o t interfere w i t h t h e d e t e r m i n a t i o n . C o l l e c t t i s s u e (e. g. f r o m l a b o r a t o r y a n i m a l s ) w i t h t h e
q u i c k - f r e e z e t o n g s (refer t o " C e l l a n d T i s s u e D i s i n t e g r a t i o n " , p. 4 0 0 ) .
Deproteinization:
See u n d e r A T P , p. 2 0 9 9 . U s e 0.1 m l . o f e x t r a c t for t h e a s s a y m i x t u r e . If t h e d e p r o t e i n i z e d a n d

n e u t r a l i z e d s a m p l e c o n t a i n s less t h a n 1 / / m o l e n u c l e o s i d e t r i p h o s p h a t e / m l . , the s a m p l e v o l u m e
m u s t be c o r r e s p o n d i n g l y i n c r e a s e d .
T h e n u c l e o s i d e t r i p h o s p h a t e s are s t a b l e for o n l y a f e w h o u r s in a p p r o x i m a t e l y n e u t r a l , p r o t e i n -
free s o l u t i o n .
Assay System
a t u r e ; m e a s u r e a g a i n s t air.
Triethanolamine/ 93 m M triethanolamine;
3-phosphoglycerate solution (Ill) 2.80 ml. 6.5 m M 3-phosphoglycerate;
2.8 m M MgS0 ; 4
1.3 m M EDTA
N A D H solution (IV) 0.05 ml. 0.2 m M NADH
Sample 0.10 ml. 0.03 to 0.15 m M X T P
M i x , read extinction E . x
G A P D H / P G K suspension (V) 0.04 ml. 2.4 U G A P D H / m l .

27 U P G K / m l .
M i x ; after r e a c t i o n h a s o c c u r r e d ( c a . 3 0 m i n . ) , r e a d E . 2
E - E = A E is u s e d in t h e c a l c u l a t i o n .
x 2
T h e i n c r e a s e in e x t i n c t i o n r e s u l t i n g f r o m t h e a d d i t i o n o f G A P D H / P G K is d e t e r m i n e d at t h e
e n d o f the r e a c t i o n b y a d d i t i o n o f a further 0 . 0 4 m l . o f s u s p e n s i o n ( V ) . T h e r e s u l t i n g e x t i n c t i o n
difference m u s t b e t a k e n i n t o a c c o u n t in E . 2
Calculations
The reaction proceeds stoichiometrically u n d e r the conditions indicated. F o r m u l a (2) on p. 312 is therefore
valid. T h e result is given as pmole nucleoside t r i p h o s p h a t e per ml. of sample. However, this value must be
multiplied by an a p p r o p r i a t e factor if the sample has been deproteinized, neutralized, or otherwise diluted.
W h e n whole blood is used, moreover, its specific gravity (ca. 1.06) a n d its fluid content (ca. 80%) must
be taken into account.
G u a n o s i n e - 5 '-triphosphate a n d Inosine-5'-triphosphate 2161
T h e concentration of the sample is therefore given by t h e following r e l a t i o n s h i p s :

AE x 4.90 AE x 4.81 AE x 8.79 [jimole/ml.]
GTP c = AE x 2.56 AE x 2.52 AE x 4.60 [mg./ml.]
ITP c = AE x 2.49 AE x 2.44 AE x 4.47 [mg./ml.]
Interference in assay technique: Inadequate purity o f the e n z y m e s (see p. 2 1 5 9 ) c a n lead t o

i n c o r r e c t results. S i d e r e a c t i o n s in w h i c h N A D H is c o n s u m e d a r e p a r t i c u l a r l y t r o u b l e s o m e ,
s i n c e t h e e n d - p o i n t o f t h e r e a c t i o n t o b e m e a s u r e d c a n n o l o n g e r b e d e t e r m i n e d w i t h sufficient
a c c u r a c y . I n this c a s e , it is a d v i s a b l e t o carry o u t a b l a n k test ( w i t h o u t s a m p l e ) .
Phosphoglycerate kinase c a n use practically all nucleoside t r i p h o s p h a t e s as t h e p h o s p h a t e d o n o r . T h e re

action rates with the purine nucleotides ( A T P , G T P , I T P ) are roughly equal, whereas those with t h e
pyrimidine nucleotides a r e considerably lower ( U T P 5 %, C T P 1 % of t h e reaction rate with A T P ) . T h e 6
deoxypyrimidine t r i p h o s p h a t e s ( d e o x y - C T P a n d deoxy-TTP) c a n n o t be d e t e r m i n e d in this w a y .

U n d e r the conditions described above, the individual triphosphates c a n n o t be distinguished. However,
this can be achieved in p a r t by simultaneous determination of A T P according to Gruber, Mollering, and
Bergmeyer . 6
By decreasing the quantity of P G K used in the assay to 1/10 of the original concentration,
it is possible to determine the purine riboside triphosphates, whereas the pyrimidine riboside t r i p h o s p h a t e s
react so slowly u n d e r these conditions that they cause only a "creep r e a c t i o n " , which can be taken into
account by extrapolation.
The nucleoside triphosphates can also be determined with the aid of the hexokinase-catalysed reaction
(with glucose-6-phosphate dehydrogenase as the indicator enzyme), possibly also with fructose-6-phosphate
kinase from yeast (see d e t e r m i n a t i o n of C T P , p . 2145) or with creatine kinase.
References
1 H. Schmitz, N a t u r w i s s . 41, 120 [1954].

3 P. Ayengar, D. M. Gibson, C. H. Lee Peng & D. R. Sanadi, J. biol. C h e m . 218, 521 [1956].
5 E. Randerath & K. Randerath, A n a l . Biochem. 12, 83 [1965].
6 W. Gruber, H. Mollering & H. U. Bergmeyer, E n z y m . biol clin. 7, 115 [1966].
Guanosine-5 -monophosphate
Marianne Grassl
As a precursor of ribonucleic acid G - 5 - M P is u b i q u i t o u s : it can be detected in the free state in m i c r o

organisms ' 1 2
and in various animal o r g a n s . G - 5 - M P can be determined by c h r o m a t o g r a p h y (ion ex
3
change c h r o m a t o g r a p h y on thin-layer p l a t e s 4
or c o l u m n s ) . These m e t h o d s frequently require prior
3
purification of the substance a n d therefore are rather time-consuming.

The determination of G - 5 - M P with the specific G - 5 - M P kinase ( A T P : ( d ) G M P phosphotransferase, E C
2.7.4.8), where G - 5 - M P is p h o s p h o r y l a t e d with A T P , is simpler. T h e resulting G D P a n d A D P are deter
mined by coupling with the reactions catalysed by pyruvate kinase, P K ( A T P : pyruvate 2 - 0 - p h o s p h o t r a n s -
ferase, E C 2.7.1.40) a n d lactate dehydro genase ( L - L a c t a t e : N A D oxidoreductase, E C 1.1.1.27).
Application of Method: In biochemistry, foodstuff chemistry a n d possibly in clinical chemistry.
Principle
(1) G-5-MP + ATP , G

~ - 5 M P
, GDP + ADP
kinase , ,
, I
I
(2) G D P + A D P + 2 P E P ., p y r u v a t e
- A T P + G T P + 2 Pyruvate
kinase i
I
(3) 2 Pyruvate + 2 N A D H + 2 H +
^Xglse' 2
L-Lactate + 2 N A D +
The decrease of N A D H , as measured by the extinction change at 340 (334, 365) n m , is p r o p o r t i o n a l

to the a m o u n t of G - 5 - M P present.
At neutral p H the equilibrium of the reactions (2) a n d (3) lies on the right side completely.
Equipment
S p e c t r o p h o t o m e t e r or s p e c t r u m - l i n e p h o t o m e t e r suitable for precise m e a s u r e m e n t s at 340,

3 3 4 o r 3 6 5 n m ; b e n c h c e n t r i f u g e (if d e p r o t e i n i z a t i o n is n e c e s s a r y ) .
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 7. Reduced nicotinamide-adenine
2. S o d i u m h y d r o x i d e , A . R . , 1 N dinucleotide, NADH
3. M a g n e s i u m sulphate, disodium salt, N A D H - N a ; commercial p r e p
2
MgS0 -7H 0, 4 2 A.R. a r a t i o n , see p . 545.

4. P o t a s s i u m c h l o r i d e , A . R . 8. L a c t a t e d e h y d r o g e n a s e , LDH
5. P h o s p h o e n o l p y r u v a t e , P E P from rabbit skeletal muscle (or pig skeletal
tricyclohexylammonium salt; commercial p r e p muscle), crystalline suspension in 3.2 M a m
a r a t i o n , see p . 548. m o n i u m sulphate s o l u t i o n : ^ 500 U / m g . (25 °C);
6. A d e n o s i n e t r i p h o s p h a t e , A T P commercial p r e p a r a t i o n , see p. 4 8 1 .
2 2 2
preparation, see p. 527.

Guanosine-5 '-monophosphate 2163
9. P y r u v a t e k i n a s e , P K 10. G u a n o s i n e - 5 ' - m o n o p h o s p h a t e k i n a s e ,
from rabbit skeletal muscle, crystalline suspens G - 5 - M P kinase
ion in 3.2 M a m m o n i u m sulphate s o l u t i o n : from pig brain, solution in 50% glycerol:
^ 2 0 0 U / m g . (25 °C); commercial p r e p a r a t i o n , ^ 10 U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n ,
see p . 509. see p. 472.
Purity of Reagents
L D H and P K must be free from G - 5 - M P kinase. T h e content of p h o s p h a t a s e , A T P a s e , myokinase a n d

nucleotide m o n o p h o s p h a t e kinase should not exceed 0 . 1 % relative to the activity of G - 5 - M P kinase.
P E P should be completely free from pyruvate. A T P must be especially p u r e ( > 1 % A D P + A - 5 - M P ) .
D i s s o l v e 1.86 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in ca. 5 0 m l . d i s t i l l e d w a t e r , a d j u s t t o
p H 7.6 w i t h 1 N N a O H a n d d i l u t e t o 1 0 0 m l . w i t h d i s t i l l e d w a t e r .
II. M a g n e s i u m s u l p h a t e / p o t a s s i u m c h l o r i d e / p h o s p h o e n o l p y r u v a t e ( 0 . 5 M M g S 0 4 ; 2 M KC1;
31.5 m M P E P ) :
D i s s o l v e 1.235 g. M g S 0 - 7 H 0 , 1 . 4 9 g. K C 1 a n d 150 m g . P E P , C H A - s a l t in 10 m l . d i s t i l l e d
4 2
water.
III. A d e n o s i n e t r i p h o s p h a t e ( 1 6 m M A T P ) :
D i s s o l v e 10 m g . A T P - N a H - 3 H 0 in 1 m l . d i s t i l l e d w a t e r .
2 2 2
IV. R e d u c e d n i c o t i n a m i d e - a d e n i n e dinucleotide (12 m M N A D H ) :

V. Lactate d e h y d r o g e n a s e / p y r u v a t e kinase, L D H / P K (2.5 m g . L D H / m l . ; 5 m g P K / m l . ) :
Dilute the stock suspensions accordingly with 3.2 M a m m o n i u m sulphate solution a n d
mix.
V I . G u a n o s i n e - 5 ' - m o n o p h o s p h a t e k i n a s e , G - 5 - M P k i n a s e (2 m g . G - 5 - M P k i n a s e / m l . ) :
Dilute the stock solution accordingly with 50% glycerol.
Store all solutions and suspensions, stoppered, in a refrigerator at 0 to 4 °C. Solutions II, III and IV are
stable for ca. 1 week, enzyme suspension V for ca. 1 year, enzyme solution VI for ca. 6 m o n t h s . Solution
I is often c o n t a m i n a t e d with bacterial growth after only a few days, therefore it should be prepared freshly
each week and stored overnight in a refrigerator.
Procedure
Collection and deproteinization:
F o r d e t a i l s o f t h e d e t e r m i n a t i o n in b l o o d a n d t i s s u e see t h e c h a p t e r o n A d e n o s i n e - 5 ' - m o n o
p h o s p h a t e , p. 2 1 2 9 . S o far w e h a v e h a d n o e x p e r i e n c e w i t h d e p r o t e n i z e d s a m p l e s .
G - 5 - M P is s t a b l e for at least a w e e k at 4 ° C in p r o t e i n - f r e e s o l u t i o n s a n d o v e r t h e p H r a n g e
4 t o 10.
Assay System
a t u r e ; read a g a i n s t b l a n k .
assay mixture
Buffer s o l u t i o n (I) 2.60 ml. 2.50 ml. 85 m M triethanolamine

Sample (neutralized) 0.10 ml. up to ca. 0.09 m M
G-5-MP
M g S 0 / K C l / P E P solution
4 (II) 0.15 ml. 0.15 ml. 25 m M MgS0 ; 4
0.1 M K C 1 ;
1.6 m M P E P
A T P solution (III) 0.10 ml. 0.10 ml. 0.53 m M A T P
N A D H solution (IV) 0.05 ml. 0.05 ml. 0.2 m M N A D H
L D H / P K suspension (V) 0.02 ml. 0.02 ml. 8.5 U L D H / m l .
6.8 U P K / m l .
M i x , f o l l o w t h e e x t i n c t i o n c h a n g e u n t i l c o n s t a n t (ca. 5 m i n . ) .
T h e n read e x t i n c t i o n E 1 for t h e b l a n k a n d s a m p l e .
G - 5 - M P kinase solution (VI) 0.02 ml. 0.02 ml. 140 m U / m l .
M i x a n d r e a d e x t i n c t i o n after 5 m i n . In c a s e t h e r e a c t i o n h a s n o t
r e a c h e d c o m p l e t i o n , r e a d t h e e x t i n c t i o n at 10 a n d 15 m i n . ; if a
"creep" reaction occurs, determine extinction E 2 for t h e b l a n k
and sample by extrapolation of these values to the time of
G - 5 - M P kinase addition (see p. 308).
(Ei - E ) 2 s a m p l e - (E1 - E ) 2 b l a n k - A E is u s e d for t h e c a l c u l a t i o n s .
Calculations
U n d e r the above conditions the reaction proceeds stoichiometrically and 2 mole N A D H are formed
for 1 mole G - 5 - M P . T h e calculation formula (2) on p . 312 applies, but the factor 2 must also appear in
the d e n o m i n a t o r . T h e results are obtained as pmole G - 5 - M P / m l . sample. If the sample has been treated
in any way (deproteinized, diluted or neutralized) the resulting value must be multiplied by the a p p r o p r i a t e
dilution factor. In the case of whole blood the specific gravity (ca. 1.06) a n d the water content (ca. 80 %)
must also be taken into account.
T h e following relationships hold for the calculation of the concentration in the s a m p l e :

c = AE x 0.875 AE x 0.858 AE x 1.57 [mg./ml.]
Guanosine-5 '-monophosphate 2165
Further D e t e r m i n a t i o n s
By addition of phosphodiesterase from beef heart, G - 5 - M P and cyclic G - 3 , 5 - M P can be determined

successively (see p . 2166).
If the enzymes are not sufficiently p u r e there are slow side reactions, which c a n n o t be corrected for exactly
by extrapolation. If the P E P contains p y r u v a t e o r the A T P c o n t a i n s t o o m u c h A D P , the N A D H c o n c e n t r a
tion is then t o o low. T h e remedy is to a d d m o r e N A D H before the a d d i t i o n of the G - 5 - M P kinase. If the
G - 5 - M P kinase contains some myokinase, m o r e myokinase (highest purity) can be a d d e d before the start
of the m e a s u r e m e n t s to completely remove A - 5 - M P .
Specificity
G - 5 - M P kinase reacts with deoxy G - 5 - M P as well as G - 5 - M P . A - 5 - M P , C - 5 - M P , I-5-MP, U - 5 - M P ,

and their c o r r e s p o n d i n g deoxy derivatives as well as d e o x y - T - 5 - M P d o not react with the enzyme. C T P ,
G T P , I T P a n d U T P can serve as p h o s p h a t e d o n o r s . T h e reaction with these c o m p o u n d s is considerably
slower . 5
So far no studies have been m a d e as to whether the N A D P H - d e p e n d e n t G - 5 - M P reductase from E n t e r o -

b a c t e r i a is suitable for the quantitative determination of G - 5 - M P . A m e t h o d for the m i c r o d e t e r m i n a t i o n
6
of guanosine nucleotides in the succinate thiokinase reaction has been published by Cha c . s . . 7
References
1 T. Okabayashi & E. Masuo, C h e m . P h a r m . Bull. (Tokoyo) 8, 370 [I960].

2 Jean Gregoire, Jana Gregoire & N. Limozin, Bull. Soc. Chim. Biol. 40, 767 [1958].
4 E. Randerath & K. Randerath, Analyt. Biochem. 12, 83 [1965].
5 M. Grafil, U. Haid & H. Schebesch, unpublished result.
6 / . Mager & B. Magasamik, J. biol. C h e m . 235, 1474 [I960].
7 S. Cha, Ch.J. Cha & J.S. Chim, Analyt. Biochem. 33, 174 [1970].
Guanosine-3': 5 -monophosphate, cyclic
Marianne Grassl
Interest in cyclic nucleotide m o n o p h o s p h a t e s continues to grow because of the widespread function of

cyclic a d e n o s i n e - 3 ' : 5 ' - m o n o p h o s p h a t e as a regulator of metabolism.
G u a n o s i n e - 3 ' : 5 ' - m o n o p h o s p h a t e , cyclic ( G - 3 : 5 - M P ) can be hydrolysed by a phosphodiesterase from beef
heart, P D E ( 3 ' : 5 ' - C y c l i c - A M P 5'-nucleotido-hydrolase, E C 3.1.4.17) to g u a n o s i n e - 5 ' - m o n o p h o s p h a t e
(G-5-MP) which can be determined enzymatically as described on p. 2162.
Application of Method: In biochemistry and p h a r m a c o l o g y .
Principle
(1) G-3:5-MP + H 0 2 G-5-MP
U n d e r the conditions described for the assay of G - 5 - M P the enzymatic hydrolysis requires ca. 1 hr.
Equipment
See p. 2162.
R e a g e n t s and S o l u t i o n s
P h o s p h o d i e s t e r a s e f r o m b e e f heart, P D E ( 1 0 m g . p r o t e i n / m l . ) ; s o l u t i o n in 5 0 % g l y c e r o l ,
p H a p p r o x . 7, 5 0 m M tris, 5 0 m M M g S 0 4 ; ^ 0 . 1 5 U / m g . (25 ° C , m e a s u r e d w i t h c y c l i c a d e n o
sine-3': 5 ' - m o n o p h o s p h a t e as substrate); c o m m e r c i a l preparation, see p. 526.
F o r o t h e r r e a g e n t s a n d s o l u t i o n s , see " G u a n o s i n e - 5 ' - m o n o p h o s p h a t e " , p. 2 1 6 2 , 2 1 6 3 .
P D E is s t a b l e for c a . 3 - 6 m o n t h s s t o r e d at 4 ° C .
Procedure
S o far t h e d e t e r m i n a t i o n o f G - 3 . 5 - M P h a s n o t b e e n carried o u t o n b i o l o g i c a l m a t e r i a l .
A q u e o u s , a p p r o x i m a t e l y n e u t r a l s a m p l e s are s t a b l e for several d a y s at 4 ° C .
Assay System
T h e a s s a y is carried o u t e x a c t l y a s d e s c r i b e d for G - 5 - M P o n p. 2 1 6 4 . O n c o m p l e t i o n o f t h e
reaction catalysed by G - 5 - M P kinase read extinction E , add 0.02 ml. P D E s u s p e n s i o n and mix.
2
G u a n o s i n e - 3 ' : 5 ' - m o n o p h o s p h a t e (cyclic) 2167
A f t e r 6 0 m i n . r e a d t h e e x t i n c t i o n at 5 m i n . i n t e r v a l s ; b y e x t r a p o l a t i o n t o t h e t i m e o f P D E
a d d i t i o n (see p . 3 0 8 ) d e t e r m i n e e x t i n c t i o n E .3
E -E
2 3 = A E is u s e d for t h e c a l c u l a t i o n s .
T h e c h a n g e in e x t i n c t i o n d u e t o P D E a l o n e is d e t e r m i n e d at t h e e n d o f t h e r e a c t i o n b y t h e
a d d i t i o n o f a further 0 . 0 2 m l . o f t h e e n z y m e . T h e e x t i n c t i o n c h a n g e is u s e d t o c o r r e c t E . 3
Calculations
W i t h adherence to the conditions used for the d e t e r m i n a t i o n of G - 5 - M P the calculations for the G - 3 , 5 - M P
concentration are the same as for G - 5 - M P .

c - AE x 0.832 AE x 0.816 AE x 1.49 [mg./ml.]
A p a r t from G - 3 : 5 - M P only d e o x y - G - 3 : 5 - M P reacts in this system.
Further D e t e r m i n a t i o n s
A similarly measuring principle has been published by Sutherland c . s . . F o r the d e t e r m i n a t i o n of very

1
small G - 3 : 5 - M P c o n c e n t r a t i o n s radiochemical m e t h o d s a n d protein binding a s s a y s are described.

2 3
References
1 J.G. Hardman, J. W. Davis & E. W. Sutherland, J. biol. C h e m . 241, 4812 [1966].

2 A. L. Steiner, Ch. W. Parker & D. M. Knipnis, J. biol. C h e m . 247, 1106 [1972].
3 F. Murad, V. Manganiello & N. Vaugham, P r o c . N a t . A c a d . Sci. U . S. 68, 736 [1971].
Inosine-5 '-monophosphate
Marianne Grassl
I n o s i n e - 5 ' - m o n o p h o s p h a t e (I-5-MP) is widely distributed in N a t u r e a n d has, for example, been d e m o n s t r a

ted in liver , t u m o r cells a n d m i c r o - o r g a n i s m s . T h e determination of this c o m p o u n d is of particular
1 2 3
interest in canned m e a t a n d fish, a n d in other p r o d u c t s of the foodstuff i n d u s t r y . Generally the estimation4
is carried out by the U V a b s o r p t i o n of I-5-MP after its separation from other nucleotides by c h r o m a t o
graphic m e t h o d s ' . A n enzymatic m e t h o d is described here, which has so far only been used to analyse
2 5
relatively p u r e p r e p a r a t i o n s of I - 5 - M P . It is based on the hydrolysis of I - 5 - M P by alkaline p h o s p h a t a s e , A P

( O r t h o p h o s p h o r i c - m o n o e s t e r p h o s p h o h y d r o l a s e (alkaline o p t i m u m ) , E C 3.1.3.1), conversion of the result
ing inosine with nucleoside phosphorylase, N P (Purine-nucleoside: o r t h o p h o s p h a t e ribosyltransferase,
E C 2.4.2.1) to h y p o x a n t h i n e which reacts with xanthine oxidase, X O D ( X a n t h i n e : oxygen oxidoreductase,
E C 1.2.3.2) to form uric acid. T h e increase in extinction at 293 n m which occurs in the latter reaction, is a
measure of the I-5-MP c o n c e n t r a t i o n . T h e d e t e r m i n a t i o n can be carried further by the addition of uricase
( U r a t e : oxygen oxidoreductase, E C 1.7.3.3). T h e allantoin formed does n o t a b s o r b at 293 n m , and therefore
a decrease in extinction is observed (see also D e t e r m i n a t i o n of Inosine, p . 1932). Cyclic inosine-3': 5'-mono
p h o s p h a t e can also be measured by this principle, if it is first converted to I-5-MP by the action of p h o s
phodiesterase . 6
Application of Method: In biochemistry a n d foodstuff chemistry.
Principle
(1) I-5-MP + H 0 2 Inosine + P }
I 1 1
'
(2) Inosine + P* H y p o x a n t h i n e + Ribose-5-P
* '
(3) Hypoxanthine + 2 0 2 + 2 H 0 - & ^ + Urate +
2 2H 0 2 2
(4) Urate + 2 H 0 2 Allantoin + H 0 2 2 + C0 2
T h e increase in extinction at 293 n m after the addition of xanthine oxidase or the decrease after addition
of uricase is p r o p o r t i o n a l to the a m o u n t of I-5-MP present.
Between p H 7 and 9 the equilibria of reactions (1), (3) a n d (4) are in favour of the reaction p r o d u c t s . It
is preferable to carry out the assay at p H 7.6, because at this p H the alkaline p h o s p h a t a s e a n d uricase are
still sufficiently active a n d the other enzymes are near o p t i m u m activity.
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at 2 9 3 n m .
Inosine-5 ' - m o n o p h o s p h a t e 2169
Reagents
1. T r i e t h a n o l a m i n e h y d r o c h l o r i d e 5. N u c l e o s i d e p h o s p h o r y l a s e , N P
2. S o d i u m hydroxide, A . R., 1 N from calf spleen, crystalline suspension in 3.2 M
3. A l k a l i n e p h o s p h a t a s e , A P ammonium sulphate solution; ^25 U/mg.
from calf intestine, suspension in 3.2 M (25 °C); commercial p r e p a r a t i o n , see p . 490.
a m m o n i u m sulphate solution; ^300 U/mg. 6. X a n t h i n e o x i d a s e , X O D
(25 °C); commercial p r e p a r a t i o n , see p . 496. from milk, suspension in 3.2 M ammonium
4. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA sulphate solution; 10 m M E D T A ; ^ 0 . 4 U / m g .
d i s o d i u m salt, E D T A - N a H - 2 H 02 2 2 (25 °C); commercial p r e p a r a t i o n , see p . 521.
7. Uricase
from pig liver. Solution in 50% glycerol;
50 m M glycine, 0.13 M N a C 0 ; ^ 8 U / m g .
2 3
(25 °C); commercial p r e p a r a t i o n , see p . 518.
Purity of Reagents
A P a n d N P m u s t be free from X O D a n d uricase, a n d X O D m u s t be free from uricase. This type of cross

c o n t a m i n a t i o n has n o t yet been observed.
D i s s o l v e 1.86 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e in c a . 5 0 m l . d i s t i l l e d w a t e r , adjust t o
p H 7.6 w i t h 1 N N a O H a n d d i l u t e w i t h d i s t i l l e d w a t e r t o 100 m l .
II. A l k a l i n e p h o s p h a t a s e , A P (5 m g . p r o t e i n / m l . ) :
III. E t h y l e n e d i a m i n e t e t r a - a c e t a t e (0.1 M ) :
D i s s o l v e 37 m g . E D T A N a H - 2 H 0 in 1 m l . distilled w a t e r .
2 2 2
I V . N u c l e o s i d e p h o s p h o r y l a s e , N P (5 m g . p r o t e i n / m l . ) :
V. Xanthine oxidase, X O D (10 mg. protein/ml.):
V I . U r i c a s e (2 m g . p r o t e i n / m l . ) :
U s e the stock solution undiluted.
Store all solutions a n d suspensions, stoppered, in a refrigerator. Solutions I a n d III should be m a d e

u p freshly ca. every 2 weeks. X O D is stable for ca. 3 - 6 m o n t h s , A P , N P a n d uricase for ca. 6 - 1 2 m o n t h s .
Procedure
T h i s m e t h o d h a s s o far o n l y b e e n u s e d o n p r o t e i n - f r e e , a q u e o u s s o l u t i o n s . I - 5 - M P is s t a b l e
for at least a w e e k at 4 ° C in p r o t e i n - f r e e s o l u t i o n s o f p H 4 - 1 0 .
Assay System
W a v e l e n g t h : 2 9 3 n m ; light p a t h : 1 c m . ; final v o l u m e : 2 . 9 2 m l . ; 37 ° C o r r o o m t e m p e r a t u r e ;
read a g a i n s t a b l a n k c u v e t t e .
Pipette into a cuvette: C o n c e n t r a t i o n in a s s a y m i x t u r e
T r i e t h a n o l a m i n e buffer (I) 2.70 ml. 90 m M

Sample 0.02 ml. 5-30 pM
A P suspension (II) 0.05 ml.
M i x a n d i n c u b a t e for 3 hr. at 37 ° C . 9 0 /zg./ml. = 2 7 U / m l .
E D T A solution (III) 0.10 ml.

N P suspension (IV) 0.02 ml.
3.4 m M
M i x and read extinction E v
X O D suspension (V) 0.01 m l .

34 /ig./ml. = 850 m U / m l .
M i x a n d o n c o m p l e t i o n o f t h e r e a c t i o n read e x
tinction E . 2
3 4 / i g . / m l . = 14 m U / m l .
Uricase solution (VI) 0.02 ml.
14^g./ml. = 112mU/ml.
M i x and o n c o m p l e t i o n of the reaction read ex
tinction E . 3
E 2 - E t = JE X O D orE 2 - E 3 = AE U r i c a s e are u s e d
T h e i n c r e a s e s in e x t i n c t i o n c a u s e d b y t h e a d d i t i o n o f X O D o r u r i c a s e a l o n e are d e t e r m i n e d
at t h e e n d o f t h e s e c o n d r e a c t i o n b y the s e p a r a t e a d d i t i o n o f the e n z y m e s . E 2 ( X O D ) or E 3
( u r i c a s e ) m u s t b e c o r r e c t e d for t h e e x t i n c t i o n c h a n g e s w h i c h o c c u r .
Calculations
U n d e r the conditions described here the reaction proceeds stoichiometrically a n d therefore the calculation
formula ( 2 ) on p . 312 applies.
e for X O D r e a c t i o n : 12.4 cm. //zmole

2
e for uricase reaction: 12.6 c m . / / m i o l e

2
The results are obtained in /miole/ml. sample. T h e following relationships h o l d :
from the X O D reaction

c = Ê X O D x 11.8 [//mole/ml.]
c = Ê X O D x 4.11 [mg./ml.]
from the uricase reaction

c = <dE uricase x 11.6 [//mole/ml.]
c = AE u r i c a s e x 4.04 [mg./ml.]
Inosine-5 ' - m o n o p h o s p h a t e 2171
Insufficient purity of the enzymes (see p . 2169) can lead to interference d u e to side reactions. Heavy
metals inhibit xanthine oxidase so that the rate of the reaction is greatly decreased. T h e slightest displace
m e n t in the wavelength setting of the s p e c t r o p h o t o m e t e r results in incorrect extinction readings. As
the a b s o r p t i o n m a x i m u m of uric acid and its salts is at 293 n m the correct wavelength setting of the instru
m e n t can be checked by measuring the extinction at adjacent wavelengths. C o n t a m i n a n t s in the sample
which have extremely high a b s o r p t i o n at 293 n m can m a k e the d e t e r m i n a t i o n extremely difficult or even
impossible.
As the actual m e a s u r e m e n t s in the assay are started with X O D , xanthine, h y p o x a n t h i n e , their ribosides
a n d xanthinosine p h o s p h a t e also react. T h e free bases a n d ribosides can, however, be estimated in a
separate assay in which alkaline p h o s p h a t a s e or nucleoside phosphorylase are omitted. Inosine-5'-diphos-
phate and t r i p h o s p h a t e can be determined by the m e t h o d s described on p . 2152 a n d 2158.
References
1 A. Tsevelever & R. E. Libinson, Biokhimiya 27, 305 [1962].

2 H. Schmitz, W. Hart & H. Ried, Z. Krebsforschung 60, 301 [1955].
3 Jean Gregoire, Jana Gregoire & Nicole Limozin, Bull. Soc. C h i m . Biol. 40, 767 [1958].
4 T. Fujita & Y. Hashimoto, N i p p o n Suisan G a k k a i s h i 26, 907 [I960].
5 A. Deutsch & K. Nilsson, A c t a chem. scand. 7, 1288 [1953].
6 M. Grassl & H. Schebesch, unpublished work.
Uridine-5 -triphosphate, Uridine-5 -diphosphate,
and Uridine-5 -monophosphate
Dietrich Keppler, Karlfried G a w e h n , and Karl D e c k e r
As precursors for the synthesis of nucleic acids a n d for the formation of u r i d i n e d i p h o s p h a t e sugars, t h e
uridinephosphates are detectable in nearly all forms of life. T h e enzymatic s p e c t r o p h o t o m e t r i c assay
m e t h o d described here is superior in its simplicity, sensitivity, a n d high specificity to the c h r o m a t o g r a p h i c
separation that has been c o m m o n l y used until n o w .
The irreversible oxidation of U D P g l u c o s e to U D P g l u c u r o n a t e , in which 2 mole of N A D are reduced per
mole of U D P g l u c o s e by U D P g l u c o s e dehydrogenase, U D P G - D H ( U D P g l u c o s e : N A D 6-oxidoreductase,
E C 1.1.1.22), is used as the indicator reaction. In the presence of glucose-1-phosphate, U T P is converted
by UDPglucose pyrophosphorylase, UDPGP (UTP: a-D-glucose-1-phosphate uridylyltransferase,
E C 2.7.7.9) into U D P g l u c o s e a n d p y r o p h o s p h a t e . T h e c o m b i n e d reactions are highly specific for uridine-
1
p h o s p h a t e . U D P can be converted by nucleosidediphosphate kinase, N D P K ( A T P : nucleosidediphosphate

phosphotransferase, E C 2.7.4.6) into U T P a n d can thus enter into the reaction. U - 5 - M P is p h o s p h o r y l a t e d
to U D P with A T P by n u c l e o s i d e m o n o p h o s p h a t e kinase, N M P K ( A T P : nucleosidemonophosphate
phosphotransferase, E C 2.7.4.4).
Principle
(1) U-5-MP+ATP <

n u c l e o s i d e m o n o
% UDP+ADP
phosphate kinase .
I
(2) U D P + A T P . ""'•"> ""- . U T P + A D P 5id
phosphate kinase
(3) U T P + Glucose-l-P , U D p
8"*°' e
. UDPglucose+PP,
pyrophosphorylase .
(4) UDPglucose+H 0 + 2 N A D 2
+
t
u p p
g l u c o s e
L UDPglucuronate+ 2 N A D H + 2 H +
v
dehydrogenase
T h e N A D reduction m e a s u r e d sequentially at 340 (334, 365) n m is p r o p o r t i o n a l to the quantity of U D P

glucose a n d / o r U T P a n d / o r U D P a n d / o r U - 5 - M P .
T h e p H o p t i m u m for the d e t e r m i n a t i o n of U - 5 - M P , U D P , a n d U T P with U D P G dehydrogenase as the

indicator enzyme is p H 8.7. In contrast with U D P G dehydrogenase, which is active even in the presence
of ethylenediaminetetra-acetate, U D P G p y r o p h o s p h o r y l a s e requires divalent c a t i o n s ; the highest activity
is exhibited by m a g n e s i u m . If a mixture of the two enzymes is used, the activity of the U D P G p y r o
2
phosphorylase can be suppressed at first by E D T A to allow the d e t e r m i n a t i o n of U D P g l u c o s e ; the determi

nation of u r i d i n e p h o s p h a t e is then m a d e possible by addition of an excess of m a g n e s i u m . 3,4
The separate determination of U T P a n d U D P with the aid of U D P G - D H containing N D P K is possible

only if the sample contains o t h e r nucleosidetriphosphates as well as U T P . Otherwise the total U T P + U D P
U r i d i n e - 5 ' - t r i p h o s p h a t e , -diphosphate, - m o n o p h o s p h a t e 2173
is found (assay mixture A ) . In this case, an additional assay mixture (assay mixture B) is necessary for the
separate determination of U T P a n d U D P , e. g. in tissue extracts.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r for m e a s u r e m e n t at 3 3 4 , 3 4 0 , o r 3 6 5 n m .
Reagents
1. P e r c h l o r i c a c i d , A . R . 7 0 % ( w / w ) , s p . g r . 11. U D P g l u c o s e d e h y d r o g e n a s e , U D P G - D H
1.67 from bovine liver, suspension in 3.2 M a m m o
2. P o t a s s i u m h y d r o g e n c a r b o n a t e , A . R . n i u m sulphate s o l u t i o n ; ^ 0 . 6 U / m g . (25 °C).
3. P o t a s s i u m h y d r o x i d e s o l u t i o n , A . R., 1 N C o m m e r c i a l p r e p a r a t i o n s , see p . 519.
4. Glycine 12. U D P G p y r o p h o s p h o r y l a s e , UDPGP
5. T r i e t h a n o l a m i n e h y d r o c h l o r i d e , A . R . from bovine liver, solution in 50% (v/v) glycerol;
6. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E D T A ;> 100 U / m g . (25 °C). C o m m e r c i a l p r e p a r a t i o n s ,
d i s o d i u m salt, E D T A - N a H - 2 H 0
2 2 2 see p . 519.
7. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , 13. N u c l e o s i d e d i p h o s p h a t e k i n a s e , N D P K
NAD, from bovine liver, solution in 50% (v/v) glycerol;
free acid, commercial p r e p a r a t i o n s , see p. 545. ^ 8 0 U / m g . (25 °C), commercial p r e p a r a t i o n s ,
8. M a g n e s i u m a c e t a t e , see p . 488.
Mg(CH C0 ) -4H 0,
3 2 2 2 A.R. 14. N u c l e o s i d e m o n o p h o s p h a t e k i n a s e ,
9. G l u c o s e - 1 - p h o s p h a t e , G - 1 - P - K - 2 H 0 , 2 2 NMPK
commercial p r e p a r a t i o n s , see p . 537. from bovine liver, lyophilized, ^ 0 . 5 U / m g .
10. A d e n o s i n e - 5 ' - t r i p h o s p h a t e , A T P (25 °C). C o m m e r c i a l p r e p a r a t i o n s , see p . 489.
crystalline d i s o d i u m salt, A T P - N a H - 3 H 0 , 2 2 2
Purity of Reagents
T h e enzymes must be substantially free from N A D H - c o n s u m i n g dehydrogenases, phosphodiesterase,

and alkaline p h o s p h a t a s e s . U D P G P must be, a n d U D P G - D H should be, substantially free from c o n t a m i n
ation with N D P K .
P r e p a r e all s o l u t i o n s w i t h f r e s h l y d i s t i l l e d w a t e r .
D i l u t e 5.2 m l . 7 0 % H C 1 0 4 to 100 ml. with water.
II. P o t a s s i u m h y d r o g e n c a r b o n a t e ( 2 M ) :
D i s s o l v e 2 0 g. K H C 0 3 in w a t e r a n d m a k e u p t o 100 m l .
III. G l y c i n e buffer ( 0 . 5 M ; p H 8 . 7 ) :
D i s s o l v e 7.51 g. g l y c i n e in a p p r o x . 8 0 m l . w a t e r a n d a d d 7.2 m l . 1 N K O H . C h e c k t h e
p H with the glass electrode and m a k e up to 200 ml. with water.
I V . T r i e t h a n o l a m i n e buffer ( 0 . 3 M t r i e t h a n o l a m i n e ; p H 7 . 5 ; 4 m M m a g n e s i u m a c e t a t e ) :
D i s s o l v e 5.6 g. t r i e t h a n o l a m i n e h y d r o c h l o r i d e a n d 86 m g . M g ( C H C 0 ) - 4 H 0 3 2 2 2 in
a p p r o x . 8 0 m l . w a t e r , a d j u s t p H t o 7.5 w i t h a p p r o x . 12 m l . 1 N K O H , a n d m a k e u p t o
100 m l . w i t h w a t e r .
2174 M e t a b o l i t e s : Nucleic Acid, Purines, Pyrimidines, Nucleosides, Coenzymes
V. G l y c i n e / N A D / E D T A (0.5 M glycine; 3 m M N A D ; 8 m M E D T A ) :
D i s s o l v e 22 m g . N A D and 30 m g . E D T A - N a H - 2 H 0 2 2 2 in g l y c i n e buffer (III) a n d
m a k e u p t o 10 m l .
V i a . G l u c o s e - 1 - p h o s p h a t e / m a g n e s i u m a c e t a t e (51 m M G - l - P ; 0.51 M M g 2 +
):
Dissolve 20 mg. G - l - P - K - 2 H 0 2 2 a n d 110 m g . M g ( C H C 0 ) - 4 H 0
3 2 2 2 in w a t e r a n d
m a k e up to 1 ml.
VIb. Glucose-1-phosphate/magnesium acetate/UDPGP (46 m M G-l-P; 0.46 M Mg 2 +
;
0.45 m g . / m l . o r 4 5 U / m l . U D P G P ) :
M i x 0.04 ml. U D P G P (XII) with 0.4 ml. solution V i a .
V i l a . A d e n o s i n e triphosphate (0.11 M A T P ) :
D i s s o l v e 68 m g . A T P - N a H - 3 H 0 a n d 100 m g . K H C 0
2 2 2 3 in g l y c i n e buffer (III) a n d
m a k e up to 1 ml.
V l l b . A T P / N D P K ( 9 6 m M A T P ; 0 . 6 5 m g . / m l . o r 52 U / m l . N D P K ) :
M i x 0.4 ml. solution V i l a with 0.06 ml. N D P K (XIII).
V I I I . N u c l e o s i d e m o n o p h o s p h a t e k i n a s e , N M P K (5 m g . p r o t e i n / m l . ) :
D i s s o l v e a p o r t i o n o f t h e l y o p h i l i z a t e in c o l d w a t e r t o g i v e a p r o t e i n c o n c e n t r a t i o n o f
a p p r o x . 5 m g . / m l . If t h e e n z y m e s o l u t i o n is t o b e u s e d o v e r a p e r i o d o f several d a y s ,
a s o l u t i o n in 5 0 % ( v / v ) g l y c e r o l is a d v a n t a g e o u s .
I X . U D P g l u c o s e d e h y d r o g e n a s e , U D P G - D H (5 m g . / m l . ) :
C e n t r i f u g e a p o r t i o n o f t h e s u s p e n s i o n in a m m o n i u m s u l p h a t e s o l u t i o n for 15 m i n .
at 1 5 , 0 0 0 g, p i p e t t e off t h e s u p e r n a t a n t , a n d d i s s o l v e t h e p r o t e i n in t h e s a m e v o l u m e o f
g l y c i n e buffer (III).
X. Glucose-1-phosphate/UDPGP/triethanolamine buffer/Mg acetate (25 m M G-l-P;
0.31 m g . / m l . o r 31 U / m l . U D P G P ; 0 . 2 8 M t r i e t h a n o l a m i n e ; 3.8 m M M g 2 +
):
D i s s o l v e 9 m g . G - l - P - K - 2 H 0 in 0.9 m l . o f t r i e t h a n o l a m i n e buffer ( I V ) a n d a d d 0 . 0 6 m l .
2 2
of U D P G P (XII).
X I . A T P / N D P K / M g a c e t a t e ( 9 6 m M A T P ; 0.65 m g . / m l . o r 52 U / m l . N D P K ; 0 . 4 7 M M g 2 +
):
D i s s o l v e 4 0 m g . M g ( C H C 0 ) - 4 H 0 in 0 . 4 m l . s o l u t i o n V l l b .
3 2 2 2
X I I . U D P G p y r o p h o s p h o r y l a s e , U D P G P (5 m g . p r o t e i n / m l . ) :
X I I I . N u c l e o s i d e d i p h o s p h a t e k i n a s e , N D P K (5 m g . p r o t e i n / m l . ) :
Solution I keeps indefinitely, solutions II a n d IV for several m o n t h s , a n d solution III for a p p r o x . 1 m o n t h

at 0 - 4 °C. Solutions V, V i a , V i l a , a n d IX should be kept at 0 - 4 °C a n d should not be used for longer
than 1 week. Solutions V I b , V l l b , X, a n d X I should be freshly p r e p a r e d before each determination.
Procedure
Collection of sample: F o r u r i d i n e p h o s p h a t e d e t e r m i n a t i o n s in t i s s u e , the s a m p l e m u s t b e

c o l l e c t e d w i t h " q u i c k - f r e e z e " t o n g s (cf. p. 4 0 0 )
Uridine-5'-triphosphate, -diphosphate, -monophosphate 2175
Deproteinization: A d d a p p r o x . 5 p a r t s b y w e i g h t o f i c e - c o l d p e r c h l o r i c a c i d (I) t o t h e s a m p l e
o r t o t h e d e e p - f r o z e n p i e c e o f t i s s u e ( 0 . 2 - 1 . 5 g.) a n d h o m o g e n i z e i m m e d i a t e l y . A l l o w t h e
h o m o g e n a t e t o s t a n d f o r 1 0 - 1 5 m i n . at 0 - 4 ° C , t h e n c e n t r i f u g e for 15 m i n . at a p p r o x .
20,000 g (0 °C). D e c a n t t h e acidic supernatant a n d adjust t o a p H value o f a b o u t 8 with
solid potassium hydrogen carbonate or with K H C 0 3 s o l u t i o n (II). C e n t r i f u g e off t h e p o t a s s i u m
p e r c h l o r a t e p r e c i p i t a t e after 15 m i n . ( a t 0 — 4 ° C ) a n d u s e t h e s u p e r n a t a n t f o r t h e d e t e r m i n a t i o n .
Stability of sample: O w i n g t o the instability o f U T P a n d U D P towards acids, temperatures

of about 0 °C must be maintained during the acid deproteinization, a n d the extracts must b e
r a p i d l y n e u t r a l i z e d . N e u t r a l a q u e o u s u r i d i n e p h o s p h a t e s o l u t i o n s k e e p f o r a t least o n e d a y a t
4 ° C , b u t s h o u l d b e d e e p - f r o z e n if t h e y are t o b e s t o r e d f o r l o n g e r p e r i o d s .
Assay System A ( U T P + U D P and U M P )
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 0 . 9 2 — 0 . 9 8 m l . ( s e m i -
m i c r o c u v e t t e s ) ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t air.
Pipette into cuvettes Concentration in assay mixture
G l y c i n e / N A D / E D T A solution (V) 0.50 ml. 272 m M glycine

1.6 m M N A D
4.3 m M E D T A
Sample (deproteinized, neutralized) 0.40 ml. c a . 3 - 1 5 0 pM
uridinephosphates
U D P G - D H solution (IX) 0.02 ml. 109 pg. UDPG-DH/ml.=
65 m U / m l .
M i x a n d allow a n y U D P g l u c o s e present in the sample

t o react ( 5 — 1 0 m i n . ) . R e a d e x t i n c t i o n E . x
G-l-P/Mg 2 +
/ U D P G P solution (VIb) 0.02 ml. 1 m M G - l - P , 10 m M M g 2+
,
9 . 6 pg. U D P G P / m l . =
960 m U / m l .
M i x a n d read extinction E 2 when extinction has in

creased t o a constant value ( 1 0 — 1 5 min.). E 2 — E ^
= A E corresponds to the U T P content o f the sample.
t
A T P / N D P K solution (Vllb) 0.02 ml. c a . 2 m M A T P , 13 pg.

NDPK/ml. = 1 U/ml.
M i x a n d read extinction E 3 after e x t i n c t i o n h a s i n

creased t o a constant value ( 1 0 — 1 5 min.). E — E 3 2 =
=A E c o r r e s p o n d s t o t h e U D P c o n t e n t o f t h e s a m p l e .
2
N M P K solution (VIII) 0.02 ml. 1 0 2 Mg./ml. = 51 m U / m l .
M i x , follow increase in extinction until a constant

v a l u e is r e a c h e d ( 1 0 — 1 5 m i n . ) ; t h e n r e a d e x t i n c t i o n E . 4
E — E = A E corresponds to the U M P content o f the

4 3 3
sample.
2176 M e t a b o l i t e s : Nucleic Acid, Purines, Pyrimidines, Nucleosides, Coenzymes
T h e i n c r e a s e in e x t i n c t i o n r e s u l t i n g f r o m t h e a d d i t i o n o f A T P / N D P K s o l u t i o n ( V l l b ) o r o f
N M P K s o l u t i o n is d e t e r m i n e d at t h e e n d o f t h e r e a c t i o n b y a r e n e w e d a d d i t i o n , a n d t h e result
is s u b t r a c t e d f r o m E 3 o r E . It is a l s o p o s s i b l e t o r e a d t h e e x t i n c t i o n ( E a n d E ) i m m e d i a t e l y
4 2 3
after t h e e n z y m e s o l u t i o n ( V I I b o r V I I I ) h a s b e e n m i x e d in. In s o l u t i o n s c o n t a i n i n g o t h e r
nucleosidetriphosphates as well as U T P (e.g. tissue extracts), solutions V I b and V l l b s h o u l d
b e m i x e d in a r a t i o o f 1:1 w h e n U D P G - D H c o n t a i n i n g N D P K is u s e d , a n d t h e d e t e r m i n a t i o n
of U T P + U D P should be started with 0.04 ml. o f this mixture.
Calculations
U n d e r the above conditions, 2 mole of N A D are reduced per mole of U D P g l u c o s e a n d / o r U T P a n d / o r

U - 5 - M P . In the analysis of tissue extracts, the result (^mole/ml. of neutral sample solution) must be
multiplied by the dilution factor resulting from deproteinization a n d neutralization. T h e following
relationships are valid for the calculation of the u r i d i n e p h o s p h a t e c o n c e n t r a t i o n of the sample.
UDPG c Êx0.189 JEx0.185 A E x 0.338 [/miole/ml.]

UTP c A E x 0.193 JExO.189 J E x 0.346 [jumole/ml.]
U D P or U T P + U D P c A E x 0.197 JEx0.193 A E x 0.353 [/imole/ml.]
U-5-MP c A E x 0.201 Êx0.197 A E x 0.371 [^mole/ml.]
Uridine-5'-triphosphate, -diphosphate, -monophosphate 2177
Assay System B ( U T P and U D P )
This determination gives the total U D P G + U T P (see p. 2172) a n d also U D P . By c o m b i n a t i o n

o f t h e results o f d e t e r m i n a t i o n s A a n d B , it is p o s s i b l e t o d e t e r m i n e U T P a n d U D P s e p a r a t e l y
in t i s s u e e x t r a c t s , e v e n w h e n t h e r e is n o N D P K - f r e e U D P G - D H a v a i l a b l e .
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 0 . 8 2 - 0 . 8 4 m l . ( s e m i -
m i c r o c u v e t t e s ) ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t air.
Pipette into cuvettes: C o n c e n t r a t i o n in i n c u b a t i o n
G-l -P/UDPGP/triethanolamine (X) 0.10 ml. 8.3 m M G-l-P

buffer 1.3 m M M g 2 +
93 m M triethanolamine
1 0 3 fig. U D P G P / m l . = 10.3 U / m l .
S a m p l e (neutral supernatant) 0.20 ml.
M i x a n d p r e i n c u b a t e for 3 0 m i n . a t r o o m t e m p e r a t u r e . C o n c e n t r a t i o n in a s s a y m i x t u r e
G l y c i n e / N A D / E D T A solution (V) 0.50 ml. 0.31 M g l y c i n e

1.8 m M NAD
4.9 m M EDTA
M i x , read extinction E.t
U D P G - D H solution (IX) 0.02 ml. 1 2 2 fig. U D P G - D H / m l . =

73 m U / m l .
M i x and read extinction E 2 after e x t i n c t i o n h a s i n

c r e a s e d t o a c o n s t a n t v a l u e ( c a . 10 m i n . ) . E - E 2 1 =
= A E c o r r e s p o n d s t o t h e s u m o f U D P G + U T P in t h e
cuvette.
ATP/NDPK/Mg 2 +
solution (XI) 0.02 ml. 2.3 m M A T P
11 m M M g 2 +
15 fig. N D P K / m l . = 1.2 U / m l .
M i x and read extinction E 3 after e x t i n c t i o n h a s i n

creased to a constant value ( 1 0 - 1 5 min.). E - E = A E 3 2 2
c o r r e s p o n d s t o t h e q u a n t i t y o f U D P in t h e c u v e t t e .
T h e i n c r e a s e s i n e x t i n c t i o n r e s u l t i n g f r o m t h e a d d i t i o n o f s o l u t i o n s I X a n d X I are d e t e r m i n e d
at t h e e n d o f t h e r e a c t i o n s b y r e n e w e d a d d i t i o n a n d s u b t r a c t e d f r o m E a n d E . It is a l s o p o s s i b l e
2 3
t o r e a d t h e e x t i n c t i o n s (E{ a n d E ) i m m e d i a t e l y after t h e e n z y m e s o l u t i o n s ( I X a n d X I re

2
spectively) h a v e been m i x e d in.
Calculations
D u r i n g the preincubation, U T P is converted into U D P G .
T h e following relationships are valid for the c o n c e n t r a t i o n s in the neutralized s u p e r n a t a n t :

UDPG+UTP c = AEx 0.336 z l E x 0.330 AEx 0.603 [^mole/ml.]
UDP c=dEx0.344 JEx0.338 JEx0.618 [/imole/ml.]
2178 M e t a b o l i t e s : Nucleic Acid, Purines, Pyrimidines, Nucleosides, C o e n z y m e s
T h e U T P c o n c e n t r a t i o n in t h e sample is found from the difference between U T P - ( - U D P (determination

A ) a n d U D P . If U D P g l u c o s e has been determined (by reading the increase in extinction d u e to U D P G -
D H in determination A o r c o r r e s p o n d i n g t o p . 2177), the following calculation can also be carried o u t :
UTP=(UDPG+UTP) - UDPG.
Repeated d e t e r m i n a t i o n s on solutions c o n t a i n i n g u r i d i n e p h o s p h a t e give a coefficient of variation of

2 % for the d e t e r m i n a t i o n of U T P , U D P , a n d U-5-MP.'
N o r m a l Values
The contents of U T P , U D P , a n d U M P ( ± s t a n d a r d deviation) in rat liver are 0 . 2 8 ± 0 . 0 5 , 0.06 + 0.01,

a n d 0.04 + 0.01 /miole per g. fresh weight. T h e r a t i o U T P / U D P was found t o be 4.8 ± 0 . 6 . The contents
in guinea-pig livers a n d m o u s e livers are n o t significantly different from those for the rat. T h e contents
of U T P + U D P + U M P a r e 0.23 + 0.02 in rat brain, 0.34 + 0.05 in rat kidney, a n d 0 . 1 1 + 0 . 0 1 5 /miole/g.
in rat skeletal m u s c l e . 4
N A D H a n d U D P g l u c u r o n a t e in high c o n c e n t r a t i o n s inhibit the U D P g l u c o s e dehydrogenase used

as the indicator e n z y m e . U D P G P is inhibited by sulphate ions. T h e u r i d i n e p h o s p h a t e contents in the
5
liver are increased after the a d m i n i s t r a t i o n of o r o t a t e 4

a n d of u r i d i n e ; D-galactosamine, 2-deoxy-D-
galactose, a n d D-glucosamine lead t o a m a r k e d d e c r e a s e . 3,6
The c o m b i n a t i o n of t w o enzymes specific for U T P a n d for U D P g l u c o s e ( U D P G P a n d U D P G - D H ) ' 2 5
m a k e s the m e t h o d extremely specific for the d e t e r m i n a t i o n of u r i d i n e p h o s p h a t e . O n the other h a n d ,

4
the nucleosidediphosphate kinase from bovine liver is non-specific. In the p h o s p h o r y l a t i o n of U D P t o

U T P , A T P can be replaced by d A T P , I T P , d T T P , C T P , or G T P . F o r the p h o s p h o r y l a t i o n of U - 5 - M P
to U D P by n u c l e o s i d e m o n o p h o s p h a t e kinase from bovine liver, only A T P or d A T P can act as the s u b s t r a t e ;
G T P , I T P , C T P , a n d d T T P are inactive. O f t h e u r i d i n e m o n o p h o s p h a t e s , only U - 5 - M P reacts in the
m e t h o d described, while 6 - a z a - U - 5 - M P does n o t r e a c t . In a d d i t i o n t o the u r i d i n e p h o s p h a t e , U D P g l u c o s e
6
(see p . 2225) a n d U D P g a l a c t o s e (see p . 2221) can also be determined in t h e same p r o c e d u r e . A sensitive

a n d specific d e t e r m i n a t i o n of glucose-1-phosphate is possible by replacement of glucose-1-phosphate with
U T P in solution VI.
References
1 H. M. Kalckar & E. P. Anderson in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, Vol. I l l ,

p . 976 [1957], A c a d e m i c Press, N e w York & L o n d o n .
2 R. G. Hansen, G. J. Albrecht, S. T. Bass & L. L. Seifert in S. P. Colowick & N. O. Kaplan: M e t h o d s in
Enzymology, Vol. V I I I , p . 248 [1966], A c a d e m i c Press, N e w York a n d L o n d o n .
3 D. Keppler & K Decker, E u r o p e a n J. Biochem. 10, 219 [1969].
4 D. Keppler, J. Rudigier & K Decker, Analyt. Biochem. 38, 105 [1970].
5 /. Zalitis & D. S. Feingold, Biochem. Biophys. R e s . C o m m . 31, 693 [1968].
6 D. Keppler, J. Rudigier, E. Bischoff 8L K. Decker, E u r o p e a n J. Biochem. 17, 246 [1970].
Flavin Mononucleotide
Herbert C. F r i e d m a n n
T h e cofactor of lactate oxidase ( L - L a c t a t e : oxygen 2-oxidoreductase, decarboxylating, E C 1.13.12.4) from

pneumococci is flavin m o n o n u c l e o t i d e ( F M N ) . T h e a p o e n z y m e of lactate oxidase is specifically activated
by F M N . T h e m e t h o d described here d e p e n d s o n this a c t i v a t i o n . T h e m e t h o d is similar t o the d e t e r m i n a t i o n
1
of flavin adenine dinucleotide ( F A D ) with D - a m i n o acid a p o - o x i d a s e . 2,3
Principle
(1) L-Lactate + 0 2
L
~ > Acetate + C 0 2 + H 0 2
T h e activity of lactate oxidase is p r o p o r t i o n a l to the a m o u n t of F M N within certain limits; it is d e t e r m i n e d

manometrically by the oxygen u p t a k e per unit time. A s t a n d a r d curve is o b t a i n e d if the reaction rates with
k n o w n c o n c e n t r a t i o n s of F M N are plotted against the F M N c o n c e n t r a t i o n a c c o r d i n g t o m e t h o d s of
Michaelis-Menten or Lineweaver-Burk . 4
T h e Michaelis c o n s t a n t , K M of the lactate apo-oxidase for F M N
is 4.8 x 1(T 7
M . 1
Equipment
A p p a r a t u s f o r Warburg manometry.
Reagents
1. S o d i u m d i h y d r o g e n p h o s p h a t e , 5. F l a v i n m o n o n u c l e o t i d e
NaH P0 H 02 4 2 s o d i u m salt, F M N - N a H • 2 H 0 ; 2 commercial
2. D i s o d i u m h y d r o g e n p h o s p h a t e , p r e p a r a t i o n , see p . 533.
Na HP0 -2H 0
2 4 2 6. L a c t a t e a p o - o x i d a s e
3. P o t a s s i u m h y d r o x i d e , 5 N p r e p a r e d a c c o r d i n g t o from Diplococcus
1
pneu
4. Lithium-DL-lactate moniae R 36 A ; ca. 2 0 0 0 U / m g . (30 °C). F o r
m e t h o d of p r e p a r a t i o n , see A p p e n d i x , p. 2181.
D i s s o l v e 1 3 8 . 0 g. N a H P 0 . H 0 a n d 1 7 8 . 0 5 g. N a H P 0 . 2 H 0 in d i s t i l l e d w a t e r a n d
2 4 2 2 4 2
m a k e up to 1 0 0 0 ml.
II. D L - L a c t a t e ( 1 . 0 M ) :
D i s s o l v e 0 . 9 6 0 1 g. l i t h i u m - D L - l a c t a t e in d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
III. F l a v i n m o n o n u c l e o t i d e , FMN
a) S t o c k s o l u t i o n ( 0 . 3 5 m M ) :
D i s s o l v e 9 m g . F M N - N a H • 2 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 5 m l . C h e c k t h e
2
concentration spectrophotometrically (extinction coefficient 5

o f F M N at 4 5 0 n m a n d
p H 7 is 1 1 . 3 c m . / j u m o l e ) .
2
b) S t a n d a r d s o l u t i o n (7 pM):
D i l u t e 0.1 m l . s o l u t i o n a) i m m e d i a t e l y b e f o r e u s e t o 5 0 m l . w i t h d i s t i l l e d w a t e r .
IV. Apoenzyme:
Preparation o f solution, see A p p e n d i x , p. 2 1 8 1 .
Store the F M N stock solution in a b r o w n bottle. T h e solutions of the a p o e n z y m e , lactate and F M N keep
for several m o n t h s at —15 °C.
Procedure
Experimental Material
T h e c o n d i t i o n s for t h e l i b e r a t i o n o f F M N are t h e s a m e as for F A D (see p . 2 1 8 3 ) .
Assay System
Warburg m a n o m e t e r s ; vessels with centre well and side-arm; gas p h a s e : air; temperature:
30 ° C . T h e f o l l o w i n g v e s s e l s are r e q u i r e d for e a c h d e t e r m i n a t i o n : 2 - 3 e x p e r i m e n t a l v e s s e l s ,
3 - 4 standards, 1 control ( F M N - f r e e ) , 1 thermobarometer.
Experimental Thermo
Prepare the vessels as f o l l o w s : Control
& Standards barometer
Main compartment Buffer ( S o l n . I) 0.1 m l . 0.1 m l .

A p o e n z y m e (Soln. IV) 0.1 m l . 0.1 m l . —
Sample or standard
(Soln. Illb) 1.7 m l . —
Distilled water — 1.7 m l . 2.0 ml.
Side-arm L a c t a t e ( S o l n . II) 0.1 m l . 0.1 m l . —
C e n t r e well 5 N K O H ( o n filter p a p e r ) 0.1 m l . 0.1 m l . —
A l l o w v e s s e l s t o s t a n d for 3 0 m i n . at r o o m t e m p e r a t u r e in t h e d a r k ( r e a c t i v a t i o n o f a p o e n z y m e
b y F M N ) . T h e n e q u i l i b r a t e all v e s s e l s for a b o u t 10 m i n . at 30 ° C . T i p in t h e c o n t e n t s o f t h e s i d e -
a r m i n t o t h e m a i n c o m p a r t m e n t , c l o s e t a p s , start s t o p w a t c h a n d r e a d m a n o m e t e r l e v e l s ( h )
e v e r y 5 or 10 m i n . C a l c u l a t e d h / m i n . a n d a v e r a g e t h e v a l u e s .
Calculations
The oxygen u p t a k e A 0 / m i n . for the experimental a n d s t a n d a r d vessels is calculated from the m a n o m e t e r

2
readings (mm. m a n o m e t e r fluid), corrected for changes in the t h e r m o b a r o m e t e r a n d control, by multipli

cation by the vessel constants k.
1 1
Plot the — for the s t a n d a r d s against the
A 0 /min.
2 F M N content
O b t a i n the F M N content of the experimental vessels from this s t a n d a r d curve.
Flavin M o n o n u c l e o t i d e 2181
O t h e r M e t h o d s for E n z y m a t i c D e t e r m i n a t i o n o f F M N
F M N can also be determined spectrophotometrically by m e a n s of its activation of the a p o e n z y m e of

N A D P H cytochrome c reductase 6
(modification ). 5
Appendix
Preparation of Lactate Oxidase 1
A d d solid a m m o n i u m sulphate u p t o 50% s a t u r a t i o n t o a n autolysate from Diplococcus pneumoniae

R 36 A at 0 - 4 °C. Centrifuge a n d discard the precipitate. To the s u p e r n a t a n t fluid a d d solid a m m o n i u m
sulphate to give 6 8 % saturation. Centrifuge a n d dissolve precipitate in a solution of 20 m M N a H P 0 2 4 +
+ 1 m M E D T A . Dialyse for 3 hr. with stirring against this solution.
Preparation of Apoenzyme 1
To 10 ml. of a solution containing 2800 units* (47 U ) lactate oxidase (specific activity: 35 units/mg.
0.6 U / m g . ) a d d 3.5 ml. s a t u r a t e d a m m o n i u m sulphate solution. Very slowly a d d 4.5 ml. 0.1 N H S 0
2 4 to
this mixture in the cold. Allow t o stand for 15 min. at 0 °C a n d then centrifuge. Wash the precipitate with
5 ml. saturated a m m o n i u m sulphate solution a n d finally dissolve in 10 ml. 0.1 M p h o s p h a t e buffer ( p H 7.2).
References
1 S. Udaka, J. Koukol & B. Vennesland, J. Bacteriol. 78, 714 [1959].

3 F. B. Straub, Biochem. J. 33, 787 [1939].
4 H. Lineweaver & D. Burk, J. A m e r . chem. Soc. 56, 658 [1934].
5 F. M. Huennekens & S. P. Felton in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c
Press, N e w York 1957, Vol. I l l , S. 950.
6 E. Haas, B. L. Horecker & T. R. Hogness, J. biol. C h e m . 136, 747 [1940].
* A u n i t is the a m o u n t of enzyme which causes an oxygen u p t a k e of 1 /zmole/hr. at 30 °C in a reaction

1
mixture containing 100 /miole p h o s p h a t e buffer ( p H 7.2) a n d 100 /miole Li-DL-lactate in a final v o l u m e
of 2.8 m l . ; i.e. 1/60 /miole/min. 1 unit therefore c o r r e s p o n d s t o 16.6 m U .
Flavin-adenine Dinucleotide
Herbert C. Friedmann
The a p o e n z y m e of D - a m i n o acid oxidase from pig kidney, D - A O D ( D - A m i n o a c i d : oxygen oxidoreductase,

deaminating, E C 1.4.3.3) is specifically reactivated by flavin-adenine dinucleotide ( F A D ) ' . The m e t h o d 1 2 3
described here is based o n this re-activation.
Principle
(1) D - A m i n o acid + H 0 + 0 2 2
D
~ A O D
> 2-Oxo-acid + N H 3 + H 0
2 2
Within certain limits the activity of D - A O D is p r o p o r t i o n a l to the F A D c o n c e n t r a t i o n ; the activity is

measured by the oxygen u p t a k e per unit time.
Proportionality between oxygen u p t a k e per unit time a n d F A D c o n c e n t r a t i o n exists u p to 25 pg. F A D / m l .

It is necessary to c o m p a r e the reactivation with an F A D s t a n d a r d , because the Michaelis c o n s t a n t of the
enzyme for F A D varies with the t e m p e r a t u r e a n d from p r e p a r a t i o n to p r e p a r a t i o n . T h e a p p a r e n t dis
sociation constant for F A D varies between 0.13 a n d 0.33 uM, i.e. over an a p p r o x i m a t e l y two a n d a
half-fold range ( s e e ) . O t h e r reasons for the use of an F A D s t a n d a r d a r e given below.
4,5
Equipment
A p p a r a t u s for Warburg manometry.
Reagents
1. S o d i u m p y r o p h o s p h a t e , 5. D i s o d i u m h y d r o g e n p h o s p h a t e ,
Na P 0
4 2 7 or N a P O 1 0 H O
4 2 7 2 Na HP0 12H 0
2 4 2
2. D L - A l a n i n e 6. P o t a s s i u m h y d r o x i d e , 2 0 % ( w / v )
3. S u l p h u r i c a c i d , 1 N 7. F l a v i n - a d e n i n e d i n u c l e o t i d e , F A D
4. S o d i u m d i h y d r o g e n p h o s p h a t e , free acid; commercial p r e p a r a t i o n , see p.532.
NaH P0 H 0
2 4 2 8. A p o e n z y m e o f D - a m i n o a c i d o x i d a s e
from pig k i d n e y s ; p r e p a r a t i o n , see p. 2184.
D i s s o l v e 5 . 3 2 g. N a P 0 4 2 7 o r 8 . 9 2 g. N a P O 1 0 H O in 1 5 0 m l . d i s t i l l e d w a t e r , a d j u s t
4 2 7 2
p H t o 8.5 w i t h c a . 4 m l . 1 N H S 0 2 4 and dilute to 200 ml. with distilled water.

II. D L - A l a n i n e ( 1 . 0 M ) :
D i s s o l v e 0 . 8 9 1 g. D L - a l a n i n e in d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
Flavin-adenine Dinucleotide 2183
III. P h o s p h a t e buffer ( 1 0 m M ; p H 7 . 0 ) :
a) D i s s o l v e 7 . 1 6 4 g. N a H P 0 1 2 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2 4 2
b) D i s s o l v e 2 . 7 6 0 g. N a H P 0 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2 4 2
M i x 61 m l . s o l u t i o n a ) w i t h 3 9 m l . s o l u t i o n b) a n d d i l u t e t o 2 0 0 m l . w i t h d i s t i l l e d w a t e r .
IV. Flavin-adenine dinucleotide, F A D :
a) S t o c k s o l u t i o n ( c a . 1.5 m M ) :
D i s s o l v e 6 m g . p u r e F A D ( d r i e d in vacuo a t 5 0 - 6 0 ° C o v e r P O ) in 5 m l . p h o s p h a t e
2 s
buffer ( s o l u t i o n III).
b) Working s o l u t i o n (ca. 3 pM):
Dilute the stock solution just before use 500-fold with distilled water.
After dilution of the stock solution determine the exact F A D c o n t e n t s p e c t r o p h o t o m e t r i c -
ally. P u r e F A D h a s a n e x t i n c t i o n c o e f f i c i e n t at 4 5 0 n m a n d p H 7 o f 1 1 . 3 c m . / / m i o l e . A 2
s o l u t i o n c o n t a i n i n g e x a c t l y 6 m g . F A D / 5 m l . is 1.5275 m M a n d after 5 0 - f o l d d i l u t i o n *
h a s a n e x t i n c t i o n at 4 5 0 n m o f 0 . 3 4 1 ( p H 7.0 a n d 1 c m . light p a t h ) .
F o r p u r e F A D p r e p a r a t i o n s t h e r a t i o o f t h e e x t i n c t i o n s at 2 6 0 a n d 4 5 0 n m ( p H 7) is e x a c t l y
3 . 2 5 . T h e p u r i t y c a n a l s o b e c h e c k e d b y p a p e r c h r o m a t o g r a p h y . F A D c a n b e freed f r o m
3
F M N , riboflavin a n d various nucleotides by electrophoresis o n W h a t m a n N o . 1 paper

followed by elution of the fluorescent material . 6
V. A p o e n z y m e o f D - a m i n o oxidase:
U s e the solution prepared according to p. 2184.
The p h o s p h a t e a n d p y r o p h o s p h a t e buffers keep for weeks if n o g r o w t h of m i c r o - o r g a n i s m s occurs. T h e

F A D and a p o e n z y m e solutions keep for several weeks at - 1 5 ° C .
Procedure
S o l u t i o n s c o n t a i n i n g free F A D c a n b e a n a l y s e d d i r e c t l y . P r o t e i n - b o u n d F A D c a n b e l i b e r a t e d
in t h e m a n o m e t e r v e s s e l b y h e a t d e n a t u r a t i o n ( p l a c e t h e m a n o m e t e r v e s s e l w i t h t h e F A D -
c o n t a i n i n g s o l u t i o n in a b o i l i n g w a t e r b a t h for 5 m i n . ; after c o o l i n g a d d t h e o t h e r r e a g e n t s ) . 3
S t r o n g l y b o u n d F A D c a n b e l i b e r a t e d b y h e a t d e n a t u r a t i o n f o l l o w e d b y p r o t e o l y s i s (see ) . 7
B i o l o g i c a l m a t e r i a l m u s t b e d i l u t e d c o n s i d e r a b l y t o o b t a i n a final F A D c o n c e n t r a t i o n o f 0.1 ^ M
t o 1 pM, a n d t h e r e f o r e i n t e r f e r i n g s u b s t a n c e s in t h e s a m p l e c a n u s u a l l y b e i g n o r e d . M u c h h i g h
er c o n c e n t r a t i o n s (0.1 m M t o 1 m M ) o f p a r t i a l s t r u c t u r a l a n a l o g u e s ( F M N , A M P , ATP,
N A D , free r i b o f l a v i n , etc.) c a n c a u s e c o n s i d e r a b l e c o m p e t i t i v e o r n o n - c o m p e t i t i v e i n h i b i t i o n . 8
In d o u b t f u l c a s e s , i n h i b i t i o n c a n b e r u l e d o u t b y u s e o f i n t e r n a l s t a n d a r d s . If p u r i f i c a t i o n o f
t h e s a m p l e is n e c e s s a r y 3 , 6
, t h e u s e o f i n t e r n a l s t a n d a r d s c o r r e c t s for a n y l o s s e s .
F A D is s e n s i t i v e t o a c i d s , b a s e s a n d light. E x c e s s i v e e x p o s u r e t o u l t r a v i o l e t light is t o b e a v o i d e d .
* F o r the s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s it m a y be necessary to dilute the solution with p h o s p h a t e

buffer (solution III) instead of with distilled water.
Assay System
Warburg m a n o m e t e r ; m a n o m e t e r vessels with centre well and side-arm; gas p h a s e : air; temper
a t u r e : 37 ° C . T h e f o l l o w i n g are r e q u i r e d : 1 - 2 e x p e r i m e n t a l v e s s e l s , 3 - 4 s t a n d a r d v e s s e l s ,
1 control (without F A D ) and 1 thermobarometer.
Experimental
Prepare vessels as f o l l o w s : Control Thermobarometer
standard
Main Buffer
compartment ( s o l n . I) 1.0 m l . 1.0 m l . —
Apoenzyme
soln. (V) 0.3 m l . 0.3 m l . —
Sample o f F A D or
standard soln. (IV b) 0.6 m l . — —
Distilled water — 0.6 m l . 2.1 m l .
Side-arm A l a n i n e ( s o l n . II) 0.1 m l . 0.1 m l . —
C e n t r e well 20% KOH
( o n filter p a p e r ) 0.1 m l . 0.1 m l . —
E q u i l i b r a t e , tip c o n t e n t s o f s i d e - a r m i n t o m a i n c o m p a r t m e n t a n d c l o s e m a n o m e t e r t a p s . Start a
s t o p w a t c h a n d read t h e m a n o m e t e r s at 5 t o 10 m i n . i n t e r v a l s . T h e i d e a l r a n g e o f o x y g e n u p t a k e
is b e t w e e n 10 a n d 4 0 / d . p e r 10 m i n .
Calculations
The oxygen c o n s u m p t i o n , A 0 / m i n . or A O / 1 0 min. of the experimental a n d s t a n d a r d vessels is obtained

2 2
from the m a n o m e t e r readings ( m m . m a n o m e t e r fluid) after correction for the changes in the t h e r m o
barometer and control. T h e values for the successive measured intervals should agree within ± 5 % a n d
are averaged . F o r the s t a n d a r d s plot
3
A 0 /min.
2 A O / 1 0 min.
2 F A D content
Obtain from this s t a n d a r d curve the F A D content corresponding to the oxygen u p t a k e of the experi
mental vessels. T h e molecular weight of F A D is 785.6. F o r low c o n c e n t r a t i o n s of F A D there is a linear
relationship between the oxygen u p t a k e a n d the a m o u n t of F A D and therefore a reciprocal plot is u n
necessary.
Appendix
Preparation of D-Amino Acid Oxidase*
Extract an acetone-dried p o w d e r of pig kidney at r o o m t e m p e r a t u r e with distilled water a n d centrifuge.

The supernatant fluid should contain ca. 10 mg. protein/ml. Carry out all the succeeding steps at 0 °C.
To 9.8 ml. of the s u p e r n a t a n t fluid a d d 3.4 ml. saturated a m m o n i u m sulphate solution. Slowly a d d ,
* The m e t h o d described h e r e is similar to t h a t of Warburg a n d Christian , except that H S 0 is used

9 1
2 4
instead of HC1. A m e t h o d employing acetic acid has been described by Huennekens and Felton . F o r 3
the effects of various anions, such as chloride a n d sulphate, on the rate constants for the dissociation
and association of the F M N - c o n t a i n i n g " o l d yellow e n z y m e " , refer t o . 1 0
Flavin-adenine Dinucleotide 2185
with stirring, 5.6 ml. 0.1 N H S 0 and then centrifuge. Discard the s u p e r n a t a n t fluid, wash the precipitate
2 4
with 4.9 ml. saturated a m m o n i u m sulphate solution and centrifuge again. Discard the s u p e r n a t a n t fluid.
Suspend the precipitate in 3.5 ml. N a - p h o s p h a t e buffer ( p H 7.2), centrifuge and discard the precipitate.
Use the s u p e r n a t a n t fluid undiluted or store at —15 °C.
References

2 F. B. Straub, Biochem. J. 33, 787 [1939].
3 F. M. Huennekens & S. P. Felton in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c
Press, N e w Y o r k 1957, Vol. I l l , p . 950.
4 K Burton in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c Press, N e w Y o r k
1955, Vol. II, p . 199.
5 E. Diamant, D. R. Sanadi & F. M. Huennekens, J. Amer. chem. Soc. 74, 5440 [1952].
6 O. Walaas & E. Walaas, Acta chem. scand. 10, 118 [1956].
7 H Kondo, H C Friedmann & B. Vennesland, J. biol. C h e m . 235, 1533 [I960].
8 E. Walaas & O. Walaas, Acta chem. scand. 10, 122 [1956].
9 / . Koukol, Dissertation, University of Chicago, D e p a r t m e n t of Biochemistry, 1959.
10 H. Theorell & A. P. Nygaard, Acta chem. scand. 8, 1649 [1954].
Thiamine Pyrophosphate
Johannes Ullrich
T h i a m i n e p y r o p h o s p h a t e ( T P P ) occurs in virtually all organisms as coenzyme for enzymes catalyzing

the decarboxylation of 2-keto acids a n d t h e transfer of C units. 2
T h e simplest m e t h o d for T P P determination (where applicable) is its chemical oxidation t o t h i o c h r o m e

p y r o p h o s p h a t e a n d t h e m e a s u r e m e n t of this by fluorimetry . However, thiamine, t h i a m i n e m o n o p h o s
1
p h a t e ( T M P ) a n d t h i a m i n e t r i p h o s p h a t e ( T T P ) also react. In addition this assay is very susceptible t o

interference b y c o n t a m i n a n t s which fluoresce o r q u e n c h , a n d often c a n n o t b e used for assay of biological
material. In such cases, T P P - d e p e n d e n t enzymes can be used t o determine T P P provided that the a p o
enzyme can be prepared. This is relatively easy for pyruvate decarboxylase, P D C (2-Oxo-acid carboxy 2
l a s e , E. C . 4.1.1.1), which occurs in large a m o u n t s in b o t t o m brewers' yeast (Saccharomyces carlsbergensis).
Application of Method: In biochemistry, clinical biochemistry, p h a r m a c e u t i c a l chemistry a n d food

chemistry.
Principle
TPP-Mg
TPP + P D C - M g ( a p o - P D C ) ^ T P P - P D C - M g ^ ^ \ / (holo-PDC)
(in excess) \ /
PDC
C H 3 C O C 0 0 H p
" 0
6
0 !;p C
2
> CH3CH0 + c o 2
(limiting)
CH3CHO + N A D H + H +
-Û CH CH OH + NAD
3 2
+
•> (excess) J z
T h e rate of oxidation of N A D H , as m e a s u r e d by t h e change in extinction at 340 o r 365 n m , is p r o p o r t i o n a l

to t h e a m o u n t of T P P , provided t h a t the saturation of P D C with T P P does n o t exceed 2 0 - 2 5 % .
P D C has a sharp r e c o m b i n a t i o n o p t i m u m at p H 6 . 8 3 , 4
and a K M for T P P of 2.4 x 1 0 ~ M ' . T h e complete
5 5 6
binding of T P P t o t h e excess of a p o P D C requires t h e following: a sufficiently long r e c o m b i n a t i o n time

in a "preliminary i n c u b a t i o n " , t h e correct p H , M g 2 +
saturation a n d a T P P c o n c e n t r a t i o n as high as
possible. After the " q u a s i - i r r e v e r s i b l e " ' r e c o m b i n a t i o n the complex c a n be diluted safely with buffer
3,4 6
at the o p t i m u m p H of 6 . 0 - 6 . 2 as required for the assay. C o n s t a n t t e m p e r a t u r e is necessary for each series

of m e a s u r e m e n t s . T h e absolute t e m p e r a t u r e is n o t so i m p o r t a n t because a s t a n d a r d curve is included in
each series. P u r e P D C is r e c o m m e n d e d for t h e d e t e r m i n a t i o n of a m o u n t s of T P P below 0.1 n m o l e , because
it h a s a very low blank activity; otherwise P D C partially purified by acetone a n d a m m o n i u m sulphate
fractionation ( a m m o n i u m sulphate paste) is sufficient.
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r , p r e f e r a b l y w i t h a l o g r e c o r d e r ; refrigerated,
h i g h - s p e e d c e n t r i f u g e ; v i b r o m i x e r * o r stirrer; p H m e t e r .
* C h e m a p A G , CH-8708, Mannedorf, Switzerland

Thiamine Pyrophosphate 2187
Reagents
U s e analytical reagents unless otherwise stated.
1. S o d i u m h y d r o x i d e , 2 N 9. D i s o d i u m h y d r o g e n p h o s p h a t e ,
2. M a g n e s i u m s u l p h a t e , M g S 0 4 • 7H 0 2 Na HP0
2 4 • 12H 0 2
3. Citric a c i d 10. S o d i u m d i h y d r o g e n p h o s p h a t e ,
4. S o d i u m pyruvate, C H C O C O O N a 3 NaH P0 2 4 • H 0 2
5. T h i a m i n e p y r o p h o s p h a t e ( c o c a r b o x y l a s e ) j11. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA
free from thiamine, T M P and T T P ; commercial disodium salt, E D T A - N a H - 2 H 0 2 2 2
preparation, see p. 553. 12. A l c o h o l d e h y d r o g e n a s e , A D H

6. R e d u c e d n i c o t i n a m i d e - a d e n i n e - d i n u c l e o - from yeast, crystalline suspension in 3.2 M
tide, N A D H a m m o n i u m sulphate solution; ^ 200 U / m g .
sodium salt, N A D H - N a ; commercial prepara
2 (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p . 428.
tion, see p. 545. 13. P y r u v a t e d e c a r b o x y l a s e , h o l o - P D C
7. P e r c h l o r i c a c i d , ca. 7 0 % ( w / w ) , sp. gr. from brewers' y e a s t 5 7 8
. P r e p a r a t i o n , see p. 2191.
1.67
8. P o t a s s i u m b i c a r b o n a t e , K H C 0 3
I. C i t r a t e buffer ( 0 . 3 M ; p H 6 . 0 ) :
D i s s o l v e 6.3 g. citric a c i d in 3 0 m l . 2 N N a O H + 30 ml. distilled water, adjust to p H
6.0 w i t h 2 N N a O H a n d d i l u t e t o 100 m l . w i t h distilled w a t e r .
II. M a g n e s i u m s u l p h a t e ( 2 0 m M ) :
Dissolve 500 mg. M g S 0 4 • 7 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2
III. P y r u v a t e (1 M ) :
D i s s o l v e 1.1 g. s o d i u m p y r u v a t e in d i s t i l l e d w a t e r a n d m a k e u p t o 10 m l .
I V . R e d u c e d n i c o t i n a m i d e - a d e n i n e - d i n u c l e o t i d e (ca. 11 m M / ? - N A D H ) :
V. A l c o h o l d e h y d r o g e n a s e , A D H (0.5 m g . p r o t e i n / m l . ) :
D i l u t e t h e s t o c k s u s p e n s i o n c o r r e s p o n d i n g l y w i t h buffer s o l u t i o n V I I I .
VI. Apo-pyruvate decarboxylase, a p o - P D C ( 1 - 3 mg. protein/ml.):

D i s s o l v e 1 5 0 - 2 0 0 m g . a p o - P D C p a s t e (see A p p e n d i x ) in 10 m l . buffer V I I I a n d centri
f u g e for 10 m i n . at h i g h s p e e d a n d 0 ° C . If n e c e s s a r y , d i l u t e t h e s u p e r n a t a n t fluid w i t h
buffer V I I I .
VII.a Thiamine pyrophosphate, T P P (10 m M ) :

D i s s o l v e 10 m g . T P P in 2 m l . distilled w a t e r .
V I L b T P P Standard solution (10 / / M ) :

D i s s o l v e 5 m g . T P P ( w a t e r c o n t e n t v a r i e s ) in 100 m l . d i s t i l l e d w a t e r , a d j u s t t h e e x t i n c t i o n
o f t h e s o l u t i o n at 2 7 2 . 5 n m (1 c m . light p a t h ) t o 0 . 7 4 0 b y d i l u t i o n w i t h distilled w a t e r .
D i l u t e 10 m l . o f t h i s s o l u t i o n t o 100 m l . w i t h d i s t i l l e d w a t e r .
V I I I . P h o s p h a t e buffer ( 5 0 m M ; p H 6 . 8 ) :
(a) D i s s o l v e 6.9 g. N a H P 0 2 4 • H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l . (b) D i s
2
s o l v e 17.9 g. N a H P 0
2 4 • 1 2 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
2
M i x 1 v o l . o f s o l u t i o n (a) w i t h 2 v o l . s o l u t i o n ( b ) , if n e c e s s a r y a d j u s t t o p H 6.8 w i t h
a s m a l l a m o u n t o f s o l u t i o n (a) o r ( b ) .
Solutions VI and VII are only stable for a day at 0 °C. Solutions III, IV and V are stable for a few days at
0 °C and several weeks at —20 °C. The other solutions are stable for months at 0 °C.
Procedure
Collection and deproteinization :
P h o s p h a t a s e s p r e s e n t in t h e s a m p l e m u s t n o t b e a l l o w e d t o h y d r o l y s e free T P P o r t o f o r m
T P P by hydrolysis of T T P before deproteinization. A d d 0.1 v o l . 7 0 % H C 1 0 4 to aqueous
s a m p l e s a n d c e n t r i f u g e at h i g h s p e e d . A d d 125 m g . K H C 0 3 to each ml. of supernatant fluid,
a l l o w t o s t a n d 1 hr. a t 0 ° C a n d a g a i n c e n t r i f u g e at h i g h s p e e d . U s e t h e r e s u l t i n g s u p e r n a t a n t
fluid directly for t h e d e t e r m i n a t i o n o f T P P ( 0 . 2 - 1 0 n m o l e T P P / m l . ; if n e c e s s a r y , c o n c e n t r a t e
by freeze-drying).
Samples which have not been deproteinized should not be stored unless absolutely necessary
a n d t h e n at < —20 °C. T h e deproteinized and neutralized samples can be stored frozen for
several d a y s w i t h o u t l o s s o f T P P . Significant l o s s e s b y h y d r o l y s i s o c c u r w i t h i n 2 4 hr. at 0 ° C .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; v o l u m e : for p r e l i m i n a r y i n c u b a t i o n
0 . 1 0 m l . ; for a s s a y 3 . 0 0 m l . ; 3 0 ° C ( c o n s t a n t t e m p e r a t u r e ) . R e a d a g a i n s t air. P r e p a r e a b l a n k
containing water instead o f T P P .
A p o - P D C solution (VI) 0.040 ml. 1 3 - 4 0 jug./ml.*

Mg 2 +
solution (II) 0.010 ml. 67 pM*
Sample 0.050 ml. 3-167 nmole*
M i x , a l l o w t o s t a n d for 3 0 m i n . ( 2 5 ° C is a l s o s u i t a b l e ) .
Distilled water 1.600 m l .

C i t r a t e buffer (I) 1.000 m l . 100 m M
N A D H solution (IV) 0.100 ml. ca. 0.17 m M
A D H solution (V) 0.100 ml. 1 7 ^ g . / m l . = 3.4 U / m l .
M i x a n d r e c o r d b l a n k activity.
Pyruvate solution (III) 0.100 ml. 33 m M
M i x , r e a d t h e e x t i n c t i o n at 3 0 sec. i n t e r v a l s for s e v e r a l
m i n u t e s o r b e t t e r still r e c o r d t h e e x t i n c t i o n a u t o m a t i
cally (chart speed 1 - 5 c m . / m i n . ) . T h e progress curve
is linear. D e t e r m i n e A E / m i n . a n d s u b t r a c t b l a n k ( s e e
below).
D e t e r m i n e the total residual capacity of the apopyruvate decarboxylase preparation. This

assay gives the m a x i m u m P D C activity obtainable with T P P saturation:
P r e i n c u b a t e 0 . 0 4 0 m l . a p o - P D C ( a l s o h o l o - P D C w h i c h a l w a y s l o s e s s o m e T P P d u r i n g its
i s o l a t i o n ) a n d 0 . 0 0 5 m l . 10 m M T P P s o l u t i o n V l l . a a n d 0 . 0 0 5 m l . M g 2 +
s o l u t i o n II a t p H
6 . 8 - 7 . 0 a n d r o o m t e m p e r a t u r e f o r 3 0 m i n . D i l u t e w i t h p h o s p h a t e buffer V I I I ( u p t o 5 0 - f o l d )
t o a c o n c e n t r a t i o n s u i t a b l e for a s s a y a n d t a k e 0 . 0 1 0 m l . o f t h e d i l u t i o n for t h e a s s a y .
T h e a s s a y p r o c e d u r e is s l i g h t l y different t o t h a t d e s c r i b e d a b o v e : p y r u v a t e is a d d e d first i n s t e a d
o f P D C a n d a n a d d i t i o n a l 0 . 0 9 0 m l . d i s t i l l e d w a t e r is i n c l u d e d ; t h e r e a c t i o n is s t a r t e d w i t h
the P D C sample (treated as just described).
T h e a c t i v i t y is c a l c u l a t e d a c c o r d i n g t o e q u a t i o n (8) o n p . 3 1 3 .
V o l u m e activity = x 3 x x— x A E / m i n . = 3 7 5 0 x—x A E/min. [U/l.]
F = d i l u t i o n f a c t o r f o r t h e P D C . T o o b t a i n t h e specific a c t i v i t y t h e m e t h o d for p r o t e i n d e t e r
m i n a t i o n d e s c r i b e d in t h e A p p e n d i x is m o s t s u i t a b l e .
Calculations
This is a kinetic m e t h o d for the determination of concentrations (see p . 131) and therefore a s t a n d a r d
curve is required for the calculations.
S t a n d a r d c u r v e : Include s t a n d a r d s in each series of m e a s u r e m e n t s . Take 0.002, 0.005, 0.010, 0.020 a n d
0.050 ml. T P P s t a n d a r d solution V l l . b , c o r r e s p o n d i n g to 20, 50, 100, 200 a n d 500 p m o l e T P P , m a k e
u p t o 0.050 ml. with distilled water a n d treat as samples. Plot the m e a s u r e d A E/min. (deducting the A E/min.
of the blank) against the a m o u n t of T P P .
* 30-fold higher c o n c e n t r a t i o n in the preliminary incubation mixture (0.1 ml.).

Subtract the b l a n k A E/min. from the values for A E/min. found for the samples. Use the corrected values
a n d read off the corresponding T P P concentrations from the s t a n d a r d curve. A n y dilution of the sample
occuring d u r i n g its p r e p a r a t i o n m u s t be taken into account.
With 20 pmole T P P a s t a n d a r d deviation of 3 p m o l e was f o u n d ; with 200 p m o l e T P P it was 12 pmole.

The coefficient of variation is 7 . 2 % a n d 3 . 8 % respectively.
Normal Values
Animal tissues usually contain a r o u n d 1-10 n m o l e T P P / g . , while plants a n d micro-organisms contain

considerably m o r e , especially yeast grown anaerobically. T h e T P P content of T P P - d e p e n d e n t enzymes
which have been purified as the holoenzyme is also very high.
I m p u r e P D C p r e p a r a t i o n s usually contain a yellow, flavin-containing protein fraction which slowly

oxidizes N A D H . T h e resulting b l a n k reaction is m e a s u r e d before the beginning of the actual assay of
activity a n d is subtracted from the results. Incomplete separation of T P P from the a p o e n z y m e during
its p r e p a r a t i o n results in a high b l a n k in the assay. Incomplete r e c o m b i n a t i o n d u r i n g the preincubation
is indicated when the reaction increases with time (consumption of p y r u v a t e favours the r e c o m b i n a t i o n ) . 3
In such cases use the steepest p a r t of the curve for the calculation of A E/min. If necessary, a second addition
of N A D H can be m a d e . T h e presence of T P P - a n t a g o n i s t s , e.g. oxythiamine p y r o p h o s p h a t e (in larger
3
a m o u n t s t h a n T P P ) interferes with the assay.
T h i a m i n e a n d T M P d o n o t react. F o r some time there was uncertainty as to whether T T P could act as

a c o e n z y m e ~ , but n o w it is certain t h a t if p h o s p h a t a s e activity is excluded T T P is i n a c t i v e . It is p r o b a b l e
9 1 2 13
that some T P P h o m o l o g u e s with coenzyme activity can be determined by this m e t h o d : 6'-methyl-4-nor- 3
T P P , 4 - n o r - T P P , 4-ethyl-4-nor-TPP, 2'-ethyl-2-nor-TPP a n d the N - l - p y r i d i n e analogue of T P P .
Other M e t h o d s of Determination of T P P
A similarly reliable coupled assay is that employing transketolase as the rate-limiting e n z y m e ' . This 1 4 1 5 1 5 3
assay is m o r e complicated t h a n t h a t described here, but has the a d v a n t a g e of the greater stability of the
apotransketolase*. It is particularly suitable when the system u n d e r study (e.g. e r y t h r o c y t e s ) already 16
contains all the other enzymes required for the assay.
* Commercial p r e p a r a t i o n from Sigma Chemical C o . , St. Louis, M o . , U S A .

Appendix
Isolation of Pyruvate Decarboxylase, 5 , 7 , 8

1
Additional Reagents
14. A m m o n i u m sulphate 17. Sephadex G-200

15. A m m o n i a solution, ca. 2 5 % (w/w), sp. gr. 18. A c e t o n e , technical
0.907 19. Brewers' yeast, air-dried
16. Glycerol 20. Glycine, N H — C H — C O O H
2 2
IX. Glycine-phosphate buffer ( p H 8.9):

Dissolve 0.6 g. g l y c i n e + 7 . 2 g. N a H P 0 1 2 H 0 + 4 mg. E D T A - N a H - 2 H 0 in ca. 90 ml. distilled
2 4 2 2 2 2
water, adjust to p H 8.9 with 2 N N a O H and m a k e u p to 100 ml. with distilled water.
X . a A m m o n i u m sulphate (2.7 M ) :
Dissolve 357 g. ( N H ) S 0 in w a r m distilled water a n d m a k e u p to 1000 ml.
4 2 4
X . b A m m o n i u m sulphate (2.7 M ; p H 8.8):

Adjust solution X . a with cone, a m m o n i a to p H 8.8
X.c A m m o n i u m s u l p h a t e (2.2 M ) :
Dilute 250 ml. solution X . a with distilled water to 300 ml.
X.d A m m o n i u m sulphate (1.8 M ) :
Dilute 200 ml. solution X . a with distilled water to 300 ml.
Method
Maceration juice: A d d slowly a n d stir (vibro mixer) 600 g. air-dried brewers' yeast in small p o r t i o n s
to a solution of 100 ml. glycerol, 1 g. E D T A - N a H - 2 H 0 and 6 g. a m m o n i u m sulphate in 2.5 1. distilled
2 2 2
water. Allow the mixture to stand overnight at 25 °C or for 3 hr. at 37 °C, then t h o r o u g h l y mix a n d centrifuge
at high speed. C a r r y o u t further o p e r a t i o n s at 0 - 3 °C.
Acetone fractionation: Allow 0.6 vol. of acetone at —15 °C to run in over 15 min. to the ice-cold m a c e r a t i o n
juice, while mixing with vibro mixer (no foam!) After 1 - 2 hr. centrifuge at high speed, a d d m o r e acetone
(15 ml./100 ml.) to the s u p e r n a t a n t fluid a n d immediately centrifuge at 2 0 0 0 - 3 000 g. Suspend the precipitate
in 600 ml. p h o s p h a t e buffer V I I I . After the addition of 10 ml. T P P solution V l l . a , 10 ml. M g S 0 4 solution
II and 0.1 g. E D T A - N a H - 2 H 0 , stir occasionally for 2 hr. and then centrifuge at high speed. Specific
2 2 2
activity of s u p e r n a t a n t fluid is 6 - 1 0 U / m g . protein.
Ammonium sulphate fractionation: To every 100 ml. of s u p e r n a t a n t fluid a d d 40 g. a m m o n i u m sulphate

in portions, while mixing with a vibro mixer (no foam!). Centrifuge off the protein precipitate at 2000 to
3000 g and wash with 300 ml. 2.7 M a m m o n i u m sulphate solution (X.a). T h e n extract the precipitate
with 300 ml. 2.2 M a m m o n i u m sulphate solution (X.c) for 2 hr. with occasional stirring. Discard the ex
tract. Re-extract the residue with 300 ml. 1.8 M a m m o n i u m sulphate solution (X.d). M o s t of the P D C goes
into solution. R e m o v e the residue by centrifugation at high speed. Precipitate the P D C from the super
n a t a n t fluid by the slow addition, with stirring, of a m m o n i u m sulphate (8 g./lOO ml.) a n d centrifuge
at high speed. D e p e n d i n g o n the quality of the starting yeast, the yield is 1 0 - 2 5 g. of a yellow, a m o r p h o u s
paste containing 1 5 - 3 0 % protein a n d a specific activity of 2 0 - 4 0 U / m g . (30 °C). It is stable for m o n t h s in
the frozen state.
Sephadex gel filtration: A p p l y 2 - 3 g. a m m o n i u m sulphate paste in 3 - 4 ml. p h o s p h a t e buffer VIII at

0 °C o n t o a column 100 x 2 cm. containing 12 g. Sephadex G-200 (soaked for 5 hr. at 100 °C and washed
free from oligosaccharides with solution VIII). Elute with solution V I I I ( 2 0 - 4 0 ml./hr., 4 0 - 5 0 cm. of
buffer above the c o l u m n a n d ca. 20 cm. below). T h e first 100 ml. of eluate is protein-free, then collect
5 or 10 ml. fractions a n d immediately m e a s u r e protein a n d P D C activity.
T h e highest specific activity (over 80 U / m g . ) is found in the fractions of ca. 110 to 150 ml. of eluate, that
is before the protein peak ( 1 6 0 - 1 8 0 ml. eluate) which contains considerable a m o u n t s of impurities. T h e
flavin-containing fraction is visible as a yellow b a n d on the c o l u m n a n d only a p p e a r s after the protein
peak, when the P D C activity is virtually zero. All fractions with > 20 U / m g . are suitable for the T P P
determination a n d give an a p o - P D C with a very low blank.
Stabilize the active fractions by addition of 0 . 1 - 0 . 2 ml. T P P solution V l l . a a n d 0 . 1 - 0 . 2 ml. M g S 0 4
solution II a n d precipitate with a m m o n i u m sulphate (4 g./lO ml.). T h e resulting suspension is stable for
m o n t h s at 0 °C, o r better at —20 °C. T h e yield per b a t c h is u p to 150 mg. P D C of high activity and
several h u n d r e d mg. of m e d i u m activity. W i t h careful t r e a t m e n t the c o l u m n can be used three times in
succession before it becomes blocked.
Determination ofprotein: T h e protein content of the P D C solution can be m e a s u r e d by one of the following
m e t h o d s which have been standardized with freeze-dried P D C a n d checked against each other.
a) By means of the U V - a b s o r p t i o n of the a r o m a t i c side c h a i n s ' 1 7 1 7 3

(not suitable for crude extracts):
measure the extinction of the P D C solution (without addition of T P P ! ; diluted as required) in a cuvette of
1 cm. light p a t h at 280 a n d 260 n m . T h e quotient E 2 8 0 /E 2 6 0 for a p o - P D C is 1.75 a n d for h o l o - P D C is 1.60-
1.65. The protein content (mg./ml.) is calculated from the E 2 8 0 by multiplication by the dilution factor
and by 1.01 for a p o - P D C a n d 0.985 for h o l o - P D C .
b) Biuret m e t h o d : A d d 5 ml. biuret reagent to 5 ml. P D C solution ( 1 - 5 mg. protein) or 5 ml. p h o s p h a t e

1 8
buffer VIII (blank). Allow to stand for 30 min. a n d read extinctions at 546 n m (light p a t h 1 cm.). Multi
plication of the difference in extinction between the sample a n d the reagent blank by 36 gives the a m o u n t
of protein in the sample in mg. Avoid errors due to high salt c o n c e n t r a t i o n (by > 0 . 1 M a m m o n i u m
sulphate or > 0 . 3 M of other salts in the sample) as follows: precipitate the protein with 0.2 vol. of 3 M
trichloroacetic acid, centrifuge (add a trace of e t h a n o l to lower the surface tension), wash the precipitate
with 0.5 M trichloroacetic acid a n d centrifuge again. Dissolve the precipitate in 5 ml. biuret reagent
a n d dilute to 10 ml. with buffer VIII.
Preparation of Apo-Pyruvate D e c a r b o x y l a s e 7,19
This m e t h o d starts with the enzyme paste obtained after a m m o n i u m sulphate p r e c i p i t a t i o n 5,7,8
. Commerc
ially available P D C p r e p a r a t i o n m a y also be used.
Dissolve 0 . 2 - 0 . 5 g. h o l o - P D C paste p r e p a r e d according t o 5 , 7 , 8
in 1 ml. water a n d mix with 50 ml. glycine-
p h o s p h a t e buffer IX. Allow to stand for 30 min., stir in 23 g. a m m o n i u m sulphate over a period of 15 min.
and maintain the p H at 8.6-8.8 by dropwise addition of half-concentrated a m m o n i a solution. M o s t
of the a m m o n i a is required at the start. Centrifuge off the precipitate at 2 0 0 0 - 3 000 g, wash twice with
alkaline 2.7 M a m m o n i u m sulphate solution (X.b) a n d twice with a m m o n i u m sulphate solution X.a,
centrifuging at high speed each time. T h e specific activity of the resulting a p o - P D C paste is usually lower
than that of the starting h o l o - P D C . Occasionally the full activity is o b t a i n e d ; the reason for this is not
k n o w n . A p o - P D C is only stable for a few days at — 20 °C. There is considerable d e n a t u r a t i o n within a
few h o u r s at 0 °C.
References
1 O. A. Bessey, O. H. Lowry & E. B. Davis, J. biol. C h e m . 195, 453 [1952].

2 C. Neuberg & L. Karczag, Biochem. Z. 36, 68 & 76 [1911].
3 A. Schellenberger, Angew. C h e m . 79, 1050 [1967].
4 £\ T . Stevn-Parve & / / . G. tf. Westenbrink. Z. Vitaminforsch. 75, 1 [1944].
5
4a A. V. Morey & £ . /ww, J. biol. C h e m . 243, 3009 [1968].

5 H. Holzer & K Beaucamp, Biochim. Biophys. A c t a 46, 225 [1961].
6 A. Schellenberger, Z . physiol. C h e m . 346, 123 [1966] a n d 34$, 491 [1967].
7 i / . i/o/zer, G. Schultz, C. Villar-Palasi & /. Juntgen-Sell, Biochem. Z. 327, 331 [1956].
8 J. Ullrich, J. H. WittorfSc C. J. Gubler, Biochim. Biophys. A c t a 113, 595 [1966].
9 M. Viscontini, G. Bonetti & P. Karrer, Helv. C h i m . A c t a 32, 1478 [1949].
10 / . Bartos, Bull. Soc. C h i m . Biol. 38, 429 [1956].
11 A. Rossi-Fanelli, N. Siliprandi, D. Siliprandi & P. Ciccarone, Arch. Biochem. Biophys. 58, 237 [1955].
12 K. H. Kiessling, Biochim. Biophys. A c t a 20, 293 [1956].
13 K. H. Kiessling, Biochim. Biophys. A c t a 46, 603 [1961].
14 A. G. Datta & E. Racker, J. biol. C h e m . 236, 624 [1961].
15 L. R. Johnson & C. / . Gubler, Biochim. Biophys. A c t a 156, 85 [1968].
15a C. / . Gubler, L. R. Johnson & /. H. Wittorf, M e t h . E n z y m o l . 18A, 120 [1970].
16 M. Brin, J. N u t r i t i o n 71, 273 [1960] a n d 78, 179 [1962].
17 O. Warburg & W. Christian, b i o c h e m . Z. 310, 384, 400 [1942].
17a H. M. Kalckar, J. biol. C h e m . 167, 461 [1947].
18 G. Beisenherz, H. J. Boltze, T. Bucher, R. Czok, K H. Garbade, E. Meyer-Arendt & G. Pfleiderer,
Z. N a t u r f o r s c h . 8b, 555 [1953].
19 H. Holzer & H. W. Goedde, Biochem. Z . 329, 192 [1957].
Pyridoxal-5-phosphate and Pyridoxamine-5-phosphate
Gerhard Schreiber
Pyridoxal-5-phosphate ( P A L P ) and pyrid oxamine-5 -p hosphate ( P A M P ) are the catalytically active

coenzyme derivatives of vitamin B . In the earlier m e t h o d s for their d e t e r m i n a t i o n (for review, see ) P A L P ,
6
1
P A M P and free vitamin B h a d first to be separated by c h r o m a t o g r a p h y or electrophoresis. T h e separated

6
products were then measured by colorimetry, s p e c t r o p h o t o m e t r y , fluorimetry or microbiological assay.

A direct determination of P A L P with tyrosine decarboxylase from Streptococcus faecalis has been reported
by Gunsalus a n d Smith . 2
The m e t h o d described here allows the separate, specific d e t e r m i n a t i o n of P A L P and P A M P with an

apoaminotransferase (from brewers' yeast ( L - A s p a r t a t e : 2-oxoglutarate aminotransferase, E C 2.6.1.1)
and treatment of the sample with p o t a s s i u m b o r o h y d r i d e . W i t h highly purified a p o e n z y m e it is possible t o 3
determine a m o u n t s as low as 1 0 - 4
^g. P A L P and P A M P .
To determine the sum of P A L P + P A M P a modification of the m e t h o d reported by Goryachenkova* is
used in which the 2-oxo acids formed in the t r a n s a m i n a t i o n reaction are determined colorimetrically
as the dinitrophenylhydrazones.
Application of Method: In biochemistry, microbiology, foodstuff chemistry a n d medicine.
P rinc iple
Coenzyme-free apoaminotransferase can be reactivated by either P A L P or P A M P . T h e degree of reactiv 5
ation can be followed spectrophotometrically at 365 (334, 340) n m by coupling the t r a n s a m i n a t i o n reaction
with malate dehydrogenase ( L - M a l a t e : N A D oxidoreductase, E C 1 . 1 . 1 . 3 7 ) " : 6 10
(1) 2-Oxoglutarate =
+ Aspartate" , a m i n o t r a n s t e r a s e
- Glutamate' + Oxaloacetate =
(2) Oxaloacetate =
+ NADH + H +
A " a l a t e
• Malate =
+ NAD " 4
v
' dehydrogenase
If malate dehydrogenase, N A D H , 2-oxoglutarate, a s p a r t a t e a n d a p o a m i n o t r a n s f e r a s e are present in excess

the a m o u n t s of P A L P or P A M P a d d e d to the assay are rate-limiting. A plot of the m e a s u r e d enzyme activity
against the coenzyme co ncentratio n in the assay mixture gives an a p p r o x i m a t e l y linear curve at low
coenzyme concentrations. By c o m p a r i s o n with authentic P A L P or P A M P p r e p a r a t i o n s (standard curves)
it is possible to determine the P A L P a n d P A M P content of u n k n o w n samples. T h e separate determination
of P A L P and P A M P is achieved by t r e a t m e n t with b o r o h y d r i d e . T h e former is reduced by K B H , possibly 4
to pyridoxol-5-phosphoric acid, a n d is n o longer active as a coenzyme. P o t a s s i u m b o r o h y d r i d e does n o t

alter the coenzyme activity of P A M P 9 1 0
.
The p H o p t i m u m of the a p o a m i n o t r a n s f e r a s e at 25 °C in tris or t r i e t h a n o l a m i n e buffer is p H 8 . 2 - 8 . 3 . At

p H 6.8 (citrate buffer) a n d at p H 9.0 (tris or d i e t h a n o l a m i n e buffer) there is still 5 0 % of the activity found at
p H 8.2 . T h e t u r n o v e r n u m b e r of the aminotransferase at 25 °C a n d p H 8.2 is 1.91 x 1 0 mole aspartate per
5 4
mole enzyme per m i n . . Small a m o u n t s of a p o e n z y m e are sufficient to carry out a large n u m b e r of determin
5
ations.
Pyridoxal-5-phosphate and Pyridoxamine-5-phosphate 2195
Equipment
334 or 365 n m ; b e n c h centrifuge.
Reagents
1. Triethanolamine 5. P y r i d o x a l - 5 - p h o s p h o r i c a c i d
2. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o commercial preparation, see p. 550.
tide, NADH 6. P y r i d o x a m i n e - 5 - p h o s p h o r i c a c i d
disodium salt, N A D H - N a ; commercial
2 7. A p o a m i n o t r a n s f e r a s e f r o m b r e w e r s ' y e a s t
preparation, see p. 545. according t o 3
3. M a l a t e d e h y d r o g e n a s e , MDH 8. L - A s p a r t i c a c i d
from pig heart, ^ 700 U/mg. (25 °C). Suspension 9. P o t a s s i u m b o r o h y d r i d e , KBH 4
in 3.2 M ammonium sulphate solution; commer 10. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA

cial preparation, see p. 485. disodium salt, E D T A - N a H • 2 H 0 2 2 2
4. 2-Oxoglutaric acid 11. Hydrochloric acid, 2 N

commercial preparation, see p. 548. 12. S o d i u m h y d r o x i d e , 2 N
Purity of Reagents
The apoaminotransferase preparation should have as low as possible residual content of coenzymes. The
coenzymes content of a 170-fold purified preparation, which was suitable for the determination of P A L P
and P A M P , was less than 1 % of that at complete saturation . 5
P r e p a r e all s o l u t i o n s w i t h f r e s h , d o u b l y d i s t i l l e d w a t e r .
D i s s o l v e 5 . 9 7 g. t r i e t h a n o l a m i n e in d i s t i l l e d w a t e r , a d j u s t t o p H 8.2 w i t h 2 N HQ
a n d d i l u t e t o a b o u t 195 m l . w i t h d i s t i l l e d w a t e r . C h e c k t h e p H a n d d i l u t e t o 2 0 0 m l . w i t h
distilled water.
II. R e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 1 2 m M /?-NADH):
D i s s o l v e 10 m g . N A D H - N a 2 ( c o n t a i n i n g 7 8 % j S - N A D H ) in 1 m l . d i s t i l l e d w a t e r .
III. M a l a t e d e h y d r o g e n a s e , M D H ( 0 . 5 m g . p r o t e i n / m l . ) :
D i l u t e the s u s p e n s i o n accordingly with 3.2 M a m m o n i u m sulphate solution.
IV. 2-Oxoglutarate (0.5 M ) :

D i s s o l v e 7 3 0 m g . 2 - o x o g l u t a r i c a c i d ( 7 4 5 m g . o f a 9 8 % p r e p a r a t i o n ) w i t h a little d i s t i l l e d
w a t e r , n e u t r a l i z e w i t h 2 N N a O H a n d d i l u t e t o 10 m l . w i t h d i s t i l l e d w a t e r .
V . P y r i d o x a l - 5 - p h o s p h o r i c a c i d , P A L P , s t a n d a r d s o l u t i o n ( 0 . 0 5 pg./ml.; 0.189 pM):

Dissolve 5 mg. crystalline pyridoxal-5-phosphate (C H 8 1 0 O NP +
6 H 0)
2 in d i s t i l l e d
w a t e r a n d m a k e u p t o 1 0 0 m l . D i l u t e 0.1 m l . o f this s o l u t i o n t o 1 0 0 m l . M i x c a r e f u l l y .
V I . P y r i d o x a m i n e - 5 - p h o s p h o r i c a c i d , P A M P , s t a n d a r d s o l u t i o n ( 0 . 0 5 jug./ml., 0 . 2 1 5 pM):
Dissolve 5 mg. pyridoxamine-5-phosphoric a c i d in d i s t i l l e d w a t e r a n d m a k e u p t o
1 0 0 m l . D i l u t e 0.1 m l . o f this s o l u t i o n t o 100 m l . w i t h d i s t i l l e d w a t e r . M i x c a r e f u l l y .
V I I . A p o a m i n o t r a n s f e r a s e (ca. 3 m g . p r o t e i n / m l . ) :
If n e c e s s a r y , d i l u t e t h e p r e p a r a t i o n o f p . 2 1 9 8 w i t h 2.3 M a m m o n i u m s u l p h a t e s o l u t i o n .
VIII. L-Aspartate (0.5 M ) :

D i s s o l v e 6 6 5 . 5 m g . L - a s p a r t i c a c i d ( 6 7 8 . 8 m g . o f a 9 8 % p r e p a r a t i o n ) in a little d i s t i l l e d
w a t e r , n e u t r a l i z e w i t h 2 N N a O H a n d d i l u t e t o 10 m l . w i t h d i s t i l l e d w a t e r .
IX. Potassium borohydride, K B H 4 (0.1 M K B H 4 in 0 . 0 2 N KOH):

D i s s o l v e 0 . 2 7 g. K B H 4 in 0 . 0 2 N K O H a n d m a k e u p t o 5 0 m l . P r e p a r e freshly e a c h t i m e .
Solutions I to IV can be c o m b i n e d in an assay mixture, which is stable for at least 48 hr. if stored in the
d a r k a n d at 4 ° C . T h e P A L P a n d P A M P s t a n d a r d solutions (V a n d VI) are stable in d a r k bottles for
3
a b o u t a week at 4 °C. T h e stability of the a p o a m i n o t r a n s f e r a s e solution (VII) d e p e n d s on the degree of

purity. T h e time required for the activity to decrease by 5 0 % is between 63.5 a n d 251 days on storage of
the enzyme p r e p a r a t i o n in 3.2 M a m m o n i u m sulphate solution at —18 ° C . T h e a s p a r t a t e solution (VIII)
5
is relatively stable at r o o m t e m p e r a t u r e , but should be stored in the cold to avoid bacterial growth. T h e
potassium b o r o h y d r i d e solution (IX) m u s t be p r e p a r e d immediately before use.
Procedure
Collection of sample and deproteinization:
W a s h 10 g. fresh b a k e r s ' y e a s t t w i c e w i t h c o l d d i s t i l l e d w a t e r , m a k e u p t o 1 0 0 m l . w i t h distilled

w a t e r w i t h stirring a n d , t o a v o i d n o n - e n z y m a t i c , metal-catalysed transamination , 1 1
add
E D T A ( t o a final c o n c e n t r a t i o n o f a b o u t 10 m M ) . B o i l for 5 m i n . a n d i m m e d i a t e l y c o o l in a n
ice b a t h ; c e n t r i f u g e t o clarify. D e t e r m i n e P A L P a n d P A M P in t h e s u p e r n a t a n t fluid.
H o m o g e n i z e a n i m a l t i s s u e s a m p l e s w i t h 5 t i m e s their w e i g h t o f d i s t i l l e d w a t e r c o n t a i n i n g
10 m M E D T A in a n U l t r a - T u r r a x ( h i g h - s p e e d b l a d e h o m o g e n i z e r ; Janke & Kunkel, Staufen
i. B r s g . , G e r m a n y ) . H e a t for 5 m i n . at 1 0 0 ° C , c o o l in a n ice b a t h a n d c e n t r i f u g e . D e t e r m i n e
P A L P a n d P A M P in t h e s u p e r n a t a n t fluid.
Reduction with potassium borohydride:
B y r e d u c t i o n w i t h K B H , P A L P is c o n v e r t e d t o a n e n z y m a t i c a l l y i n a c t i v e c o m p o u n d , w h i l e
4
t h e a c t i v i t y o f P A M P a s a c o e n z y m e for t h e t r a n s a m i n a t i o n is n o t a f f e c t e d . F o r t h e r e d u c t i o n
a d d 0.6 m l . a n d 0.5 m l . K B H 4 s o l u t i o n ( I X ) in d a r k test t u b e s t o 0.1 a n d 0.2 m l . , r e s p e c t i v e l y ,
o f t h e s a m p l e t o b e a n a l y s e d . S t o p p e r a n d i n c u b a t e for 5 m i n . at 37 ° C . A f t e r a l l o w i n g t o c o o l
in ice w a t e r a d d 0.3 m l . 0.1 N H S 0 2 4 to destroy the excess borohydride. H e a t the tightly stopper
e d t u b e s for 5 m i n . at 9 0 - 1 0 0 ° C a n d c o o l in ice w a t e r a g a i n . T a k e 5 t o 10 t i m e s t h e a m o u n t
o f r e d u c e d s a m p l e a s c o m p a r e d t o t h e n o n - r e d u c e d s o l u t i o n for t h e s p e c t r o p h o t o m e t r i c a s s a y
because of the dilution due to the various additions.
Pyridoxal-5-phosphate a n d P y r i d o x a m i n e - 5 - p h o s p h a t e 2197
A l l p y r i d o x a l d e r i v a t i v e s are l i g h t - s e n s i t i v e , e s p e c i a l l y in n e u t r a l o r a l k a l i n e s o l u t i o n . In
a c i d s o l u t i o n a n d in t h e s o l i d s t a t e t h e s e n s i t i v i t y is less. T h e ester b o n d o f t h e 5 - p h o s p h a t e s is
relatively difficult t o h y d r o l y s e , b u t t h a t o f P A M P is h y d r o l y s e d m o r e e a s i l y t h a n t h a t o f P A L P .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; l i g h t p a t h : 1.00 c m . ; final v o l u m e : 3 . 0 m l . ; t e m p e r a t u r e :
25 ° C ( c o n s t a n t t e m p e r a t u r e c u v e t t e ) ; r e a d a g a i n s t distilled w a t e r o r air.
D i l u t e t h e s a m p l e s o t h a t t h e rate o f t h e r e a c t i o n A E / m i n . after s u b t r a c t i o n o f t h e c o n t r o l
v a l u e ( w a t e r i n s t e a d o f c o e n z y m e ) lies o n t h e l i n e a r part o f t h e s t a n d a r d c u r v e . T h e a s s a y
system should be checked by including a sample with a k n o w n content o f P A L P and P A M P
w i t h e a c h series o f m e a s u r e m e n t s ( s e e " S t a n d a r d c u r v e s " ) .
F r o m e a c h s a m p l e ( P A L P -f P A M P ) a n d f r o m e a c h r e d u c e d s a m p l e ( P A M P ) t a k e s e v e r a l
different a m o u n t s .
Reagent mixture:
T r i e t h a n o l a m i n e buffer (I) 26.1 m l .

N A D H solution (II) 1.0 m l .
M D H suspension (III) 0.3 m l .
2-Oxoglutarate solution (IV) 0.2 ml.
27.6 ml.
assay mixture
Reagent mixture 2.76 ml. 1 7 4 m M buffer

0.4 m M NADH
3.3 m M 2 - O x o G
5 pg. M D H / m l . = 3 . 6 U / m l .
Sample 0.20 ml.
Apoaminotransferase
0.01 m l . 10 pg./ml
suspension (VII)
M i x , i n c u b a t e for 5 m i n . a n d r e a d t h e e x t i n c t i o n at
3 0 sec. i n t e r v a l s u n t i l it n o l o n g e r c h a n g e s o r u n t i l
a n y s m a l l d e c r e a s e in e x t i n c t i o n is c o n s t a n t . D e t e r m i n e
A E J m i n . (mean value).
Aspartate solution (VIII) 0.03 ml. 5 mM
R e a d t h e e x t i n c t i o n d e c r e a s e at 3 0 sec. i n t e r v a l s for
5 t o 10 m i n . D e t e r m i n e A E / m i n . 2
Calculations
T h e t r a n s a m i n a s e reaction should be linear over a certain range. F r o m the m e a n values A E ^ m i n . a n d

A E / m i n . calculate the difference A E / m i n . - A E ^ m i n . This gives A E
2 2 s a m p l e / m i n . and A E c o n t r o l /min.
Ê s a m p l e /min. - J E c o n t r o l /min. = ^ E P A L P + P A M P /min.

2198 M e t a b o l i t e s : Nucleic Acids. Purines, Pyrimidines, Nucleosides, C o e n z y m e s
Similarly, A E P A M P / m i n . is obtained for the reduced sample. These values are characteristic for the rate of the
transaminase reaction which is dependent on the concentration of pyridoxal-5-phosphate a n d pyridoxamine-
5-phosphate. T h e coenzyme concentrations corresponding to A E/min. (corrected for the control) are read
off from the s t a n d a r d curve. T h e difference A E P A L P + P A M P /min. - AE P A M P / m i n . gives the a m o u n t of P A L P .
Standard Curves
Prepare s t a n d a r d curves for P A L P ( a b o u t 0.63 to 6.3 n M ) and for P A M P ( a b o u t 0.72 to 14.4 n M ) . F o r this
add 0.01 ml. ( = 0.0005 jig) toO.lOml. ( = 0.005 pg) of s t a n d a r d solution V ( P A L P ) a n d 0.01 ml. ( = 0.0005 pg.)
to 0.20 ml. ( = 0.01 pg.) of s t a n d a r d solution VI ( P A M P ) to the assay system. Plot rate A E/min. corrected
for the control on the o r d i n a t e and the concentration ( n M ) of coenzyme in the assay mixture on the
abscissa.
We obtained values for the coefficient of variation for the sum ( P A L P + P A M P ) of 2.9 to 3 . 3 % . T h e
values for P A L P , because they are obtained by difference, depend on the ratio of ( P A L P + P A M P ) : P A M P
in the sample. T h e error becomes larger the m o r e the value to be subtracted a p p r o a c h e s that for the sum.
Normal Values 1 0
In b a k e r s ' yeast the m e a n content of P A L P was 0.5 pg./g. fresh wt., the m e a n content of P A M P 3.1 pg./g.
fresh wt.
The following values have been measured in various rat tissues (related to g. fresh wt.). Liver: 0.9 pg. P A L P
and 7.5 pg. P A M P ; kidney: 0.8 pg. P A L P and 4.5 pg. P A M P ; b r a i n : 0.2 pg. P A L P a n d 2.4 pg. P A M P .
T h e assay is slightly inhibited by pyridoxal and o r t h o p h o s p h a t e , a n d strongly inhibited by pyridoxol

p h o s p h a t e and isonicotinic acid hydrazide. T h e inhibition by pyridoxol p h o s p h a t e can cause interference
when the sample contains large a m o u n t s of P A L P . P A L P is reduced by K B H , p r o b a b l y t o pyridoxol
1 0
4
p h o s p h a t e , consequently a t o o low result for the content of P A M P in the sample is o b t a i n e d . This inhibition
is only significant with high P A L P concentrations. F o r the quantitative estimation of the inhibition, s e e . 10
The m e t h o d is specific for P A L P and P A M P ' 5 9 1 0

.
Appendix
Apoaminotransferase from Brewers' Yeast 3
Carry out all steps, unless otherwise stated, at 0 - 4 °C. M a c e r a t i o n juice: Stir one p a r t dried brewers' yeast
(about 2 0 0 - 3 0 0 g.) with 3 parts distilled water for 3 hr. at 37 °C and then allow to stand for several h o u r s
at 0 °C. Centrifuge t o obtain a clear s u p e r n a t a n t fluid. T h e protein content of the s u p e r n a t a n t fluid is a b o u t
3 0 - 7 0 mg./ml.
Partial heat d e n a t u r a t i o n : H e a t the m a c e r a t i o n juice to 53 °C - 54 °C over 4 min., hold for 8 min. at this
temperature and then cool in an ice b a t h .
Pyridoxal-5-phosphate a n d P y r i d o x a m i n e - 5 - p h o s p h a t e 2199
F r a c t i o n a t i o n with a m m o n i u m s u l p h a t e : Adjust the s u p e r n a t a n t fluid to a b o u t 40 mg. protein/ml., slowly

bring to 1.23 M with solid a m m o n i u m sulphate a n d allow t o stand for 20 m i n . in a n ice b a t h . Centrifuge off
the precipitated protein, adjust the s u p e r n a t a n t fluid to 1.73 M a m m o n i u m sulphate, allow to stand for
20 min. in an ice b a t h a n d then centrifuge. T h e sediment contains a mixture of aminotransferase a n d
apoaminotransferase.
Gel filtration t h r o u g h Sephadex G 100: Take u p the precipitated protein in a little 1 M tris buffer, p H 7.3
and add to a Sephadex-G 100 c o l u m n (6 cm. x 40 cm.) equilibrated with 10 m M tris buffer. C o l u m n s filled
60 cm. with gel still give a g o o d flow r a t e ; with higher c o l u m n s the longer time the enzyme is o n the c o l u m n
results in a decreased yield, because the enzyme is not very stable at this stage. Elute with 10 m M tris
buffer, p H 7.3 a n d follow the extinction of the eluate at 280 n m . Several peaks are o b t a i n e d . T h e enzyme
appears in the second half of the first peak from the c o l u m n .
C h r o m a t o g r a p h y o n D E A E - c e l l u l o s e : Immediately (!) a d d the enzyme fraction purified o n Sephadex G 100
a n d freed from a m m o n i u m sulphate to a DEAE-cellulose c o l u m n (e.g. c o l u m n v o l u m e 270 ml., height of
packing after settling 44 cm., equilibrated with 10 m M tris buffer, p H 7.3). After the enzyme is a b s o r b e d
wash the c o l u m n with two c o l u m n volumes of 10 m M tris buffer, p H 7.3 a n d then elute with 50 m M tris
buffer, p H 7.3. M e a s u r e the enzyme activity and extinction at 280 n m of the eluate. C o m b i n e the fractions
containing the enzyme, bring to 2.6 M with solid a m m o n i u m sulphate a n d centrifuge off the precipitate. Take
u p the sediment in a little 1 M tris buffer, p H 7.3, bring t o 2.6 M with solid a m m o n i u m sulphate a n d store at
—18 °C. T h e a p o e n z y m e content of P A L P + P A M P is < 1% of t h a t of the enzyme when saturated with
coenzyme. T h e stability d e p e n d s on the p u r i t y . After incubation of a p o e n z y m e solutions with P A L P , it is
5
b o u n d again t o the a p o e n z y m e a n d c a n n o t be removed by dialysis . 5
References
1 A. E. Braunstein, T h e Enzymes, A c a d e m i c Press, N e w Y o r k 1960, 2nd. edn., Vol. II, p . 136.

2 /. C. Gunsalus & R. A. Smith in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c
Press, N e w Y o r k 1957, Vol. I l l , p. 963.
3 G. Schreiber, M. Eckstein, A. Oeser & H. Holzer, Biochem. Z. 340, 13 [1964].
4 E. V. Goryachenkova, Biochimiya (russ.) 28, 565 [1963].
5 G. Schreiber, M. Eckstein, G. Maass & H. Holzer, Biochem. Z. 340, 21 [1964].
6 J. S. LaDue, F. Wroblewski & A. Karmen, Science (Washington) 120, 497 [1954].
7 A. Karmen, F. Wroblewski & J. S. LaDue, J. clin. Invest. 34, 131 [1955].
8 H Holzer, U. Gerlach, G. Jacobi & Af. Gnoth, Biochem. Z. 329, 529 [1958].
9 H. Holzer & G. Schreiber, Proceedings of the S y m p o s i u m on Chemical a n d Biological Aspects of
Pyridoxal Catalysis, R o m e 1962. Oxford, L o n d o n , N e w Y o r k , Paris, P e r g a m o n Press 1963, p . 523.
10 G. Schreiber, M. Eckstein, A. Oeser & H. Holzer, Biochem. Z. 340, 35 [1964].
11 E. E. Snell, Fed. Proc. Suppl. 20, N o . 10, 81 [1961].
Coenzyme-B 12
Robert H. Abeles
T h e enzyme p r o p a n e d i o l dehydratase from Aerobacter aerogenes 1

(1,2-Propanediol hydro-lyase, E C 4.2.1.28)
in the presence of coenzyme B 1 2 (dimethyl-benzimidazole c o b a m i d e coenzyme, D B C C ) catalyses the
oxidation of p r o p a n e d i o l to p r o p i o n a l d e h y d e . T h e enzyme is also active in the presence of other B 1 2
coenzymes, e.g. with adenyl-cobamide coenzyme and benzimidazolyl coenzyme. As the V m a x with all
these coenzymes is the same, the m e t h o d described here should also be applicable with these coenzymes.
It is also i m p o r t a n t to r e m e m b e r that some extracts of biological material can contain unidentified forms
of the coenzyme, which m a y be less active. We have used the m e t h o d described here continuously and so
far the results have been reproducible and in agreement.
Principle
<D C H 3 - C H - C H 2 ^ ^ C H 3 - C H 2 - C ^ + H 0
2
I I H
OH OH
With low concentrations of the coenzyme the rate of p r o p i o n a l d e h y d e formation is directly p r o p o r t i o n a l

to the coenzyme concentration. The a m o u n t of p r o p i o n a l d e h y d e formed per unit time is measured in a
colorimetric assay with 2,4-dinitrophenylhydrazine . 3
Vitamin B 1 2 and related derivatives inhibit. To decrease this inhibition a 2- to 4-fold excess of enzyme is
used. The experimental results are related to a s t a n d a r d curve. Inhibitions due to u n k n o w n c o m p o u n d s in
biological extracts are allowed for by addition of s t a n d a r d solution to the sample and observing the
recovery.
Equipment
Potter-Elvehjem h o m o g e n i z e r . R o t a r y v a c u u m evaporator; centrifuge (up to ca. 1 2 0 0 0 g ) ;

p h o t o m e t e r for m e a s u r e m e n t s at c a . 5 4 0 n m .
Reagents
1. D i p o t a s s i u m h y d r o g e n p h o s p h a t e 5. Dioldehydratase
K HP0 ,
2 4 A.R. from Aerobacter aerogenes ( A T C C 8724) ac
2. P o t a s s i u m d i h y d r o g e n p h o s p h a t e cording t o ; purification as far as step E-3 is
2
K H P 0 , A.R.
2 4
sufficient.
3. 2,4-Dinitrophenylhydrazine 6. Dimethyl-benzimidazolylcobamide
4. Pyridine coenzyme, coenzyme B 1 2
dried over K O H e. g. from Glaxo

Coenzyme-B 12
2201
7. D L - l , 2 - P r o p a n e d i o l , 9. Methanol,
redistilled A . R . , redistilled over N a B H 4
8. B o v i n e s e r u m a l b u m i n 10. P o t a s s i u m h y d r o x i d e , K O H
11. H y d r o c h l o r i c acid, 2 N
12. E t h a n o l , 9 6 % ( w / v ) , A . R .
U s e o n l y fresh d i s t i l l e d w a t e r .
a) D i s s o l v e 3 4 . 8 g. K H P 0 2 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
b ) D i s s o l v e 2 7 . 2 g. K H P 0 2 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 0 m l .
M i x b o t h s o l u t i o n s s o t h a t p H 8.0 is a c h i e v e d .
II. S u b s t r a t e s o l u t i o n ( 2 6 0 m M p r o p a n e d i o l ) :
Dissolve 20 ml. D L - l , 2 - p r o p a n e d i o l , 50 ml. K H P 0 2 4 s o l u t i o n ( l a ) a n d 1 g. a l b u m i n i n
distilled water a n d m a k e u p t o 1 0 0 0 ml.
III. D i o l d e h y d r a t a s e ( 1 0 - 1 5 U / m l . ) :
D i l u t e t h e e n z y m e p r e p a r a t i o n a c c o r d i n g l y w i t h s u b s t r a t e s o l u t i o n (II). D e t e r m i n e t h e
activity according t o . 2
IV. C o e n z y m e - B , D B C C standard solution ( 0 . 0 1 0 - 0 . 0 1 5

1 2 fig./ml):
W e i g h o u t 1 0 - 1 5 m g . D B C C in s u b d u e d l i g h t a n d d i s s o l v e in 10 m l . d i s t i l l e d w a t e r ;
p r o t e c t this s o l u t i o n f r o m direct light. T h i s s t o c k s o l u t i o n c a n b e s t o r e d f r o z e n for a b o u t
1 m o n t h . F r o m t h i s s o l u t i o n p r e p a r e further d i l u t i o n s a s s t a n d a r d s . D e t e r m i n e the
c o e n z y m e c o n t e n t b y m e a s u r e m e n t o f t h e e x t i n c t i o n at 5 2 0 n m . T h e e x t i n c t i o n c o e f f i c i e n t
is 8.0 c m . / ^ m o l e , t h e m o l e c u l a r w e i g h t 1 5 8 0 . A l t e r n a t i v e a s s a y : d i l u t e t h e s t o c k s o l u t i o n
2
in K C N s o l u t i o n t o g i v e a final c o n c e n t r a t i o n o f 0.1 M K C N a n d d e t e r m i n e t h e e x t i n c t i o n
o f t h e d i c y a n o c o b a l a m i n at 3 6 0 n m . T h e e x t i n c t i o n c o e f f i c i e n t is 3 0 . 4 c m . / ^ m o l e .
2
V. Pyridine (80%):
M i x 4 0 0 ml. pyridine a n d 100 ml. distilled water.
V I . M e t h a n o l i c - K O H (ca. 1.4 N ) :
M i x 8 0 m l . m e t h a n o l a n d 2 0 m l . 4 0 % K O H ( 4 0 g. K O H in d i s t i l l e d w a t e r m a d e u p t o
100 m l . ) .
V I I . 2 , 4 - D i n i t r o p h e n y l h y d r a z i n e , 2 , 4 - D N P H (8 m M ) :
A d d 2 5 m g . 2 , 4 - D N P H t o 10 m l . m e t h a n o l ; a d d 0 . 2 m l . c o n e . H C 1 . A f t e r c o m p l e t e
s o l u t i o n a d d a further 15 m l . m e t h a n o l . P r e p a r e t h i s s o l u t i o n f r e s h l y e a c h d a y .
Procedure
Carry out the following m a n i p u l a t i o n s in the d a r k ; a 3 volt l a m p as the only light source.
Mince a n d homogenize 300 mg. of liver in 5 ml. water in a Potter-Elvehjem H o m o g e n i z e r . P o u r e the
h o m o g e n a t e then into 25 ml. boiling ethanol a n d heat for 5 m i n . ; cool and filter t h r o u g h filter paper. A d d the
precipitate again to 12 ml. boiling ethanol a n d heat for 3 m i n . ; cool and filter again. Bring the c o m b i n e d
filtrates virtually to dryness in a r o t a t o r y e v a p o r a t o r in vacuo a n d dissolve the residue in 3 ml. distilled
water. Centrifuge this solution for 1 hr. at 12000 g. It is necessary to o b t a i n a solution as clear as possible.
The extract must n o t be exposed to light; it is stable for several weeks in the frozen state.
Assay System
I n c u b a t i o n t e m p e r a t u r e : 37 ° C ; i n c u b a t i o n v o l u m e : 1.0 m l . ; final v o l u m e : 8.1 m l . ; r o o m

t e m p e r a t u r e ; w a v e l e n g t h : 5 4 0 ( H g 5 4 6 ) n m . R e a d a g a i n s t a c u v e t t e c o n t a i n i n g 0.8 m l . distilled
w a t e r + 0 . 2 m l . buffer (I). S e v e r a l e x t r a c t s c a n b e m e a s u r e d s i m u l t a n e o u s l y , b u t t a k e t w o dif
ferent a m o u n t s (0.1 a n d 0.2 m l . ) f r o m e a c h extract. F o r o n e extract e i g h t t u b e s are r e q u i r e d :
n o . 1 t o 4 for s t a n d a r d a n d b l a n k , n o . 7 for s t a n d a r d w i t h s a m p l e , n o . 8 for b l a n k w i t h s a m p l e .
N o . 5 a n d 6 are t h e a c t u a l e x p e r i m e n t a l t u b e s . F o r e a c h series o f m e a s u r e m e n t s p r e p a r e at least
o n e blank with sample ( t u b e 8 ) : a s for e x p e r i m e n t a l t u b e w i t h 0.2 m l . s a m p l e , b u t distilled w a t e r
i n s t e a d o f e n z y m e (III).
Pipette into centrifuge t u b e s : Tube 5 Tube 6
assay mixture
Buffer (I) 0.2 m l . 0.2 m l . 40 m M

Distilled water 0.5 m l . 0.4 ml.
E q u i l i b r a t e t o t e m p e r a t u r e . C a r r y o u t all o t h e r a d d i t i o n s in
t h e d a r k w i t h a 3 v o l t l a m p a s t h e o n l y light s o u r c e .
Sample 0.1 m l . 0.2 m l . u p t o 0.08 pg./ml
M i x a n d e q u i l i b r a t e at 37 ° C for 5 m i n .
Dioldehydratase solution (III) 0.2 m l . 0.2 ml. 2 - 3 U/ml.
M i x a n d i n c u b a t e at 37 ° C for e x a c t l y 6 0 m i n .
2NHC1 0.1 m l . 0.1 m l . 0.025 N
S h a k e a n d carry o u t all further o p e r a t i o n s at r o o m t e m p e r a t u r e

a n d at n o r m a l i l l u m i n a t i o n .
2 , 4 - D N P H solution (VII) 1.0 m l . 1.0 m l . 1 mM
M i x a n d a l l o w t o s t a n d for 3 0 m i n .
Pyridine solution (V) 5.0 m l . 5.0 m l . 50%

Mix
Methanolic-KOH (VI) 1.0 m l . 1.0 m l . 0.17 N
M i x carefully w i t h a g l a s s s p a t u l a a n d a l l o w t o s t a n d for 5 m i n .
If n e c e s s a r y , c e n t r i f u g e t o clear a n d t h e n m e a s u r e t h e e x t i n c t i o n s .
Standard values (tubes 1-3): A s for e x p e r i m e n t a l t u b e s , b u t i n s t e a d o f s a m p l e + distilled

w a t e r t a k e 0 . 1 , 0.2 m l . a n d 0 . 4 m l . D B C C s t a n d a r d s o l u t i o n ( I V ) + distilled w a t e r ; read a g a i n s t
b l a n k ( t u b e 4 ) w i t h 0.6 m l . distilled w a t e r + 0.2 m l . buffer (I) + 0 . 2 m l . e n z y m e (III). T h e
m e a s u r e d e x t i n c t i o n s s h o u l d b e in t h e ratio o f 1 : 2 : 4 ; significant d e v i a t i o n i n d i c a t e s that t h e
a s s a y is n o t correct.
A standard with sample ( t u b e 7) tests for t h e p r e s e n c e o f i n h i b i t o r s in t h e s a m p l e : as for t h e
e x p e r i m e n t a l t u b e w i t h 0 . 2 m l . s a m p l e , b u t in a d d i t i o n 0 . 2 m l . D B C C s t a n d a r d s o l u t i o n I V .
Coenzyme-B 12 2203
Calculations
F o r tube 6 with 0.2 ml. sample the a m o u n t of p r o p i o n a l d e h y d e formed in 60 min. is given b y :
^sample —
(^B + S + E ),
B
F o r tube 5 with 0.1 ml. s a m p l e :
^ s a m p l e ~~ Cll E B + S + E ). B
E B = extinction of b l a n k (tube 4)
E B + S = extinction of b l a n k + sample (tube 8)
These t w o values should give a 1 : 2 ratio.

If the sample contains n o inhibitor, the extinction of 0.2 ml. sample + extinction of 0.2 ml. s t a n d a r d
solution ( - e x t i n c t i o n of blank) should be the same as the extinction of the s t a n d a r d + sample. If there is
significant deviation from this situation, the sample contains inhibitors; calculate the following correction
factor:
E —E
Correction factor f = — —
Est + s Es —
E s t = extinction of 0.2 ml. s t a n d a r d (tube 2)
Est+s =
extinction of s t a n d a r d with sample (tube 7)
E s = extinction of 0.2 ml. sample
The coenzyme-B 12 c o n c e n t r a t i o n is calculated as follows:
fig. D B C C / m l . extract = E
s ~ E
b+s - E B x c
hSt —L B i
c = fig. B D C C / m l . s t a n d a r d solution
In most cases the correction factor is n o t required.
With m a n y extraction m e t h o d s it is difficult to determine h o w quantitative the extraction p r o c e d u r e is.

M o r e intensive extraction does not significantly increase the yield of coenzyme. In some studies rats were
injected with [ Co]-labelled v i t a m i n - B ; 24 hr. after injection the a n i m a l s were killed a n d the livers
60
1 2
removed. Sixty to seventyfive per cent of the labelled coenzyme was extracted. It is not certain whether dif
ferences in the extraction p r o c e d u r e give different forms of v i t a m i n - B . 12
References
1 R. H. Abeles, C. Myers & T. A. Smith, A n a l . Biochem. 15, 192 [1966].

2 H. A. Lee & R. H. Abeles, J. biol. C h e m . 238, 2367 [1963].
3 H. Bohme & O. Z. Winkler, A n a l . C h e m . 142, 1 [1954].
Adenosine-5 -diphosphoglucose
Jack Preiss a n d Elaine Greenberg
Adenosine-5'-diphosphoglucose (ADP-glucose) is found in acid a n d alcoholic extracts of plants a n d

bacteria. Until n o w n o specific m e t h o d for the d e t e r m i n a t i o n of A D P - g l u c o s e existed. A D P - g l u c o s e can
be converted with inorganic p y r o p h o s p h a t e (PPi) and A D P - g l u c o s e p y r o p h o s p h o r y l a s e * to A T P a n d
G - l - P . This specific reaction is the basis for the m e t h o d described here for the d e t e r m i n a t i o n of A D P -
glucose.
Principle
(1) PPi + A D P - g l u c o s e t
A
- g
D p
^ l u c o s e
ATP + a-Glucose-l-P
pyrophosphorylase
(2) a-Glucose-l-P , P h o s
P °g h
°- l u c
A Glucose-6-P
mutase**
I
(3) Glucose-6-P + N A D P +
«"*•»«-«-'', 6-Phosphogluconolactone + N A D P H + H +
dehydro genase * * *
The increase of N A D P H , as measured by the change in extinction at 340 (334, 365) n m , is p r o p o r t i o n a l to

the a m o u n t of A D P - g l u c o s e present.
T h e p H o p t i m a of the enzymes catalysing reactions ( l ) - ( 3 ) are between 7.5 a n d 8.5: therefore the m e a s u r e
ments should be m a d e in this range. A D P - g l u c o s e p y r o p h o s p h o r y l a s e from spinach leaves is activated by 1
3-phosphoglycerate; V m a x is increased a n d K M decreased 1 , 2

. We therefore a d d 3-phosphoglycerate to the
assay system. Inorganic p h o s p h a t e i n h i b i t s ; the assay system should contain less t h a n 1 m M p h o s p h a t e .
1,2
Equipment
334 or 365 n m ; p H meter.
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 4. S o d i u m p y r o p h o s p h a t e ,
2. M a g n e s i u m c h l o r i d e , M g C l * 6 H 0 , A . R
2 2 Na P O 10H O,
4 2 7 2 A.R.
3. Albumin
crystalline from bovine p l a s m a
* A T P : a - D - g l u c o s e - l - p h o s p h a t e adenylyltransferase, EC 2.7.7.27.
** a - D - G l u c o s e - l , 6 - b i s p h o s p h a t e : a-D-glucose-1-phosphate p h o s p h o t r a n s f e r a s e , EC 2.7.5.1.
*** D - G l u c o s e - 6 - p h o s p h a t e : N A D P 1-oxidoreductase, EC 1.1.1.49.
ADP-glucose 2205
5. 3 - P h o s p h o g l y c e r a t e , 3 - P G 8. G l u c o s e - 1 , 6 - d i p h o s p h a t e , G-l,6-P , 2
trisodium salt, 3 - P G - N a ; commercial p r e p

2
cyclohexylammonium salt, G-1,6-P -(CHA) -
2 4
aration, see p . 540. • 4 H 0 ; commercial preparation, see p. 537.

2
6. P h o s p h o g l u c o m u t a s e , P G l u M 9. A D P - g l u c o s e p y r o p h o s p h o r y l a s e ,
from rabbit muscle, crystalline suspension in for isolation, see Appendix, p. 2207.
3.2 M a m m o n i u m sulphate solution: ^200 10. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e
U / m g . (25 ° C ) ; 10 m g . / m l . ; commercial p r e p phosphate, N A D P ,
aration, see p . 499. disodium salt, N A D P - N a H ; commercial prep
2
7. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , aration, see p. 546.

G6P-DH 11. Hydrochloric acid, 1 N , A . R.
from yeast, suspension in 3.2 M a m m o n i u m 12. H y d r o c h l o r i c a c i d , 3 N , A . R .
sulphate solution: ^ 140 U / m g . (25 ° C ) ;
13. S o d i u m h y d r o x i d e , 0.01%, A . R .
10 mg./ml. commercial p r e p a r a t i o n , see p . 458.
Purity of Reagents
The enzymes should be free from inorganic p y r o p h o s p h a t a s e s . Traces can be completely inhibited by
inclusion of 10 m M N a F in the assay system. T h e enzymes must not c o n t a i n any N A D P H oxidase. T h e
m e t h o d described in the A p p e n d i x for the purification of A D P - g l u c o s e p y r o p h o s p h o r y l a s e results in enzyme
preparations which are free from N A D P H oxidases a n d inorganic p y r o p h o s p h a t a s e .
P r e p a r e all s o l u t i o n s w i t h d i s t i l l e d w a t e r w h i c h h a s b e e n further purified b y p a s s a g e o v e r a n

i o n e x c h a n g e resin.
D i s s o l v e 12.1 g. tris i n 6 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 7.5 w i t h 3 N H C 1 a n d d i l u t e
II. 3 - P h o s p h o g l y c e r a t e ( 6 . 2 5 m M 3 - P G ; 3 7 . 5 m M M g C l ) : 2
D i s s o l v e 17.8 m g . 3 - P G a n d 7 6 m g . M g C l 2 • 6 H 0 in 10 m l . d i s t i l l e d w a t e r
2
III. P y r o p h o s p h a t e (0.1 M ; p H 8 . 0 ) :
D i s s o l v e 4 . 4 6 g. N a P 04 2 7 • H 0 in 7 0 m l . d i s t i l l e d w a t e r , a d j u s t t o p H 8.0 w i t h 1 N H C 1
2
I V . G l u c o s e - 1 , 6 - d i p h o s p h a t e (0.1 m M G - l , 6 - P ; 10 m g . a l b u m i n / m l . ) :
2
D i s s o l v e 0.8 m g . G - 1 , 6 - P ( C H A ) - 4 H 0 a n d 100 m g . a l b u m i n in 10 m l . d i s t i l l e d w a t e r .
2 4 2
V. P h o s p h o g l u c o m u t a s e , P G l u M (2 mg. protein/ml.):
Dilute the stock suspension accordingly with distilled water.
VI. A D P - g l u c o s e p y r o p h o s p h o r y l a s e (ca. 6 U / m l . ) :
U s e the e n z y m e preparation prepared according to p. 2207 undiluted.
VII. Nicotinamide-adenine dinucleotide phosphate, N A D P (10 m M ) :
D i s s o l v e 2 5 m g . N A D P - N a H w i t h 2.5 m l . c o l d d i s t i l l e d w a t e r , a d j u s t t o c a . p H 5.5 w i t h
2
0.1 N N a O H ; d i l u t e t o 3 m l . w i t h d i s t i l l e d w a t e r .
VIII. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , G 6 P - D H (2 m g . p r o t e i n / m l . ) :
Dilute the stock suspension accordingly with distilled water.
Solutions I, III, V a n d VIII, well-stoppered, are stable indefinitely at 0 - 4 °C. Solutions II, IV and VII,
well-stoppered, are stable indefinitely at — 12 °C. T h e ADP-glucose p y r o p h o s p h o r y l a s e is stable for a b o u t
1 - 2 m o n t h s at 0 - 4 °C.
Procedure
Nucleoside phosphates can be extracted by m a n y m e t h o d s from p l a n t s 3 - 6

and micro-organ
i s m s . U s u a l l y b o i l i n g a l c o h o l o r c o l d d i l u t e a c i d s are u s e d . F u r t h e r p u r i f i c a t i o n c a n b e carried
7
o u t b y p a p e r c h r o m a t o g r a p h y o r i o n e x c h a n g e c h r o m a t o g r a p h y . F o r t h e e n z y m a t i c a s s a y it
is sufficient t o e x t r a c t t h e s a m p l e , e . g . a l g a e , w i t h b o i l i n g 8 0 % e t h a n o l .
Assay System

a t u r e ; read a g a i n s t b l a n k c o n t a i n i n g d i s t i l l e d w a t e r i n s t e a d o f s a m p l e .
If t h e s a m p l e c o n t a i n s a - g l u c o s e - 1 - p h o s p h a t e a n d / o r g l u c o s e - 6 - p h o s p h a t e , this s h o u l d b e
d e t e r m i n e d o r a l l o w e d t o react first in t h e c u v e t t e : o m i t p y r o p h o s p h o r y l a s e s o l u t i o n ( V I ) ,
f o l l o w t h e e x t i n c t i o n u n t i l t h e r e a c t i o n is c o m p l e t e ; AE = E 2 — E t gives the s u m o f G - l - P
a n d G - 6 - P . T h e n m i x in t h e p y r o p h o s p h o r y l a s e s o l u t i o n ( V I ) ; t h e n e w i n c r e a s e in e x t i n c t i o n
c o r r e s p o n d s t o t h e A D P - g l u c o s e c o n t e n t a n d t h e A E is u s e d for t h e c a l c u l a t i o n s .
Sample 0.72 ml. 10-200 ^M ADP-glucose

Tris buffer (I) 0.04 ml. 4 0 m M tris
3-PG/MgCl 2 solution (ID 0.16 ml. 1 m M 3-PG
6 m M MgCl 2
PPi solution (III) 0.02 ml. 2 m M PPi

G-l,6-P 2 solution (IV) 0.02 ml. 2fiM G-l,6-P 2
2 0 0 jug. a l b u m i n / m l .
P G l u M solution (V) 0.01 m l . 20 ^ g . / m l . = 4 U / m l .
Pyrophosphorylase solution (VI) 0.02 ml. 0.15-0.75 U/ml.
M i x carefully a n d i n c u b a t e f o r 15 m i n .
N A D P solution (VII) 0.10 ml. 1 mM NADP
M i x carefully; follow the extinction change until
constant ( 2 - 5 min.). Read extinction E . x
G 6 P - D H solution (VIII) 0.01 m l . 2 0 / i g . / m l . = 2.8 U / m l .

R e a d t h e e x t i n c t i o n after 1, 2, 6 a n d 10 m i n . D e t e r
m i n e t h e final e x t i n c t i o n E 2 by extrapolation A E =
= E 2 — E x is u s e d for t h e c a l c u l a t i o n s .
Calculations
(2) on p . 312 applies. T h e results are obtained as pinole A D P - g l u c o s e per ml. sample. This value must be
ADP-glucose 2207
multiplied by a factor if the sample has been deproteinized, neutralized or diluted in any way. F o r this
m e t h o d the following relationships h o l d :

c = 0.228 x AE 0.223 x AE 0.403 x AE [/miole/ml.]
In the analysis of A D P - g l u c o s e s t a n d a r d p r e p a r a t i o n s a precision of 3 % was o b t a i n e d .
The presence of N A D P H oxidase, glucose p h o s p h a t e p h o s p h a t a s e (unspecific p h o s p h a t a s e ) a n d inorganic

p y r o p h o s p h a t a s e in the enzymes used causes t o o low values. N A D P H oxidase in the reaction can be
detected by the decrease in extinction in the absence of substrate. I n o r g a n i c p y r o p h o s p h a t a s e can be
detected by the formation of o r t h o p h o s p h a t e from inorganic p y r o p h o s p h a t e . Glucose p h o s p h a t e p h o s
phatase is measured by addition of k n o w n a m o u n t s of G - l - P or G-6-P to the assay system; the theoretical
increase in extinction should be attained, if the p h o s p h a t a s e is absent.
ADP-glucose is the only nucleotide sugar which reacts in this system. T h e following c o m p o u n d s d o n o t
react: UDP-glucose, TDP-glucose, G D P - g l u c o s e , CDP-glucose, G D P - m a n n o s e , A D P - m a n n o s e and
ADP-galactose. A n y glucose-1-phosphate and glucose-6-phosphate in the sample must be determined
before the measurement of ADP-glucose (see p. 1233 and p. 1238). N a t u r a l l y the G - l - P a n d G-6-P values
must be subtracted from the A D P - g l u c o s e values.
Appendix
Purification of ADP-glucose Pyrophosphorylase
7. Step: R e m o v e ribs from fresh spinach leaves, wash leaves and h o m o g e n i z e in a mixer at the highest
speed for 3 min. with the same volume 50 m M tris buffer ( p H 7.5; 2 m M glutathione, 2 m M E D T A ) .
Filter the suspension t h r o u g h a Biichner funnel, centrifuge the filtrate in the cold for 30 min. at 15000 g;
fractionate the s u p e r n a t a n t fluid.
2. Step: To the opalescent s u p e r n a t a n t fluid add sufficient 1 M p h o s p h a t e buffer, p H 7.0, so that the
p h o s p h a t e concentration is 20 m M . H e a t the solution with stirring in 300 ml. p o r t i o n s in a 1000 ml. Erlen-
meyer flask in a water b a t h at 7 2 - 7 5 °C. W h e n the t e m p e r a t u r e reaches 6 2 - 6 4 °C ( 4 - 5 min.) heat for a
further 4 - 5 min. in a water b a t h at 6 5 - 6 7 °C a n d then rapidly cool in ice-water. Centrifuge off the protein
precipitate; the s u p e r n a t a n t fluid contains all the activity. F r a c t i o n a t e with solid a m m o n i u m sulphate.
Dissolve the fraction between 35 and 60% ( N H ) S 0 4 2 4 saturation in 0.1 M tris-succinate buffer ( p H 7.2,
1 m M G S H and 1 m M E D T A ) a n d dialyse overnight against 20 m M tris-succinate buffer ( p H 7.2; 1 m M
G S H and 1 m M E D T A ) .
3. Step: A d s o r b the dialysed fraction on a DEAE-cellulose c o l u m n (5 x 40 c m . ) ; the c o l u m n is equilibrated

with 10 m M p h o s p h a t e buffer, p H 7.4. Wash the c o l u m n with 800 ml. 10 m M p h o s p h a t e buffer, p H 7.4
and elute the enzyme with a linear g r a d i e n t : 4 litre 10 m M p h o s p h a t e ( p H 7.4) in the mixing vessel a n d 4 litre
50 m M p h o s p h a t e ( p H 5.8) + 0.7 M N a C l in the reservoir. Collect 150 ml. fractions a n d m e a s u r e the enzyme
activity and combine the active fractions. Precipitate the enzyme by addition of solid a m m o n i u m sulphate
to 60% saturation, centrifuge, dissolve the precipitate in 0.1 M tris-succinate buffer ( p H 7.2; 1 m M G S H
and 1 m M E D T A ) and dialyse overnight against 3 litre 20 m M tris-succinate ( p H 7.2; 1 m M G S H and 1 m M
G S H and 1 m M EDTA).
T h e enzyme can be purified further, but the purity of the DEAE-cellulose fraction is sufficient for the assay
m e t h o d described here. This enzyme fraction has a specific activity of 4.3 U / m g . protein measured u n d e r
the conditions described a b o v e .
1
References
1 H. P. Ghosh & J. Preiss, J. biol. C h e m . 241, 4491 [1966].

2 J. Preiss, H. P. Ghosh & /. Wittkop in T. Goodwin: Biochemistry of Chloroplasts. Academic Press,
L o n d o n and N e w Y o r k 1967, Vol. 2, p . 131.
3 C. V. Cole & C. Ross, A n a l . Biochem. 77, 526 [1966].
4 R. R. Selvedran & F. A. Isherwood, Biochem. J. 105, 723 [1967].
5 H. Sandermann & H. Grisebach, Biochim. Biophys. Acta 156, 435 [1968].
6 7 . C. Su & W. Z . Hassid, J. biol. C h e m . 235, Pc 36 [1968].
7 E. Cabib & L. F. Leloir,J. biol. C h e m . 206, 779 [1954].
Cytidine-5 -diphosphoglucose
R a r d o n D . Bevill
Cytidine-5'-diphosphoglucose (CDP-glucose) occurs in a n u m b e r of m i c r o - o r g a n i s m s , where it serves as

a direct or indirect precursor or polysaccharides. C D P - g l u c o s e is quantitatively converted to C D P - 4 - k e t o -
6-deoxyglucose by the enzyme C D P - g l u c o s e d e h y d r a t a s e ( C D P glucose 4,6-hydro-lyase, E C 4.2.1.45)
from Salmonella typhimurium. Catalytic a m o u n t s of exogenous N A D are required for this reaction.
Application of Method: In biochemistry a n d microbiology.
Principle
(1) CDP-glucose C D P
~ g l u c
N °^ e
D
d
+
e h y d r a t a s e
) CDP-4-keto-6-deoxyglucose+H 0 2
Treatment of the keto sugar formed with alkali gives an enediol with an extinction m a x i m u m at 318 n m .
T h e increase m extinction at 318 n m is a m e a s u r e of the a m o u n t of C D P - g l u c o s e present. This m e t h o d
was first described by Matsuhashi et a l . . 1
T h e enzymatic reaction should be carried out between p H 8.0 a n d 9.0 to o b t a i n a m a x i m u m rate of reaction.
T h e enzyme is inhibited by high c o n c e n t r a t i o n s of p h o s p h a t e and is sensitive t o heavy m e t a l s ; for this
reason the m e a s u r e m e n t s are carried out in tris buffer with addition of E D T A .
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for a c c u r a t e m e a s u r e m e n t s at 3 1 8 n m ; w a t e r b a t h at 37 ° C .
Reagents
1. T n s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 5. S o d i u m h y d r o x i d e , A . R., 0 . 2 N
2. Ethylenediaminetetra-acetate, EDTA 6. C y t i d i n e - 5 ' - d i p h o s p h o g l u c o s e , C D P -
disodium salt, E D T A - N a H - 2 H 0
2 2 2
glucose
3. H y d r o c h l o r i c a c i d , A . R . , 2 N commercial p r e p a r a t i o n , see p. 529.
4. N i c o t i n a m i d e - a d e n i n e dinucleotide, N A D 7. C D P - g l u c o s e o x i d o r e d u c t a s e ,
free acid; commercial p r e p a r a t i o n , see p . 545. prepared from Salmonella typhimurium
See A p p e n d i x p . 2212.
T h e CDP-glucose d e h y d r a t a s e p r e p a r e d according to p . x x is sufficiently pure. C r u d e a m m o n i u m

sulphate fractions which have n o t been c h r o m a t o g r a p h e d o n D E A E - S e p h a d e x can be used if one takes the
trouble to determine the b l a n k exactly.
I. Tris buffer ( 5 0 m M tris, p H 8 . 6 ; 2 m M EDTA):
D i s s o l v e 6 . 0 6 g. tris a n d 0 . 7 4 4 g. E D T A - N a H -2 H 0 in 2 0 0 m l . d i s t i l l e d w a t e r , a d j u s t
2 2 2
t o p H 8.6 w i t h 2 N H C 1 a n d d i l u t e t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r .
II. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( c a . 2 0 m M ) :
D i s s o l v e 2 0 m g . N A D in 1.40 m l . d i s t i l l e d w a t e r .
III. C y t i d i n e - 5 - d i p h o s p h o g l u c o s e ( 1 2 m M C D P - g l u c o s e ) :
D i s s o l v e 2 5 . 0 m g . C D P - g l u c o s e in 3 m l . distilled w a t e r . ( T h e s o l u t i o n is u s e d t o d e t e r m i n e
the e n z y m e a c t i v i t y ) .
IV. C D P - g l u c o s e d e h y d r a t a s e ( 5 - 8 m g . p r o t e i n / m l . ) :
D i l u t e t h e e n z y m e s o l u t i o n (see p . 2 2 1 2 ) if n e c e s s a r y w i t h tris buffer ( s o l u t i o n I).
The N A D and C D P solutions are stable for at least 2 - 3 m o n t h s at —10 °C to —15 °C. T h e enzyme solution
loses less t h a n 3 % of its activity within 2 m o n t h s at - 1 0 °C to - 1 5 °C.
Procedure
Collection and treatment:
R a p i d l y c o o l g r o w i n g b a c t e r i a , c o l l e c t b y c e n t r i f u g a t i o n at 4 ° C a n d e x t r a c t t h e c o l d c e l l s
with boiling 7 0 % alcohol. T h e n c o o l again and centrifuge. Repeat the extraction and c o m b i n e
the e x t r a c t s . T h e y c o n t a i n t h e s o l u b l e n u c l e o t i d e s a n d n u c l e o t i d e s u g a r s o f t h e c e l l s .
O n s t o r a g e in t h e c o l d ( 4 ° C ) t h e n u c l e o t i d e s u g a r s d o n o t d e c r e a s e s i g n i f i c a n t l y in 2 4 h r . ;
in the f r o z e n state t h e y are s t a b l e for at least o n e w e e k .
CDP-glucose 2211
Assay System
W a v e l e n g t h : 3 1 8 n m ; l i g h t p a t h : 1 c m . ; i n c u b a t i o n v o l u m e : 0.5 m l . ; i n c u b a t i o n t e m p e r a t u r e :
37 ° C ; final v o l u m e : 1.0 m l . ; r o o m t e m p e r a t u r e ; r e a d a g a i n s t b l a n k .
Pipette into cuvettes: Experimental Blank
assay mixture
Tris buffer (I) 0.38 ml. 0.38 ml. 19 m M t r i s ;

0.76 m M EDTA
N A D solution (II) 0.02 ml. 0.02 ml. 0.4 m M NAD
Enzyme solution (IV) 0.05 ml. 0.05 ml. 0.5-0.8 mg.
protein/ml.
(ca. 1 3 5 - 2 2 0 m U / m l . )
I n c u b a t e for 1 m i n . at 37 ° C .
N a O H , 0.2 N 0.50 ml.

M i x a n d i n c u b a t e for 3 0 m i n . at 37 ° C .
N a O H , 0.2 N 0.50 ml. — 0.1 N
M i x a n d i n c u b a t e for 15 m i n . at 37 ° C . C o o l t o r o o m t e m p e r a t u r e ,
read extinction of experimental cuvette against blank (Ê).
T h i s v a l u e is u s e d for t h e c a l c u l a t i o n s .
Calculations
(1) on p . 312 applies. T h e m o l a r extinction coefficient for CDP-4-keto-6-deoxyglucose in 0.1 N N a O H
is 6.5. c m . / ^ m o l e . T h e following relationships therefore h o l d :
2
AE
Cuvette c o n t e n t s : c = - — [^mole/ml.]
6.5
Sample c = AE x 3.08 [^mole/ml.]
W i t h a m e a n value of 0.10 umole a s t a n d a r d deviation of 0.004 /rniole was found. T h e coefficient of

variation is 4 . 3 % .
T h e main source of error is failure to take into account the appreciable a b s o r p t i o n at 318 n m d u e to the
enzyme protein. Exactly the same a m o u n t of enzyme solution must be pipetted into the blank and ex
perimental cuvettes.
CDP-glucose dehydratase is specific for CDP-glucose a n d deoxy-CDP-glucose. T h e presence of other

sugars, sugar phosphates and nucleoside d i p h o s p h o - s u g a r s does not affect the assay.
Appendix
CDP-glucose Dehydratase
G r o w Salmonella typhimurium, strain G-30 on proteose-peptone-beef extract m e d i u m until the middle of

the log-phase. Wash the cells; they can be stored in the frozen state for u p to 6 m o n t h s without loss of
activity. Carry out all operations at 0 - 4 °C. H o m o g e n i z e 64 g. of G-30 frozen paste in 320 ml. tris buffer
(solution I) for 1 min. in a mixer. P o u r 25 ml. p o r t i o n s into vessels for sonication. Sonicate the contents
of each vessel 3 times for 10 sec. with full p o w e r of a Branson sonifier then centrifuge for 20 min. at 30000 g.
C o m b i n e the s u p e r n a t a n t fluids to give the " c r u d e e x t r a c t " : volume 270 m l . ; 30 mg. protein/ml.
Dilute the crude extract with tris buffer (I) to give a protein concentration of 11.0 mg./ml. (738 ml.). A d d
221 ml. 0.5% p r o t a m i n e sulphate in buffer I with stirring a n d continue stirring the milky suspension for a
further 10 min. Centrifuge suspension for 20 min. at 30000 g a n d discard the precipitate. To the s u p e r n a t a n t
fluid (950 ml.) add 192 ml. p o t a s s i u m p h o s p h a t e buffer (1 M . p H 7.5), then bring to 32% saturation with
202.4 g. solid a m m o n i u m sulphate. After stirring for 20 min. centrifuge for 20 min. at 30000 g and discard
supernatant fluid. To the s u p e r n a t a n t fluid a d d a further 148 g. solid a m m o n i u m sulphate (to 54% saturat
ion), stir the suspension for 15 min., then centrifuge for 20 min. at 30000 g. Dissolve precipitate in 10 m M
tris buffer, p H 7.5 (95 ml.). A d d 9.5 ml. p o t a s s i u m p h o s p h a t e buffer (1 M , p H 5.5) followed by 14.1 g.
solid a m m o n i u m sulphate (25% saturation). Stir solution for 10 min., then centrifuge 20 min. at 30000 g
and discard precipitate. To the s u p e r n a t a n t fluid a d d a further 8.8 g. solid a m m o n i u m sulphate (to 40%
saturation) and stir the mixture for 15 min. Collect the precipitate by centrifugation at 30000 g and dissolve
in 10 m M tris buffer, p H 7.5 (17.4 ml.). Dialyse the a m m o n i u m sulphate fraction overnight against 2 1.
potassium p h o s p h a t e buffer (25 m M , p H 6.5; 0.2 M KC1) and then transfer to a D E A E - S e p h a d e x - A - 5 0
column (1 cm. x 11 cm.). Equilibrate the c o l u m n before use with the last m e n t i o n e d buffer until the ex
tinction at 280 n m falls below 0.080. Elute the enzyme with a KC1 gradient, which increases from 0.2 M
to 0.4 M (400 ml. total volume). C o m b i n e active fractions, which are eluted with ca. 0 . 2 6 - 0 . 3 0 M KC1 a n d
dialyse for 24 hr. against saturated a m m o n i u m sulphate solution. Collect the precipitate by centrifugation
for 45 min. at 30000 g and dissolve in 50 m M tris buffer, p H 8.0. This p r e p a r a t i o n has a specific activity
of 270 m U / m g . a n d represents a 200-fold purification over the c r u d e extract.
T h e enzyme can also be purified from a sonicate of Pasteurella pseudotuberculosis according to the proce
dure of Matsuhashi et a l . .
1
References
1 S. Matsuhashi, M. Matsuhashi, J. G. Brown & J. L. Strominger, J. biol. C h e m . 241, 4283 [1966].

Guanosine-5-diphosphomannose
Jack Preiss
G u a n o s i n e - 5 ' - d i p h o s p h o m a n n o s e ( G D P - m a n n o s e ) occurs in m a m m a l i a n cells, bacteria a n d plants.

Until n o w there was n o m e t h o d for the direct d e t e r m i n a t i o n of this c o m p o u n d . G D P - m a n n o s e has been
determined by isolation a n d s p e c t r o p h o t o m e t r i c analysis of the guanosine moiety. G D P - m a n n o s e is
oxidized to G D P - m a n n u r o n i c acid by G D P - m a n n o s e d e h y d r o g e n a s e * a n d N A D . This enzyme catalysed
1
reaction is specific and can be used for the direct determination of G D P - m a n n o s e in tissue extracts.
Principle
(1) GDP-mannose + 2NAD " + H 0 4
2
G D
h
p
: m a n n o s e
> G D P - m a n n u r o n i c acid + 2 N A D H + 2H +
v 7 1
dehydrogenase '
The increase of N A D H concentration, as measured by the change of extinction at 334, 340 or 365 n m , is
an indication of the G D P - m a n n o s e content. Two moles of N A D are reduced per mole of G D P - m a n n o s e .
Reaction (1) is irreversible a n d therefore the conversion of G D P - m a n n o s e is quantitative. T h e p H o p t i m u m

of the reaction lies between 7.9 a n d 8.3.
Equipment
334 or 365 n m ; p H - m e t e r .
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 4. N i c o t i n a m i d e - a d e n i n e dinucleotide, N A D
K H P 0 , A.R.
2 4 free acid; commercial p r e p a r a t i o n , see p . 545.
2. D i p o t a s s i u m h y d r o g e n p h o s p h a t e , 5. G D P - m a n n o s e d e h y d r o g e n a s e ,
K H P 0 , A.R.
2 4 from Arthrobacter viscosus ' , 2 3
ca. 0.4 U / m g . ;
3. A l b u m i n isolation, see A p p e n d i x , p . 2216.
crystalline from bovine p l a s m a
Purity of Reagents
T h e m e t h o d of isolation described on p . 2216 guarantees an enzyme p r e p a r a t i o n which is free from N A D H

oxidase and G D P - m a n n o s e p y r o p h o s p h a t a s e .
* G D P m a n n o s e : N A D 6-oxidoreductase, E C 1.1.1.132
U s e o n l y d i s t i l l e d w a t e r , w h i c h h a s b e e n finally d e i o n i z e d w i t h i o n e x c h a n g e r e s i n s .
D i s s o l v e 1 7 . 4 g. K H P 0
2 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l . ; d i s s o l v e 1 3 . 6 g.
KH P0 2 4 in d i s t i l l e d w a t e r a n d m a k e u p t o 1 0 0 m l . ; a d d sufficient o f K H P 0 2 4 solution
to the K H P 0 2 4 s o l u t i o n t o g i v e p H 7.9.
II. A l b u m i n ( 1 0 m g / m l . ) :
D i s s o l v e 100 m g . a l b u m i n in 10 m l . d i s t i l l e d w a t e r .
III. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e ( 1 0 m M / ? - N A D ) :
D i s s o l v e 21 m g . N A D in cold d i s t i l l e d w a t e r , a d j u s t p H t o 5 - 6 . 5 ( i n d i c a t o r p a p e r ) a n d
dilute t o 3 ml. with distilled water.
IV. G D P - m a n n o s e d e h y d r o g e n a s e (ca. 1 m g . / m l . ) :
Dilute the e n z y m e solution obtained according to p. 2216 with 1 % a l b u m i n solution.
The buffer solution (I) is stable indefinitely in the cold. Solutions I I - I V are stable for at least two months
in the frozen state.
Procedure
Nucleoside phosphate sugars can be extracted by m a n y m e t h o d s from p l a n t s 4 - 7

, micro
organisms 8
a n d a n i m a l t i s s u e s " . U s u a l l y b o i l i n g a l c o h o l o r c o l d d i l u t e a c i d s are u s e d .
9 1 1
Further purification c a n b e carried out b y i o n e x c h a n g e or paper c h r o m a t o g r a p h y . F o r e n z y m

atic a s s a y s it is u s u a l l y sufficient t o e x t r a c t t h e s a m p l e ( e . g . a l g a e ) w i t h b o i l i n g 8 0 % e t h a n o l .
GDP-mannose 2215
Assay System

ature; read against a cuvette containing water instead of sample.
Sample 0.83 ml. 5 - 5 0 fiM GDP-mannose

P h o s p h a t e buffer (I) 0.05 ml. 50 m M
Albumin solution (II) 0.01 m l . 100 fig./ml.
N A D solution (III) 0.10 ml. 1 mM
M i x a n d follow extinction until constant ( 2 - 5 min.).

Read extinction E.
t
GDP-mannose dehydrogenase
(IV) 0.04 ml. 20-40 mU/ml.
M i x a n d r e a d e x t i n c t i o n after 15, 2 0 a n d 2 5 m i n . D e
termine extinction E . A E = E 2 2 — E t is u s e d for t h e
calculations.
Calculations
(2) on p . 312 applies. It should be n o t e d t h a t 2 m o l e of N A D are reduced per m o l e of G D P - m a n n o s e . T h e
results are obtained as /rniole G D P - m a n n o s e / m l . This value must be multiplied by a factor if the sample
has been deproteinized, neutralized or diluted in any way. F o r this m e t h o d the following relationships hold.

A precision of 3 % was found in the assay of G D P - m a n n o s e s t a n d a r d p r e p a r a t i o n s .
T h e enzyme p r e p a r a t i o n m u s t not contain any N A D H oxidase or G D P - m a n n o s e p y r o p h o s p h a t a s e .

These two c o n t a m i n a n t s cause low results. T h e presence of N A D H oxidase is tested for by addition of
N A D H to the assay system; the extinction should not decrease. P y r o p h o s p h a t a s e is detected as follows:
add G D P - m a n n o s e to the assay system a n d observe whether there is any increase in extinction.
The enzyme p r e p a r a t i o n m u s t give a clear solution, because turbidity can interfere with the determination.
G D P - m a n n o s e a n d d e o x y - G D P - m a n n o s e are the only c o m p o u n d s which react with the dehydrogenase.

T h e following c o m p o u n d s are inactive: a - m a n n o s e - 1 - p h o s p h a t e ; C D P - m a n n o s e , T D P - m a n n o s e , I D P -
mannose, ADP-mannose, U D P - g l u c o s e , T D P - g l u c o s e , CDP-glucose, GDP-glucose, ADP-glucose,
UDP-galactose a n d T D P - g a l a c t o s e . N A D H c a n n o t replace N A D as the coenzyme. T h e Michaelis constants
of the enzyme for G D P - m a n n o s e a n d d e o x y - G D P - m a n n o s e are 0.017 m M a n d 0.084 m M respectively.
Appendix
Isolation of GDP-mannose Dehydrogenase
G r o w t h of Arthrobacter viscosus*: C u l t u r e m e d i u m : 1% glucose, 0.5% Difco b a c t o p e p t o n e , 0 . 3 % Difco

yeast extract a n d 0 . 3 % Difco malt extract. Inoculate 62 ml. culture m e d i u m with a l o o p of bacterial culture
from a malt-yeast agar slope. After 2 4 - 4 8 hr. at 25 °C transfer the whole culture to 1.5 1. culture m e d i u m ,
which contains 3 % glucose, 0 . 3 % casein hydrolysate (Nutritional Biochemical C o r p o r a t i o n ) , 0.4% K H P 0 , 2 4
0.08% M g S 0 - 7 H 0 , and 0.005% M n S 0

4 2 4 (adjusted to p H 7.0 with H S 0 ) . I n c u b a t e for 1 6 - 2 4 hr. at
2 4
25 °C and harvest with a Sharpies centrifuge. Wash the cells with cold 0.9% N a C l solution (15 ml./g. cells)
and store as a paste at - 1 5 °C.
Purification method: T h a w 15 g. of frozen cells and suspend in 45 ml. 50 m M tris buffer ( p H 7.5; containing
5 m M G S H and 10 m M M g C l ) . Disintegrate cells in a French press at 20000 p.s.i., centrifuge off the cell
2
debris at 30000 g for 10 min. a n d discard the sediment. Centrifuge for 1 hr. at 105000 g and use the super
n a t a n t fluid for further fractionation.
Slowly add with c o n t i n u o u s stirring 34 ml. 1% p r o t a m i n e sulphate solution to the s u p e r n a t a n t fluid, after
10 min. centrifuge the suspension for 10 min. at 26000 g and discard the s u p e r n a t a n t fluid. Extract the pre
cipitate twice with 43 ml. 0.3 M p o t a s s i u m p h o s p h a t e buffer ( p H 7.0; 10 m M glutathione) each time.
C o m b i n e the extracts (80 ml.) a n d a d d 84 g. solid a m m o n i u m sulphate. T h e d e h y d r o g e n a s e precipitates.
Centrifuge the suspension for 10 min. at 26000 g a n d dissolve the precipitate in 20 m M p o t a s s i u m p h o s p h a t e
buffer ( p H 7.0; 10 m M G S H ) . Dialyse this solution overnight against 500 ml. p h o s p h a t e buffer/GSH. T h e
purification is summarized in the following T a b l e : 2
Volume Protein Activity

ml. mg./ml. U/ml. U/mg.
C r u d e extract 43 11.8 0.23 0.02

P r o t a m i n e sulphate 11 1.2 1.47 0.39
References
1 / . Preiss, J. biol. C h e m . 239, 3127 [1964].

2 / . Preiss in E. F. Neufeld & V. Ginsburg: M e t h o d s in Enzymology, A c a d e m i c Press, N e w Y o r k , Vol.
VIII, p . 271 [1966].
3 J. Preiss in E. F. Neufeld & V. Ginsburg: M e t h o d s in Enzymology, A c a d e m i c Press, N e w Y o r k , Vol.
VIII, p . 285 [19661.
4 C. V. Cole & C Ross, A n a l . Biochem, 17, 526 [1966].
5 R. R. Selvendran & F. A. Isherwood, Biochem. J. 105, 723 [1967].
6 H. Sandermann & H. Grisebach, Biochim. Biophys. Acta 156, 435 [1968].
7 J. C Su & W. Z . Hassid, J. biol. C h e m . 235, Pc36 [I960].
8 E. Cabib & L. F. Leloir, J. biol. C h e m . 206, 779 [1954].
9 O. Gabriel &G. Ashwell, J. biol. C h e m . 237, 1400 [1962].
10 K. P. Wong & T. L. Sourkes, Anal. Biochem. 21, 444 [1967].
11 A. Kobata, J. Biochem. 53, 167 [1963].
* N R R L B 1973; the culture can be obtained from D r . R. F . Andersen, N o r t h e r n Regional Research

Laboratories, U. S. Dept. of Agriculture, Peoria, Illinois, U S A .
Deoxythymidine-5 -diphosphoglucose
R a r d o n D . Bevill
Deoxythymidine-5'-diphosphoglucose ( d T D P G ) is widely distributed in m i c r o - o r g a n i s m s a n d plants.

This c o m p o u n d is a precursor of d T D P - L - r h a m n o s e a n d d T D P - g a l a c t o s e , which act as glycoside d o n o r s
in the biosynthesis of polysaccharides. d T D P G is quantitatively converted to dTDP-4-keto-6-deoxyglucose
by an enzyme d T D P G dehydrase ( d T D P glucose 4,6-hydro-lyase, E C 4.2.1.46) from E. coli B. This m e t h o d
was first described by Gilbert et a l . ' . 1 2
Application of Method: In biochemistry a n d microbiology.
Principle
(1) dTDPG d ^f s e > dTDP-4-keto-6-deoxyglucose + H 02
In contrast to the enzyme which reacts with C D P - g l u c o s e this enzyme does n o t require exogenous N A D ,
because it contains p r o t e i n - b o u n d N A D . Treatment of the k e t o sugar with alkali gives an enediol with a
m a x i m u m extinction at 318 n m . T h e increase in extinction at 318 n m is therefore m e a s u r e d .
The p H of the reaction mixture should be between p H 8.0 a n d 9.0 to achieve m a x i m u m rates. T h e enzyme
is inhibited by high c o n c e n t r a t i o n s of p h o s p h a t e (0.2 M ) a n d is sensitive to heavy m e t a l s ; the assay is
therefore carried out in tris buffer containing E D T A .
Equipment
S p e c t r o p h o t o m e t e r s u i t a b l e for p r e c i s e m e a s u r e m e n t s at 3 1 8 n m ; 37 ° C w a t e r b a t h .
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 5. D e o x y t h y m i d i n e d i p h o s p h a t e g l u c o s e ,
2. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA dTDP-glucose
disodium salt, E D T A - N a H - 2 H 0 2 2 2
3 . H y d r o c h l o r i c a c i d , 2 N , A . R. 6. d T D P - g l u c o s e d e h y d r a s e
4 . S o d i u m h y d r o x i d e , 0 . 2 N , A . R. from E. coli B, for p r e p a r a t i o n , see A p p e n d i x ,
p . 2220.
d T D P dehydrase p r e p a r e d according to the m e t h o d of Gilbert et a l . is sufficiently p u r e at the stage of

1
hydroxyapatite c h r o m a t o g r a p h y for accurate m e a s u r e m e n t s of d T D P - g l u c o s e .

I. Tris buffer ( 5 0 m M tris, p H 8 . 6 ; 2 m M E D T A ) :

D i s s o l v e 6.06 g. tris in 2 0 0 m l . d i s t i l l e d w a t e r , a d d 0 . 7 4 4 g. E D T A - N a H 2 2 • 2 H 0 , adjust t o
2
p H 8.6 w i t h 2 N HC1 a n d d i l u t e t o 1 0 0 0 m l . w i t h distilled w a t e r .

II. dTDP-glucose(12mM):
D i s s o l v e 16.2 m g . d T D P - g l u c o s e in 2 m l . distilled w a t e r .
III. d T D P - g l u c o s e d e h y d r a s e (ca. 2 0 m g . p r o t e i n / m l . ) :
U s e the a m m o n i u m s u l p h a t e f r a c t i o n II o f t h e p r e p a r a t i v e p r o c e d u r e (see A p p e n d i x )
u n d i l u t e d . It c o n t a i n s a b o u t 6.7 U / m l . (38 ° C ) ; t a k e 0 . 0 5 - 0 . 0 8 U ( a b o u t 0.01 m l . ) for t h e
assay.
Stability of Solutiosn
The dTDP-glucose solution (II) is stable for at least 2 - 3 m o n t h s at —10° to — 15 °C. The dTDP-glucose
dehydrase (III) is stable for at least 5 weeks at —10° to — 15 °C. Freezing of the solutions in small portions
is recommended.
Procedure
T h e s a m p l e s s h o u l d b e in a q u e o u s s o l u t i o n , p H 7 t o 8. If c u l t u r e s o f m i c r o - o r g a n i s m s are b e i n g
s t u d i e d , c o o l rapidly, c e n t r i f u g e at 4 ° C a n d e x t r a c t the cell p a s t e w i t h b o i l i n g 70 % e t h a n o l . C o o l
a g a i n , c e n t r i f u g e a n d r e p e a t t h e e x t r a c t i o n . C o m b i n e t h e e x t r a c t s ; t h e y c o n t a i n the s o l u b l e
n u c l e o t i d e a n d n u c l e o t i d e s u g a r s o f t h e cells. C o n c e n t r a t e t h e a l c o h o l i c s o l u t i o n s t o a s m a l l
v o l u m e a n d t h e n d i l u t e w i t h w a t e r . If n e c e s s a r y , c e n t r i f u g e t o clarify. In t h e c a s e o f p l a n t
m a t e r i a l h o m o g e n i z e t h e s a m p l e in i c e - c o l d 2 % p e r c h l o r i c a c i d , c e n t r i f u g e t o r e m o v e t h e cell
d e b r i s a n d t h e n adjust t o p H 7 w i t h c o l d 2 N K O H . A l l o w t h e m i x t u r e t o s t a n d for 1 hr. at
0 ° C , r e m o v e t h e K C 1 0 b y c e n t r i f u g a t i o n a n d u s e s u p e r n a t a n t fluid for a n a l y s i s .
4
GDP-mannose 2219
Assay System
I n c u b a t i o n t e m p e r a t u r e : 37 ° C ; i n c u b a t i o n v o l u m e : 0.5 m l . R e a d at r o o m t e m p e r a t u r e a g a i n s t
b l a n k ; w a v e l e n g t h : 318 n m ; light p a t h : 1 c m . ; final v o l u m e : 1.0 m l .
Pipette into experimental C o n c e n t r a t i o n in

Experimental Blank
and blank cuvettes: assay mixture
Tris buffer (I) 0.44 ml. 0.44 ml. 4 4 m M tris 1.76 m M

EDTA
d T D P G dehydrase solution (III) 0.01 m l . 0.01 m l . 0.4 m g . p r o t e i n / m l .
ca. 0 . 1 - 0 . 1 6 U / m l .
0.2 N N a O H 0.50 ml. 0.1 N

Sample solution 0.05 ml. 0.05 ml.
0.2 N N a O H 0.50 ml. — 0.1 N
I n c u b a t e for 15 m i n . at 37 ° C . R e a d e x t i n c t i o n a g a i n s t b l a n k .
Calculations
(2) on p . 312 applies. T h e extinction coefficient 1
for dTDP-4-keto-6-deoxyglucose in 0.1 N N a O H is
e
3i8 = 4.8 c m . / / m i o l e . Hence it follows that for:
2
Cuvette c o n t e n t s : c = AE x 0.208 [/miole/ml.]

Sample: c = AE x 4.16 [/miole/ml.]
With a m e a n value of 0.10 //mole d T D P - g l u c o s e in the sample a s t a n d a r d deviation of 0.003 ^ m o l e was

obtained. T h e coefficient of variation is 4.1 %.
The main source of error lies in the failure to correct for the extinction of the enzyme protein at 318 n m .
Exactly the same a m o u n t of enzyme must be a d d e d to the experimental a n d blank cuvettes.
The enzyme is specific for d T D P - g l u c o s e . Other sugars, sugar p h o s p h a t e s or nucleoside d i p h o s p h a t e

sugars in the sample do not interfere with the assay.
Appendix
Isolation of dTDP-glucose Dehydrase 1,3
Carry out all steps at ca. 3 °C.

Suspend E. coli cells in 25 m M tris buffer ( p H 7.6) (ca. 10 ml. buffer/g. fresh wt.) and sonicate with a water-
cooled R a y t h e o n 10 kc ultrasonic generator for 20 min. Centrifuge the suspension at 30000 g for 30 min.
and discard the precipitate.
Prepare a 0.5% p r o t a m i n e sulphate solution and adjust to p H 7.5 with 5 N K O H . To 319 ml. crude extract
(14.2 mg. protein/ml.) a d d 260 ml. p r o t a m i n e sulphate solution. Centrifuge at 5 000 g, discard the precipitate
and add a further 36 ml. p r o t a m i n e sulphate solution to the s u p e r n a t a n t fluid. Centrifuge again, collect the
precipitate and extract with 70 ml. p o t a s s i u m p h o s p h a t e buffer (50 m M , p H 7.6) for 30 min. using a magnetic
stirrer. Centrifuge and discard the precipitate.
To 67 ml. of the enzyme extract a d d an equal volume of potassium p h o s p h a t e buffer (50 m M , p H 7.6).
A d d 37.1 g. solid a m m o n i u m sulphate (45% saturation) over 20 min. with c o n t i n u o u s stirring. Centrifuge
and discard the precipitate. A d d a further 18.0 g. solid a m m o n i u m sulphate as described above (65%
saturation). Centrifuge a n d take u p the precipitate in 8 ml. 25 m M tris buffer ( p H 7.6).
Dilute 1.5 ml. of the enzyme solution with 4.6 ml. distilled water and then stir in 6.45 ml. calcium p h o p h a t e
gel suspension (20 mg./ml.). Allow to stand for 10 min. with occasional stirring, centrifuge and discard
the precipitate.
To 11 ml. enzyme solution add 0.55 ml. potassium p h o s p h a t e buffer (50 m M , p H 7.6). Stir in 2.90 g. solid
a m m o n i u m sulphate (45% saturation), centrifuge, discard the precipitate. Stir in a further 1.17 g. solid
a m m o n i u m sulphate (60% saturation). Centrifuge a n d dissolve the precipitate in 0.45 ml. tris buffer
(25 m M , p H 7.6).
The purification at this stage is 38-fold a n d the yield is a b o u t 37% (taking into account the fact that the
calcium p h o s p h a t e gel treatment was only carried out with a p o r t i o n of the total material. T h e final solution
contains a b o u t 20 mg. p r o t e i n / m l . ; the specific activity is ca. 0.3 U / m g . (38 °C).
References
1 / . M. Gilbert, M. Matsuhashi & J. L. Strominger, J. biol. C h e m . 240, 1305 [1965].

2 M. Matsuhashi, J. M. Gilbert, S. Matsuhashi, J. G. Brown, & J. L. Strominger, Biochem. Biophys. Res.
C o m m . 15, 55 [1964].
3 S. Wang & O. Gabriel, J. biol. C h e m . 244, 3430 [1969].
Uridine-5 -diphosphogalactose
Dietrich Keppler and Karl Decker
UDP-Galactose can be formed in various micro-organisms and in animal tissues, especially liver, by
epimerization from UDP-glucose or -by uridylyl transfer to galactose-1-phosphate. UDP-galactose is
involved in the synthesis of lactose, glycolipids and glycoproteins.
A sensitive and specific determination of UDP-galactose is obtained by conversion to U D P - g l u c o s e . 1
Uridylyl transferase, U T ( U D P - g l u c o s e : a-D-galactose-1-phosphate uridylyltransferase, EC 2.7.7.12),

in the presence of glucose-1-phosphate, catalyses the formation of galactose-1-phosphate and U D P -
glucose from UDP-galactose. UDP-glucose is irreversibly oxidized to UDP-glucuronate with N A D and
UDP-glucose dehydrogenase, U D P G - D H (UDP-glucose: N A D 6-oxidoreductase, EC 1.1.1.22).
Application of Method: In biochemistry and in clinical chemistry.
Principle
(1) UDP-Galactose + Glucose-1 -P t

U T
- UDP-glucose + Galactose-l-P
. I
I
UDP-Glucose + H 0 + 2 N A D 2
+ U P P G
~ D H
> UDP-Glucuronate + 2 N A D H + 2H +
The increase in extinction due to reduction of N A D , as measured at 340 (334, 365) nm, is proportional
to the amount of UDP-glucose or UDP-galactose present.
The pH optimum of the combined reaction is pH 8.7. UDP-glucose dehydrogenase and uridylyltrans
ferase do not require divalent cations and are active in the presence of E D T A . U T is activated 30 to 50%
by mercaptoethanol and other SH-reagents . 2
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r for m e a s u r e m e n t s at 3 3 4 , 3 4 0 o r 3 6 5 n m .
Reagents
1. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , 8. M e r c a p t o e t h a n o l , A . R., s p . gr. 0 . 8 4
s p . gr. 1.67 9. U D P - G l u c o s e dehydrogenase, UDPG-
2. P o t a s s i u m h y d r o g e n c a r b o n a t e , A . R. DH
3. P o t a s s i u m h y d r o x i d e , A . R., 1 N from beef liver, for analytical purposes, 5 mg./
4. Glycine ml., ^ 0 . 6 U/mg. (25 °C); commercial prepara
5. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA tion, see p . 519.
disodium salt, A. R., E D T A - N a H - 2 H 0 2 2 2 10. U r i d y l y l t r a n s f e r a s e , UT
6. N i c o t i n a m i d e - a d e n i n e dinucleotide, from calf liver, for analytical purposes, lyo
NAD philized, ca. 1 U / m g . (25 °C); commercial
free acid, commercial preparation, see p. 545. preparation, see p. 521.
7. G l u c o s e - 1 - p h o s p h a t e , G-l-P
crystalline dipotassium salt, G-1-P-K -2H 0, 2 2
commercial preparation, see p. 537.

Purity of Reagents
The enzymes must be free from N A D H - d e p e n d e n t dehydrogenases.
Prepare all s o l u t i o n s w i t h fresh d i s t i l l e d w a t e r .

I. P e r c h l o r i c a c i d ( 0 . 6 M ) :
D i l u t e 5.2 m l . 7 0 % H C 1 0 4 t o 100 m l . w i t h d i s t i l l e d w a t e r .
II. P o t a s s i u m h y d r o g e n c a r b o n a t e (2 M ) :
D i s s o l v e 2 0 g. K H C 0 3 in 100 m l . d i s t i l l e d w a t e r .
III. G l y c i n e buffer ( 0 . 5 M ; p H 8 . 7 ) :
D i s s o l v e 7.51 g. g l y c i n e in ca. 180 m l . d i s t i l l e d w a t e r a n d a d d 7.2 m l . 1 N K O H . C h e c k
the p H w i t h a g l a s s e l e c t r o d e a n d d i l u t e t o 2 0 0 m l . w i t h d i s t i l l e d w a t e r .
IV. G l y c i n e (0.1 M ; p H 7 . 9 ) :
D i s s o l v e 0.75 g. g l y c i n e in ca. 9 0 m l . d i s t i l l e d w a t e r , adjust t o p H 7.9 w i t h ca. 0.19 m l .
1 N K O H ( g l a s s e l e c t r o d e ) a n d d i l u t e t o 100 m l . w i t h d i s t i l l e d w a t e r .
V. G l y c i n e / N A D / E D T A / G - 1 - P (0.5 M glycine; 3 m M N A D ; 8 m M E D T A ; 2 m M G - l - P ) :
D i s s o l v e 2 2 m g . N A D , 30 m g . E D T A - N a H • 2 H 0 a n d 7.5 m g . G - 1 - P - K - 2 H 0
2 2 2 2 2 in
10 m l . g l y c i n e buffer (III).
V I . U D P - g l u c o s e d e h y d r o g e n a s e , U D P G - D H (5 m g . p r o t e i n / m l . ) :
V I I . U r i d y l y l t r a n s f e r a s e , U T (5 m g . p r o t e i n / m l . ) :
D i s s o l v e l y o p h i l i z a t e c o n t a i n i n g 5 m g . p r o t e i n in 1 m l . 0.1 M g l y c i n e s o l u t i o n ( I V ) a n d
a d d 5 p\. m e r c a p t o e t h a n o l .
Store all solutions, stoppered, at 0 - 4 °C. Solution V is stable for ca. 1 week. T h e U D P G - D H suspension
is active for several m o n t h s at 4 °C or it can be stored deep-frozen. T h e U T solution (VII) is active for
ca. 1 week.
Procedure
T i s s u e s a m p l e s s h o u l d b e o b t a i n e d b y t h e f r o z e n - s t o p m e t h o d (see p. 4 0 0 ) .
Deproteinization :
A d d ca. 5 v o l u m e s b y w e i g h t o f i c e - c o l d p e r c h l o r i c a c i d ( s o l u t i o n I) t o a p i e c e o f f r o z e n t i s s u e
( 0 . 3 - 1 . 5 g.) a n d i m m e d i a t e l y h o m o g e n i z e . A l l o w t h e h o m o g e n a t e t o s t a n d for c a . 15 m i n . a n d
t h e n c e n t r i f u g e for 15 m i n . at c a . 2 0 0 0 0 g (0 ° C ) . D e c a n t t h e s u p e r n a t a n t fluid ( 1 . 5 m l . ) a n d
immediately bring to a b o u t p H 8 with solid K H C 0 3 o r 0.5 m l . s o l u t i o n II. A f t e r ca. 3 0 m i n .
c e n t r i f u g e off (at 0 - 4 ° C ) t h e p o t a s s i u m p e r c h l o r a t e . S e p a r a t e t h e s u p e r n a t a n t fluid c a r e f u l l y
f r o m t h e p r e c i p i t a t e o f p o t a s s i u m p e r c h l o r a t e a n d u s e for t h e a s s a y .
UDP-galactose 2223
U D P - G a l a c t o s e is a l k a l i a n d a c i d - l a b i l e , t h e r e f o r e it is i m p o r t a n t t o carry o u t the p e r c h l o r i c
a c i d d e p r o t e i n i z a t i o n at a b o u t 0 ° C a n d t o r a p i d l y n e u t r a l i z e .
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 0 . 9 4 m l . ( s e m i - m i c r o
assay mixture
Sample (deproteinized, neutralized) 0.40 ml. u p t o ca. 150 pM UDP-galactose

G l y c i n e / N A D / E D T A / G - 1 -P
solution (V) 0.50 ml. 0.27 M glycine
1.6 m M NAD
4.3 m M EDTA
1.1 m M G-l-P
U D P G - D H suspension (VI) 0.02 ml. 109 /xg./ml. = 6 5 m U / m l .
M i x a n d a l l o w a n y U D P - g l u c o s e p r e s e n t in t h e s a m p l e
to react (ca. 10 m i n . ) . R e a d e x t i n c t i o n E.
1
U T solution (VII) 0.02 ml. 106 ^ g . / m l . = 106 m U / m l .
Mix, follow the increase in e x t i n c t i o n and when

constant ( 1 0 - 1 5 min.) read extinction E . 2
E 2 - E j = A E is u s e d for t h e c a l c u l a t i o n s .
D e t e r m i n e the i n c r e a s e in e x t i n c t i o n d u e t o a d d i t i o n o f U T s o l u t i o n ( V I I ) a l o n e b y a further
a d d i t i o n at t h e e n d o f t h e r e a c t i o n a n d c o r r e c t A E a c c o r d i n g l y .
Calculations
T h e concentration of U D P - g a l a c t o s e in the sample (^mole/ml.) m u s t b e multiplied by t h e a p p r o p r i a t e

dilution factor for deproteinization and neutralization to obtain ^ m o l e / g . tissue. As 2 mole N A D are
reduced per mole U D P - g a l a c t o s e or U D P - g l u c o s e the following relationships hold for the concentration
in the neutral tissue extract:

Replicate determinations (5) on U D P - g a l a c t o s e - c o n t a i n i n g solutions gave a coefficient of variation of

2.3%.
N o r m a l Values
In rat liver the U D P - g a l a c t o s e c o n t e n t is 0.09 + 0.01 /miole/g. fresh w t ; the ratio of U D P - g l u c o s e :

1
U D P - g a l a c t o s e was 3.4 ( ± 0 . 3 ) : 1. In h u m a n liver a value of 0.62 /imole/g. for the sum of U D P - g l u c o s e

a n d UDP-galactose determined c h r o m a t o g r a p h i c a l l y has been f o u n d ; this c o r r e s p o n d s to a U D P -
3
galactose content of ca. 0.14 //mole/g. T h e content in rat brain is 0.014 + 0.003 /imdle/g. and in rat kidney
is 0.049 ± 0.003 /imole/g . 1
If after neutralization the precipitate of p o t a s s i u m perchlorate is n o t carefully removed from the sample,
a significant inhibition of the uridylyltransferase reaction is observed. If the enzymes used contain U D P -
glucose p y r o p h o s p h o r y l a s e ( U T P : a-D-glucose-1-phosphate uridylyltransferase, E C 2.7.7.9), an excess
of E D T A is necessary in the assay t o inhibit the m a g n e s i u m - d e p e n d e n t activity of this enzyme.
The "hepatic U D P - g a l a c t o s e content is increased after administration of D - g a l a c t o s e , orotic acid
4 1
or
uridine; a strong depletion is induced by D-galactosamine, 2-deoxy-D-galactose, a n d D - g l u c o s a m i n e . 5
A p a r t from U D P - g a l a c t o s e , U D P - g a l a c t o s a m i n e reacts with uridylyltransferase, although with lower

affinity . Other k n o w n nucleosidediphosphate sugars are not d e t e r m i n e d because of the specificity
6
of the two enzymes used.
References
1 D. Keppler, J. Rudigier & K. Decker, Analyt. Biochem. 38, 105 [1970].

2 J. .S. Mayes & R. G. Hansen in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, A c a d e m i c
Press. New York 1966, Vol. IX. p . 708.
3 K Papenberg, Klin. Wschr. 39, 739 [1961].
4 D. Keppler, J. Rudigier & K. Decker, F E B S Letters 11, 193 [1970].
5 D. Keppler, J. Rudigier, E. Bischoff 8i K. Decker, E u r o p e a n J. Biochem. 17, 246 [1970].
6 D. Keppler & K. Decker, E u r o p e a n J. Biochem. 10, 219 [1969].
Uridine-5-diphosphoglucose
Dietrich Keppler and Karl Decker
UDP-glucose ( U D P G ) is the precursor of glucose-containing oligo- a n d polysaccharides, glycoproteins

and glycolipids in animal tissues a n d in some micro-organisms. As p r e c u r s o r also of U D P - g a l a c t o s e a n d
U D P - g l u c u r o n a t e this active form of glucose is i m p o r t a n t for the synthesis of heteropolysaccharides a n d
glucuronides.
U D P - g l u c o s e is oxidized to U D P - g l u c u r o n a t e with N A D a n d U D P G - d e h y d r o g e n a s e , UDPG-DH
( U D P - g l u c o s e : N A D 6-oxidoreductase, E C 1.1.1.22) . In this specific, irreversible reaction 2 mole N A D
1
are reduced per mole U D P - g l u c o s e , so that a very sensitive s p e c t r o p h o t o m e t r i c assay is possible.
Principle
(1) UDP-glucose + H 0 + 2 N A D 2
+ U D P G
~ P H
> UDP-glucuronate + 2 N A D H + 2 H +
T h e increase of extinction at 340 (334, 365) n m due to the reduction of N A D is p r o p o r t i o n a l to the a m o u n t

of U D P G present.
T h e p H o p t i m u m for the oxidative reaction is p H 8.7; at p H 7.8 only ca. 50% of the m a x i m u m rate is
obtained. Because the enzyme is competitively inhibited by low c o n c e n t r a t i o n s of N A D H , it is a d v a n 2
tageous to work with high c o n c e n t r a t i o n s of N A D . If U D P G - D H p r e p a r a t i o n s contain U D P - g l u c o s e

pyrophosphorylase ( U T P : a-D-glucose-1-phosphate uridylytransferase, E C 2.7.7.9) the activity of the
latter can be completely inhibited by addition of E D T A , without affecting the activity of U D P G - D H .
T h e m a x i m u m rate of the reaction is significantly higher in glycine buffer t h a n in t r i e t h a n o l a m i n e buffer.
Equipment
365 n m .
Reagents
1. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , 6. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e , N A D
s p . gr. 1.67 free acid, commercial p r e p a r a t i o n , see p. 545.
2. P o t a s s i u m h y d r o g e n c a r b o n a t e , A . R. 7. U D P - g l u c o s e d e h y d r o g e n a s e , UDPG-DH
3. P o t a s s i u m h y d r o x i d e , A . R . , 1 N from ox liver, suspension in 3.2 M a m m o n i u m
4. Glycine sulphate s o l u t i o n ; 5 mg./ml., ca. 1 U / m g . (25 ° C ) ;
5. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , EDTA commercial p r e p a r a t i o n , see p. 519.
disodium salt, A . R . , E D T A - N a H • 2 H 0 2 2 2
Purity of Reagents
U D P G - D H must be free from dehydrogenases reacting with N A D H (e. g. lactate dehydrogenase, E C

1.1.1.27), in case their substrates (e. g. pyruvate) are contained in the sample.
P r e p a r e all s o l u t i o n s w i t h fresh d i s t i l l e d w a t e r .
D i l u t e 5.2 m l . 7 0 % H C 1 0 4 t o 1 0 0 m l . w i t h distilled w a t e r .
II. P o t a s s i u m h y d r o g e n c a r b o n a t e (2 M ) :
D i s s o l v e 2 0 g. K H C 0 3 in 1 0 0 m l . distilled w a t e r .
III. G l y c i n e buffer (0.5 M ; p H 8 . 7 ) :
D i s s o l v e 7.51 g. g l y c i n e in c a . 180 m l . distilled w a t e r a n d a d d 7.2 m l . 1 N K O H . C h e c k
the p H o n a glass electrode and dilute to 200 ml. with distilled water.
IV. G l y c i n e / N A D / E D T A ( 0 . 5 M g l y c i n e ; 8 m M E D T A ; 3 m M N A D ) :
D i s s o l v e 3 0 m g . E D T A - N a H - 2 H 0 a n d 2 0 m g . N A D in 10 m l . g l y c i n e buffer ( s o l u
2 2 2
t i o n III).
V . U D P - g l u c o s e d e h y d r o g e n a s e , U D P G - D H (5 m g . / m l . ) :
Suspend the e n z y m e protein and use undiluted.
Store all solutions, stoppered, at 0 - 4 °C. Solution III is stable for 1 m o n t h , solution IV for ca. 1 week.
The enzyme suspension is active for several m o n t h s at 4 ° C ; it can be deep-frozen without significant loss
of activity.
Procedure
T i s s u e s a m p l e s s h o u l d b e c o l l e c t e d b y t h e f r e e z e - s t o p m e t h o d (see p . 4 0 0 ) .
Deproteinization:
A d d 5 parts b y w t . o f i c e - c o l d p e r c h l o r i c a c i d ( s o l u t i o n I) t o t h e f r o z e n p i e c e o f t i s s u e ( 0 . 3 - 1 . 5 g.)
a n d i m m e d i a t e l y h o m o g e n i z e . A l l o w t h e h o m o g e n a t e t o s t a n d for 1 0 - 1 5 m i n . at 0 ° C a n d
t h e n c e n t r i f u g e for 15 m i n . at 1 0 0 0 0 - 2 0 0 0 0 g (0 ° C ) . D e c a n t t h e a c i d s u p e r n a t a n t fluid (1.5 m l . )
a n d w i t h o u t d e l a y b r i n g t o a b o u t p H 8 w i t h p o t a s s i u m h y d r o g e n c a r b o n a t e (either s o l i d o r
s o l u t i o n I I ; 0.5 m l . ) . A f t e r a l l o w i n g t o s t a n d for 15 m i n . at 0 - 4 ° C , c e n t r i f u g e off t h e p r e c i p i t a t e
of potassium perchlorate and decant.
U D P - g l u c o s e is alkali a n d a c i d l a b i l e , t h e r e f o r e p e r c h l o r i c a c i d d e p r o t e i n i z a t i o n at t e m p e r
3
a t u r e s a r o u n d 0 ° C a n d r a p i d n e u t r a l i z a t i o n are vital. S i m i l a r l y , p H v a l u e s a b o v e 10 are t o b e

a v o i d e d . T h e n e u t r a l i z e d s a m p l e s , s t o r e d at 4 ° C , s h o u l d b e a n a l y s e d w i t h i n a d a y or s t o r e d
deep frozen.
UDP-glucose 2227
Assay System
W a v e l e n g t h : 3 4 0 ( H g 3 3 4 , H g 3 6 5 ) n m ; light p a t h : 1 c m . ; final v o l u m e : 0 . 7 2 m l . ( s e m i - m i c r o -
G l y c i n e / N A D / E D T A solution (IV) 0.50 ml. 0.35 M glycine

2.1 m M NAD
5.6 m M E D T A
Sample (deproteinized, neutralized) 0.20 ml. c a . 3 t o 1 5 0 pM UDP-glucose
M i x and read extinction E . t
U D P G - D H suspension (V) 0.02 ml. 1 4 0 /xg./ml. = 1 4 0 m U / m l .
M i x , w h e n e x t i n c t i o n i n c r e a s e is c o n s t a n t (ca. 10 m i n . ) ,
read e x t i n c t i o n E . E 2 2 — E j = AE is u s e d for t h e
calculations.
D e t e r m i n e t h e i n c r e a s e in e x t i n c t i o n d u e t o a d d i t i o n o f t h e U D P G - D H s u s p e n s i o n ( V ) b y a
further a d d i t i o n o f 0 . 0 2 m l . s o l u t i o n V at t h e e n d o f t h e r e a c t i o n . S u b t r a c t t h e c h a n g e in e x
t i n c t i o n f r o m E . A l t e r n a t i v e l y , r e a d E , i m m e d i a t e l y after a d d i t i o n o f t h e e n z y m e .
2
Calculations
To obtain /miole/g. fresh wt. multiply the /tmole U D P - g l u c o s e / m l . sample by the dilution factor d u e to
deproteinization a n d neutralization. As 2 mole N A D are reduced per mole U D P - g l u c o s e , the following
relationships hold for the calculation of the c o n c e n t r a t i o n in the neutral tissue e x t r a c t :

F r o m replicate d e t e r m i n a t i o n s on various U D P - g l u c o s e - c o n t a i n i n g tissue extracts a n d solutions a coef

ficient of variation of 2 . 5 % is o b t a i n e d from the s t a n d a r d deviation of t h e individual values. T h e U D P -
glucose content of liver has been determined by this m e t h o d a n d by the m e t h o d involving U D P - g l u c o s e :
a-D-galactose-1-phosphate uridylyltransferase ( E C 2.7.7.12) a n d d e t e r m i n a t i o n of the glucose-1-phos
phate formed. T h e differences between the individual values of the two m e t h o d s were between 0 a n d 2 % .
Normal Values
T h e U D P - g l u c o s e c o n t e n t of rat liver was found to be 0.32 + 0.04 /miole/g , a n d of rat kidney 0.16 + 0.01
4
/miole/g. . Values for rat brain a n d muscle are 0.05 + 0.01 a n d 0.02 + 0.005 /miole/g. respectively .
5 4
The U D P - g l u c o s e content of liver can be increased by administration of orotic acid or u r i d i n e , a n d 4
markedly depressed by a d m i n i s t r a t i o n of D - g a l a c t o s a m i n e , D-glucosamine or 2-deoxy-D-galactose .

5 6
Interference in the assay technique: T h e p H of the cuvette contents should be between p H 8.2 a n d 9.2.
T h e p r o d u c t s of the reaction, N A D H a n d U D P - g l u c u r o n a t e , are powerful inhibitors of the e n z y m e . 2
A p a r t from U D P - g l u c o s e , which is b o u n d to U D P G - d e h y d r o g e n a s e with high affinity ( K M = 13 /.iM),

d U D P - g l u c o s e a n d d T D P - g l u c o s e is also oxidized. At a concentration of 2 m M the rate of the reaction
4
is 8.5% of that with U D P - g l u c o s e . T h e following either d o not react or react at a rate which is less t h a n
2
0.4% of that with UDP-glucose: UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-

galactose, U D P - m a n n o s e , GDP-mannose, G D P - g l u c o s e , C D P - g l u c o s e , A D P - g l u c o s e , glucose and
g l u c o s e - 1 - p h o s p h a t e ' . D e a m i d o - N A D a n d acetylpyridine-adenine dinucleotide, but not N A D P , are
1 2
reduced by U D P - g l u c o s e . 1
References
1 J. L. Strominger, E. S. Maxwell, J. Axelrod & H. M. Kalckar, J. biol. C h e m . 224, 79 [1957].

2 J. Zalitis & D. S. Feingold, Biochem. Biophys. Res. C o m m . 31, 693 [1968].
3 R. Caputto, L. F. Leloir, C. E. Cardini & A. C. Paladini, J. biol. C h e m . 184, 333 [1950].
4 D. Keppler, J. Rudigier & K Decker, Analyt. Biochem. 38, 105 [1970].
5 D. Keppler & K. Decker, E u r o p e a n J. Biochem. 10, 219 [1969].
6 D. Keppler, J. Rudigier, E. Bischoff & K. Decker, E u r o p e a n J. Biochem. 17, 246 [1970].
Inorganic Phosphate
Fluorimetric Method
Janet V. P a s s o n n e a u and D e m o y W . Schulz
Inorganic p h o s p h a t e is a substrate or p r o d u c t of m a n y enzyme reactions a n d affects the activity of enzymes,

e.g. h e x o k i n a s e , p h o s p h o f r u c t o k i n a s e
1 2
a n d glucose-6-phosphate d e h y d r o g e n a s e . Several
3
excellent
m e t h o d s for the d e t e r m i n a t i o n of p h o s p h a t e exist, b u t each has its disadvantages. In the m e t h o d of Fiske
a n d Subbarow* acid-labile p h o s p h a t e is hydrolysed; it can be modified, b u t then the analysis is time-
c o n s u m i n g a n d less sensitive. T h e enzymatic m e t h o d described here is carried out at n e u t r a l p H . Tissue
extracts m a y be p r e p a r e d with acids or tissue enzymes can be inactivated with alkali, but the m e t h o d can
can also be used without preliminary h a r s h t r e a t m e n t of the tissue. T h e sensitivity of the m e t h o d is limi
ted only by the p h o s p h a t e c o n t a m i n a t i o n of the reagents. T h e m e t h o d d e p e n d s on the p h o s p h o r o l y t i c
cleavageof glycogen by p h o s p h o r y l a s e a ( l ,4-a-D-Glucan: o r t h o p h o s p h a t e a-glucosyltransferase, E C 2.4.1.1).
Application of Method: In biochemistry a n d clinical biochemistry. - F o r m e a s u r e m e n t of inorganic

p h o s p h a t e in tissues. By direct fluorimetry 1 0 " mole can be determined.
9
Principle
(1) Glycogen + P {
p h o s p h o r y l a s e a
> Glucose-l-P
(2) Glucose-l-P P G l u M
* > Glucose-6-P
(3) Glucose-6-P + N A D P + G
~ ~
6 P P H
* * > 6-Phosphogluconolactone + N A D P H + H +
T h e increase of N A D P H , as measured by the fluorescence, is p r o p o r t i o n a l to the a m o u n t of Pj.
U n d e r the conditions of the assay with P < 0.1 m M the a p p a r e n t K { M for glycogen is a b o u t 0.004 % 5
or
0.25 m M (as glucosyl units). T h e r e c o m m e n d e d co ncentration is 20-fold higher a n d therefore not critical.
O n the other h a n d with the stipulated concentratio n of 0 . 0 8 % glycogen the K M for Pj is a b o u t 1 m M so
that with all Pj values used in the fluorometric p r o c e d u r e the reaction is 1 st order. A - 5 - M P is a d d e d only
in small a m o u n t s because it decreases the K M for P j 7-fold. Higher c o n c e n t r a t i o n s can increase the blank,
5
because Pj m a y be liberated from A M P by enzyme c o n t a m i n a n t s in the p h o s p h o r y l a s e p r e p a r a t i o n .

E G T A is a d d e d in excess c o m p a r e d to M g 2 +
so as to inhibit the A T P a s e (in o u r experience commercial
phosphorylase p r e p a r a t i o n s are strongly c o n t a m i n a t e d with A T P a s e ) . T h e M g 2 +
requirement of A T P a s e
is greater t h a n that of p h o s p h o g l u c o m u t a s e , therefore one can practically eliminate all A T P a s e activity a n d
still maintain sufficient P G l u M activity.
* P G l u M , p h o s p h o g l u c o m u t a s e (a-D-Glucose-1,6-bisphosphate: a-D-glucose-1 - p h o s p h a t e p h o s p h o t r a n s
ferase, E C 2.7.5.1).
** G 6 P - D H , glucose-6-phosphate dehydrogenase ( D - G l u c o s e - 6 - p h o s p h a t e : N A D P 1-oxidoreductase,
E C 1.1.1.49).
2230 M e t a b o l i t e s : Miscellaneous Substrates a n d Effectors
Equipment
Filter f l u o r i m e t e r w i t h p r i m a r y filter for e x c i t a t i o n at 3 6 0 n m . ( C o r n i n g N o . 5 8 4 0 ) a n d s e c o n d a r y

filter for e m i t t e d light o f a b o u t 4 6 0 n m ( C o r n i n g filters N o . 4 3 0 3 a n d 3 3 8 7 in c o m b i n a t i o n ) ;
3 m l . test t u b e s , 10 m m . x 75 m m .
Reagents
1. I m i d a z o l e 9. P o t a s s i u m d i h y d r o g e n p h o s p h a t e ,
e.g. from Sigma Chemical C o . , grade III (low K H P 0 , A . R.
2 4
fluorescence) 10. P h o s p h o r y l a s e a
2. H y d r o c h l o r i c a c i d , A . R . twice recrystallized; ^20 U/mg. (25 °C);
3. M a g n e s i u m a c e t a t e , commercial p r e p a r a t i o n , see p . 505.
M g ( C H C O O ) - 4 H 0 , A . R.
3 2 2
11. P h o s p h o g l u c o m u t a s e , P G l u M
4. Ethyleneglycol-bis-(/?-amino-ethylether) from muscle, suspension in a m m o n i u m sulphate
N , N ' tetra-acetic acid, E G T A solution; ^ 500 U / m g . (25 °C); commercial
5. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p r e p a r a t i o n , see p . 499.
phate, N A D P 12. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ,
disodium salt, N A D P - N a H ; commercial prep
2
G6P-DH
aration, see p . 546. from yeast, suspension in a m m o n i u m sulphate
6. A d e n o s i n e - 5 - m o n o p h o s p h a t e , A M P solution; ^ 140 U / m g . (25 ° C ) ; commercial
crystalline disodium salt, AMP-Na -6H 0; 2 2
commercial p r e p a r a t i o n , see p . 526. 13. Dithiothreitol, C O S H 4 2 2 1 0
7. A l b u m i n f r o m b o v i n e p l a s m a
8. Glycogen
from rabbit liver; commercial p r e p a r a t i o n , see
p . 540.
Purity of Reagents
All reagents must be free as possible from o r t h o p h o s p h a t e . C o m m e r c i a l p r e p a r a t i o n s of phosphorylase a

m a y be c o n t a m i n a t e d with P ^ purify by dialysis of a 2 . 5 % suspension against 1000 volumes 20 m M
imidazole buffer, p H 7.0 (containing 1 m M E G T A , 0.5 m M dithiothreitol a n d 10 pM A M P ) overnight at
4 °C. Similarly, glycogen from rabbit liver contains a m o u n t s of P which can interfere; purify by dialysis of
4
a n 8 % solution against 200 volumes 25 m M acetate buffer, p H 4.6 overnight. A N A D P p r e p a r a t i o n free

from measurable Pj should be chosen. P h o s p h o g l u c o m u t a s e a n d glucose-6-phosphate dehydrogenase
frequently contain a m o u n t s of Pj which interfere. In such cases centrifuge off the protein at 0 - 4 °C, wash the
precipitate by resuspension in 2 volumes 4 M ( N H ) S 0 solution, centrifuge again a n d repeat the washing
4 2 4
procedure twice m o r e . Suspend in the original volume of 3.2 M ( N H ) S 0 . 4 2 4
T h e r e c o m m e n d e d imidazole is suitable for use. O t h e r p r e p a r a t i o n s m u s t be treated with charcoal to remove

the high fluorescence.
P r e p a r e all s o l u t i o n s w i t h freshly p r e p a r e d , d o u b l y distilled o r d e i o n i z e d w a t e r .
I. I m i d a z o l e buffer (1 M ; p H 7 . 0 ) :
D i s s o l v e 6.8 g. i m i d a z o l e in a little distilled w a t e r , a d d 3.3 m l . 12 N HC1 a n d d i l u t e t o
100 m l . w i t h d i s t i l l e d w a t e r ; s t o r e f r o z e n .
Inorganic Phosphate 2231
I I . M a g n e s i u m a c e t a t e (1 M ) :
D i s s o l v e 2 1 . 4 5 g. M g ( C H C O O ) - 4 H 0 in 100 m l . d i s t i l l e d w a t e r ; s t o r e at r o o m t e m p e r
3 2 2
ature.
I I I . E t h y l e n e g l y c o l - b i s - (/?-amino ethyl ether) N , N ' - t e t r a - a c e t i c a c i d (0.1 M ) :
D i s s o l v e 3.8 g. E G T A in 100 m l . d i s t i l l e d w a t e r ; s t o r e at r o o m t e m p e r a t u r e .
I V . N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e (0.1 M ) :
Dissolve ca. 80 mg. N A D P - N a 2 in 1 ml. distilled water; store frozen.
V . A d e n o s i n e m o n o p h o s p h a t e , A M P (0.1 M ) :
D i s s o l v e 5 0 m g . A M P - N a - 6 H 0 in 1.0 m l . d i s t i l l e d w a t e r ; s t o r e f r o z e n .
2 2
V I . A l b u m i n (10 % w / v ) :
Stir 1 m g . a l b u m i n w i t h a little d i s t i l l e d w a t e r t o f o r m a p a s t e a n d d i l u t e w i t h d i s t i l l e d
w a t e r t o 10 m l . ; s t o r e f r o z e n
V I I . G l y c o g e n (0.5 M ; glucosyl units):
D i s s o l v e 162 m g . g l y c o g e n in 2 m l . distilled w a t e r . D i a l y s e as d e s c r i b e d u n d e r " P u r i t y
o f R e a g e n t s " ; s t o r e f r o z e n . Just b e f o r e u s e h e a t for 10 m i n . at 6 0 ° C ; t h i s reverses t h e
aggregation o f glycogen w h i c h occurs o n freezing and thawing.
V I I I . P h o s p h a t e s t a n d a r d s o l u t i o n (1 m M ) :
D i s s o l v e 136 m g . K H P 0 2 4 in 1 0 0 0 m l . d i s t i l l e d w a t e r ; s t o r e f r o z e n .
I X . P h o s p h o r y l a s e a (1 m g . p r o t e i n / m l . ) :
Dialyse the e n z y m e preparation as described under "Purity o f R e a g e n t s " ; dilute accor
d i n g l y w i t h 2 0 m M i m i d a z o l e buffer, p H 7.0 ( c o n t a i n i n g 1 m M E G T A ; 0 . 0 2 % a l b u m i n
a n d 0.5 m M d i t h i o t h r e i t o l ) ; s t o r e at 0 - 4 ° C .
X . P h o s p h o g l u c o m u t a s e , P G l u M (1 m g . p r o t e i n / m l . ) :
W a s h free o f p h o s p h a t e a s d e s c r i b e d u n d e r " P u r i t y o f R e a g e n t s " , d i l u t e a c c o r d i n g l y
w i t h 9 v o l u m e s 2 0 m M i m i d a z o l e buffer, p H 7.0 ( c o n t a i n i n g 1 m M m a g n e s i u m a c e t a t e ;
0.1 m M E G T A a n d 0 . 0 2 % a l b u m i n ) ; s t o r e at 0 - 4 ° C .
X I . G l u c o s e - 6 - p h o s p h a t e dehydrogenase, G 6 P - D H (0.5 m g . p r o t e i n / m l . ) :
D i l u t e t h e s t o c k s u s p e n s i o n (5 m g . / m l . ) a c c o r d i n g l y w i t h 9 v o l u m e s 2 0 m M tris buffer,
p H 8.0 ( c o n t a i n i n g 0 . 0 2 % a l b u m i n ) .
X I I . D i t h i o t h r e i t o l (0.1 M ) :
D i s s o l v e 1 5 . 4 g. d i t h i o t h r e i t o l in 1 m l . d i s t i l l e d w a t e r ; s t o r e f r o z e n .
All solutions are stable for several m o n t h s u n d e r the stated c o n d i t i o n s ; the dilute enzyme solutions are
stable for 1 week at 0 - 4 ° C .
Procedure
Perchloric acid extraction: F r e e z e t h e a n i m a l o r t h e t i s s u e in l i q u i d n i t r o g e n , d i s s e c t at - 1 5 ° C

and p o w d e r in a mortar with addition o f liquid nitrogen. A d d 50 or 100 m g . s a m p l e s t o 150 or
3 0 0 fil. 3.3 N H C 1 0 , w h i c h h a s b e e n f r o z e n in 8 m l . test t u b e s in d r y ice. P l a c e test t u b e s in
4
- 1 0 ° C a l c o h o l b a t h , s h a k e f o r s e v e r a l m i n u t e s , t h e n a d d 0 . 5 o r 1 m l . 1 m M E G T A at 4 ° C .
C e n t r i f u g e ; a d d t o e a c h m l . s u p e r n a t a n t fluid 0 . 3 2 m l . 2 N K O H ( 0 . 4 M i m i d a z o l e ; 0 . 4 m M KC1).
C e n t r i f u g e off t h e K C 1 0 4 p r e c i p i t a t e a n d s t o r e t h e s u p e r n a t a n t fluid at — 6 0 ° C .
2232 Metabolites: Miscellaneous Substrates and Effectors
Tissue suspensions in methanol: H o m o g e n i z e t h e t i s s u e p o w d e r p r e p a r e d as d e s c r i b e d a b o v e in

a g l a s s h o m o g e n i z e r in — 2 0 ° C a l c o h o l b a t h w i t h 50 v o l u m e s m e t h a n o l . N o further t r e a t m e n t is
r e q u i r e d . S t o r e t h e h o m o g e n a t e at — 6 0 ° C .
Stability of sample: T h e s a m p l e s are s t a b l e at - 6 0 ° C . S a m p l e s c a n b e t h a w e d , a p o r t i o n t a k e n

and then they can be re-frozen.
Assay System
Primary wavelength: 360 n m ; secondary wavelength: 460 n m ; light p a t h : 1 cm. (standardized

test t u b e s ) ; r o o m t e m p e r a t u r e ; final v o l u m e : c a . 1 m l .
F o r e a c h series o f m e a s u r e m e n t s p r e p a r e a r e a g e n t b l a n k c o n t a i n i n g w a t e r i n s t e a d o f s a m p l e
a n d s t a n d a r d s ; for t h e latter a d d 1 t o 10 pi. P s t a n d a r d s o l u t i o n ( V I I I ) , c o r r e s p o n d i n g t o 1 t o
10 n m o l e , i n s t e a d o f s a m p l e .
Reagent mixture: P i p e t t e i n t o a 100 m l . m e a s u r i n g c y l i n d e r :
I m i d a z o l e buffer (I) 5.00 ml.

Mg-acetate solution (II) 0.05 ml.
E G T A solution (HI) 1.00 m l .
N A D P solution (IV) 0.03 ml.
A M P solution (V) 0.01 m l .
Albumin solution (VI) 0.20 ml.
Glycogen solution (VII) 1.00 m l .
P G l u M solution (X) 0.30 ml.
G 6 P - D H solution (XI) 0.50 ml. T h i s m i x t u r e is
Dithiothreitol solution (XII) 0.50 ml. s t a b l e for 2 t o 3
distilled water to 100 ml. d a y s at 0 - 4 ° C
S o a r r a n g e the i n s t r u m e n t that the e x p e c t e d r e a d i n g s c a u s e a v i r t u a l l y full scale d e f l e c t i o n o f t h e

i n d i c a t o r . C h e c k t h e c o r r e c t f u n c t i o n i n g o f t h e auxiliary a n d i n d i c a t o r e n z y m e d a i l y : 2 o r
5 pi. G - l - P in t h e a s s a y s h o u l d react w i t h i n 2 m i n . ; it is h o w e v e r sufficient t o h a v e a h a l f t o a
third o f this activity.
Inorganic P h o s p h a t e 2233
P i p e t t e i n t o 3 m l . test t u b e s : C o n c e n t r a t i o n in a s s a y m i x t u r e
Reagent mixture 1 ml. 50 m M buffer

0.5 m M M g 2 +
1 mM EGTA
0.03 m M N A D P
0.5 m M d i t h i o t h r e i t o l
3 pg. P G l u M / m l . = 1.5 U / m l .
2.5 pg. G 6 P - D H / m l . = 3 5 0 m U / m l .
0.01 m M A M P
5 m M glycogen
0.02% albumin
Sample or standard solution (VIII) 1-50 pi
M i x . A f t e r a f e w m i n . G - l - P a n d G - 6 - P in t h e s a m p l e
h a v e r e a c t e d . R e a d fluorescence F t
Phosphorylase a solution (IX) 5 pi 50 pg./ml = 1 U/ml.
M i x a n d after ca. 2 0 - 3 0 m i n . ( c o m p l e t i o n o f r e a c t i o n )
read fluorescence F 2
Calculations
U n d e r the above conditions the reaction proceeds stoichiometrically and the results are calculated from the
Pj standard.
The fluorimeter should be accurate to ± 0 . 3 % . 1 0 m o l e or 0.096 ug. P can be determined.

_ 9
}
Measurements ofa p h o s p h a t e s t a n d a r d containing 5.1 x 1 0 ~ m o l e g a v e a s t a n d a r d deviation of 0.24 x 1 0 "

1 1 1 1
mole. Coefficient of variation is 4 . 7 % . In mouse brain we found a mean of 1.71 ± 0 . 2 7 ^mole/kg. fresh wt.;
coefficient of variation is 15.8%.
N o r m a l Values
We have measured the following values in mouse brain, muscle, liver and h e a r t : 1.78 ± 0.13; 3.82 ± 0.22;
2.68 ± 0.30; and 2.96 ± 0.16 jmiole/g. fresh wt. respectively.
Specificity
G l y c o g e n p h o s p h o r y l a s e reacts w i t h a r s e n a t e , b u t n o c o m m o n c o m p o n e n t c o n t a i n e d in
tissues interferes.
To o b t a i n t h e full sensitivity o f this fluorimetric m e t h o d it is n e c e s s a r y t o a v o i d p o s s i b l e

c o n t a m i n a t i o n w i t h i n o r g a n i c p h o s p h a t e . A l l g l a s s w a r e s h o u l d b e c l e a n e d c a r e f u l l y : rinse t h e
fluorimeter t u b e s , b o i l for s e v e r a l m i n u t e s in 5 0 % H N Q , rinse t h r e e t i m e s w i t h distilled w a t e r
3
a n d t w i c e w i t h d o u b l y d i s t i l l e d w a t e r . R i n s e a g a i n i m m e d i a t e l y b e f o r e u s e . R i n s e all o t h e r
g l a s s w a r e several t i m e s , e s p e c i a l l y if it h a s b e e n c l e a n e d w i t h d e t e r g e n t s .
A s e c o n d m a j o r s o u r c e o f erro r lies in t h e p r e p a r a t i o n o f t h e t i s s u e e x t r a c t s . If t h e t i s s u e is n o t
f r o z e n sufficiently r a p i d l y o r if it t h a w s d u r i n g t h e e x t r a c t i o n e r r o n e o u s l y h i g h v a l u e s are f o u n d .
C a r e m u s t b e t a k e n t o a v o i d c o n t a m i n a t i o n w i t h P j . In t h e p r e p a r a t i o n o f t i s s u e s w i c h are
dissected from b o n e (muscle, brain) care must be taken to exclude b o n e fragments otherwise
t o o h i g h v a l u e s will result.
F o r i n f o r m a t i o n o n t h e r e q u i r e d p u r i t y o f t h e e n z y m e s , see p . 2 2 3 0 . A T P a s e o f t h e s a m p l e a n d
in t h e p h o s p h o r y l a s e p r e p a r a t i o n c a n b e i n h i b i t e d , see u n d e r " O p t i m u m C o n d i t i o n s for
Measurements".
References
1 /. A. Rose, J. V. B. Warms & E. L. O'Connell, Biochem. Biophys. Res. C o m m u n . 75, 33 [1964].

2 J. V. Passonneau & O. H. Lowry, Biochem. Biophys. Res. C o m m u n . 7, 10 [1962].
3 H. Theorell, Biochem. Z. 275, 416 [1935].
4 C. H. Fiske & Y. Subbarrow, J. biol. C h e m . 81, 629 [1929].
5 O. H. Lowry, D. W. Schulz 8c J. V. Passonneau, J. biol. C h e m . 239, 1 9 4 7 [1964].
UV-Spectrophotometric Method
Karlfried G a w e h n
Inorganic p h o s p h a t e is the substrate or p r o d u c t of m a n y enzyme reactions a n d also influences the activities

of certain enzymes (see also Passonneau a n d Schulz, p . 2229). T h e m e t h o d for the enzymatic d e t e r m i n a t i o n
of Pj is based on the phosphorylysis of glycogen by phosphorylase a(l ,4-a-D-glucan: o r t h o p h o s p h a t e a-gluco-
syltransferase, E C 2.4.1.1).
Principle
(1) Pi + Glycogen p h o s p h o r y l a s e
> Glucose-l-P
a
i 1
(2) Glucose-l-P — P G l u M
* > Glucose-6-P
(3) Glucose-6-P + N A D P + G 6 P P H
* * > 6-Phosphogluconolactone + N A D P H + H +
The formation of N A D P H is p r o p o r t i o n a l to the a m o u n t of P present. {
* P G l u M , P h o s p h o g l u c o m u t a s e ( a - D - G l u c o s e - l , 6 - b i s p h o s p h a t e : a-D-glucose-1-phosphate p h o s p h o
transferase, E C 2.7.5.1).
** G 6 P - D H , Glucose-6-phosphate dehydrogenase ( D - G l u c o s e - 6 - p h o s p h a t e : N A D P 1-oxidoreductase,
E C 1.1.1.49).
Inorganic Phosphate 2235
See p. 2229.
Equipment
365 n m ; b e n c h c e n t r i f u g e .
Reagents
1. I m i d a z o l e , A . R . aration, see p. 505. The preparation of Boeh

2. H y d r o c h l o r i c a c i d , 2 N , A . R. ringer Mannheim contains 5 mg. of enzyme
3. M a g n e s i u m acetate, protein, 25 mg. lactose, 50 /rniole glycerol-2-
M g ( C H C O O ) - 4 H 0 , A . R.
3 2 2 phosphate and 1 /imole E D T A in 40 mg.
4. E t h y l e n e d i a m i n e t e t r a - a c e t a t e , E D T A lyophilizate.
disodium salt, E D T A - N a H • 2 H 02 2 2 9. P h o s p h o g l u c o m u t a s e , P G l u M
5. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e from muscle, suspension in 3.2 M ammonium
phosphate, N A D P sulphate solution; ^ 200 U/mg. (25 °C); com
disodium salt, N A D P - N a H ; commercial prep
2 mercial preparation, see p. 499.
aration, see p. 546. 10. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ,
6. A d e n o s i n e - 5 ' - m o n o p h o s p h a t e , A M P G6P-DH
crystalline disodium salt, AMP-Na -6H 0; 2 2 from yeast, suspension in 3.2 M ammonium
commercial preparation, see p. 526. sulphate solution; ^ 140 U/mg. (25 °C); com
7. Glycogen mercial preparation, see p. 458.
from rabbit liver; commercial preparation, see 11. Dithiothreitol, C O S H 4 2 2 1 0
p. 540. ClelancTs reagent

8. P h o s p h o r y l a s e a 12. P e r c h l o r i c a c i d , A . R . , 70% (w/w), sp.
crystalline from rabbit muscle, lyophilizate; gr. 1.67
^ 20 U/mg. protein (25 °C); commercial prep-
Purity of Reagents
All reagents must be as free as possible from orthophosphate, otherwise they should be purified. Commer
cial preparations of phosphorylase a, P G l u M and G 6 P - D H may contain P ; this can be removed by 5
dialysis overnight at 4 °C.
I. I m i d a z o l e buffer ( 5 0 m M ; p H 7 . 0 ) :
D i s s o l v e 3 4 0 m g . i m i d a z o l e in d i s t i l l e d w a t e r , a d j u s t t o p H 7.0 w i t h 2 N H C 1 a n d d i l u t e
II. M a g n e s i u m a c e t a t e ( 1 5 m M ) :
D i s s o l v e 3 2 2 m g . ( M g ( C H C O O ) - 4 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
3 2 2
III. E t h y l e n e d i a m i n e t e t r a - a c e t a t e ( 3 0 m M ) :
D i s s o l v e 1.1 g. E D T A - N a H • 2 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
2 2 2
IV. N i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( 1 0 m M ) :
D i s s o l v e 9.3 m g . N A D P - N a H in 1.0 m l . distilled w a t e r .
2
V . A d e n o s i n e - 5 ' - m o n o p h o s p h a t e (3 m M ) :
D i s s o l v e 2.2 m g . A M P - N a - 6 H 0 in 1.0 ml. distilled w a t e r .
2 2
VI. G l y c o g e n (60 m g . / m l . ) :
D i s s o l v e 6 0 m g . g l y c o g e n in 1.0 m l . d i s t i l l e d w a t e r .
VII. Dithiothreitol (15 m M ) :
D i s s o l v e 2.3 m g . d i t h i o t h r e i t o l in 1.0 m l . distilled w a t e r .
V I I I . P h o s p h o r y l a s e a (5 m g . p r o t e i n / m l . ) :
D i s s o l v e 4 0 m g . l y o p h i l i z a t e in 0.5 m l . distilled w a t e r . D i a l y s e o v e r n i g h t at 4 ° C a g a i n s t
1 0 0 0 m l . 50 m M i m i d a z o l e buffer ( p H 7.0) a n d t h e n d i l u t e t o 1 m l . w i t h distilled w a t e r .
( P h o s p h o r y l a s e a c r y s t a l l i z e s o u t at 4 ° C . )
I X . P h o s p h o g l u c o m u t a s e , P G l u M (5 m g . p r o t e i n / m l . ) :
D i a l y s e s t o c k s u s p e n s i o n ( 1 0 m g . / m l . ) o v e r n i g h t at 4 ° C a g a i n s t 1 0 0 0 v o l u m e s 2 0 m M
a c e t a t e buffer ( p H 5.3) a n d t h e n d i l u t e t o 5 m g . p r o t e i n / m l . w i t h t h e s a m e buffer.
X . G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , G 6 P - D H (2 m g . p r o t e i n / m l . ) :
D i a l y s e s t o c k s u s p e n s i o n (5 m g . / m l . ) o v e r n i g h t at 4 ° C a g a i n s t 1 0 0 0 v o l u m e s 5 0 m M
i m i d a z o l e buffer ( p H 7.0) a n d t h e n d i l u t e t o 2 m g . p r o t e i n / m l . w i t h t h e s a m e buffer.
X I . P e r c h l o r i c a c i d (5.8 N ) :
M i x 50 ml. c o l d distilled w a t e r a n d 5 0 ml. p e r c h l o r i c a c i d .
Store solutions I, I V - V I I I , stoppered, in a refrigerator; p r e p a r e solutions I, I V - V I I freshly every week.

Enzyme suspension VIII is stable for ca. 3 m o n t h s . Enzyme solutions IX a n d X are stable in the frozen
state at — 2 0 °C for ca. 3 m o n t h s . Solutions II a n d III are stable indefinitely.
Procedure
S a m p l e s l o w in p r o t e i n c a n b e u s e d directly for t h e a s s a y w i t h o u t d e p r o t e i n i z a t i o n .
Deproteinization :
If n e c e s s a r y , a d d 0.1 v o l u m e p e r c h l o r i c a c i d ( X I ) t o b i o l o g i c a l fluids; h o m o g e n i z e t i s s u e w i t h
4 t i m e s the a m o u n t ( w / v ) o f c o l d d i l u t e p e r c h l o r i c a c i d ( d i s t i l l e d w a t e r + perchloric acid
(XI) = 10 + 1) for 5 m i n . in a b l e n d o r a n d c e n t r i f u g e off t h e p r e c i p i t a t e . N e u t r a l i z e t h e s u p e r
n a t a n t fluid w i t h K O H a n d filter off t h e p r e c i p i t a t e o f p e r c h l o r a t e after a l l o w i n g t o s t a n d for
5 m i n . in a n ice b a t h . U s e t h e filtrate for t h e a s s a y .
I n o r g a n i c p h o s p h a t e is s t a b l e in s o l u t i o n .
Inorganic P h o s p h a t e 2237
Assay System
a t u r e : r e a d a g a i n s t air. B l a n k as for s a m p l e e x c e p t distilled w a t e r r e p l a c e s s a m p l e .
T h e c h a n g e in e x t i n c t i o n w h i c h o c c u r s o n a d d i t i o n o f G 6 P - D H s o l u t i o n c a n be d e t e r m i n e d b y
the further a d d i t i o n o f 0.01 m l . s o l u t i o n ( X ) at t h e e n d o f t h e r e a c t i o n . S u b t r a c t t h e r e s u l t i n g
extinction change.
I m i d a z o l e buffer (i) 2.40 ml. 39.5 m M

M g acetate solution en) 0.10 ml. 0.5 m M
E D T A solution an) 0.10 ml. 1 mM
N A D P solution (IV) 0.10 ml. 0.3 m M
A - 5 - M P solution (V) 0.01 m l . 0.01 m M
Glycogen solution (VI) 0.10 ml. 2 mg./ml.
Dithiothreitol solution (VII) 0.10 ml. 0.5 m M
P G l u M solution (IX) 0.01 m l . 16.5 /zg./ml. = 3.3 U / m l .
Phosphorylase a suspension (VIII) 0.01 m l . 16.5 / i g . / m l . = 3 3 0 m U / m l .
Sample (deproteinized and neutralized) 0.10 ml.
u p to 0.2 m M POl~
Mix, follow t h e c h a n g e in e x t i n c t i o n until constant
and read extinction E.
1
G 6 P - D H solution (X) 0.01 m l . 6.6 /xg./ml. = 9 2 0 m U / m l .
M i x , f o l l o w c h a n g e in e x t i n c t i o n until c o n s t a n t ( c a .
30 m i n . ) a n d r e a d e x t i n c t i o n E . 2
E 2 — E1 = AE S a m . S u b t r a c t f r o m this t h e A E o f t h e
blank: ^ E S a m — AE mk = AE is u s e d for t h e c a l c u l a -
tions.
Calculations
(2) on p . 312 applies. T h e results are obtained in yumole P per ml. sample. This value must be multi
}
plied by a factor if the sample has been deproteinized, neutralized or diluted in any way. T h e following
relationships h o l d :

c= AE x 4.98 AE x 4.89 AE x 8.81 [/miole/ml.]
Sources of Error
Insufficient purity of the reagents a n d the distilled water, in particular with regard to inorganic p h o s p h a t e ,
can give incorrect results (see also p . 2233).
Glycogen phosphorylase also reacts with arsenate, but n o G - l - P , a n d therefore n o N A D P H , is formed.

The assay is virtually specific.
Guynn et a l . a n d Scopes* have recently s h o w n t h a t inorganic p h o s p h a t e can be d e t e r m i n e d with glyceralde-

3
hyde-3-phosphate dehydrogenase (D-Gly cerald ehyde-3 -p hosphate: N A D oxidoreductase, phosphorylating,

EC 1.2.1.12), which also serves as the indicator enzyme, on the basis of the following r e a c t i o n s :
(1) Fructose-l,6-P 2
A L P
> DAP + GAP
(la) [DAP T 1 M
**>GAP]
I ^
(2) Pi + G A P + N A D * - g ^ P i L , 1,3-Diphosphoglycerate + N A D H + H +
(3) 1,3-Diphosphoglycerate + A D P P G K +
> 3-Phosphoglycerate + A T P
• I
I ~
(4) A T P + Fructose — • Fructose-6-P + ADP
T h e formation of N A D H is p r o p o r t i o n a l to the a m o u n t of Pj present. F o r further details, s e e ' .

3 4
References
1 E. N. Fawaz, L. Roth & G. Fawaz, Biochem. Z. 344, 212 [1966].

2 D. W. Schulz, J. V. Passonneau & O. H. Lowry, A n a l . Biochem. 19, 300 [1967].
3 R. W. Guynn, D. Veloso & R. L. Veech, A n a l . Biochem. 45, 277 [1972].
4 R. K. Scopes, A n a l . Biochem. 49, 88 [1972].
* A L D , Aldolase (Fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, E C 4.1.2.13).

** T I M , Triosephosphate isomerase (D-Glyceraldehyde-3-phosphate ketol-isomerase, E C 5.3.1.1).
P G K , Phosphoglycerate kinase ( A T P : 3-phospho-D-glycerate 1-phosphotransferase, E C 2.7.2.3).
+
+ +
H K , Hexokinase ( A T P : D-hexose 6-phosphotransferase, E C 2.7.1.1).
Inorganic Pyrophosphate
Karlfried G a w e h n
T h e determination of inorganic p y r o p h o s p h a t e in the presence of inorganic p h o s p h a t e is of i m p o r t a n c e

b o t h in biochemistry a n d in foodstuff chemistry.
Bailey 1
has described a m e t h o d for the d e t e r m i n a t i o n of inorganic p y r o p h o s p h a t e in p u r e solution, b u t
the m e t h o d does not distinguish between inorganic p h o s p h a t e a n d p y r o p h o s p h a t e .
Colorimetric Assay
The present m e t h o d based o n that of Josse , 2
allows the d e t e r m i n a t i o n of inorganic p h o s p h a t e in the pre
sence of p y r o p h o s p h a t e , followed by hydrolysis of the latter with inorganic p y r o p h o s p h a t a s e , P P a s e
(Pyrophosphate p h o s p h o h y d r o l a s e , E C 3.6.1.1) in a second assay. F o r further information on the m e c h a n
ism of action of the enzyme, s e e . 3
Application of Method: In biochemistry a n d in foodstuff chemistry.
Principle
(1) P 0 ~ + H 0 -25ss^ 2 H P O J -
2
4
2
The o r t h o p h o s p h a t e formed is determined photometrically at 578 n m by the m e t h o d of Fiske a n d Subbarow

as modified by Josse . 2
T h e hydrolysis of inorganic p y r o p h o s p h a t e (PPi) by PPase is quantitative a n d irreversible in the presence

of M g * . C a
2 2 +
, Co 2 +
and M n 2 +
inhibit the reaction. U n d e r o p t i m u m c o n d i t i o n s for the reaction ( p H 7.0;
3 - 4 m M PPi a n d 3 m M M g * ) the hydrolysis of PPi follows the kinetics of a 1st. order reaction until
2
ca. 90% is converted. T h e t u r n o v e r n u m b e r of the enzyme is d e p e n d e n t o n the ratio of PPi : Mg " "; u n d e r 2 1
o p t i m u m conditions it is 6 x 1 0 mole/min. F o r further properties of the enzyme, s e e .

4 3,4
Equipment
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r s u i t a b l e for m e a s u r e m e n t s at 5 7 8 n m ; b e n c h
centrifuge.
Reagents
1. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 7. S u l p h u r i c a c i d , A . R., 5 N
K H P 0 , crystalline, pure
2 4 8. A m m o n i u m m o l y b d a t e ,
2. P e r c h l o r i c a c i d , A . R . , 7 0 % ( w / w ) , (NH ) Mo 0
4 6 7 2 4 - 4 H 0 , A . R.
2
s p . gr. 1.67 9. 4-Methylaminophenolsulphate

3. P o t a s s i u m h y d r o x i d e , A . R . , 2 N 10. S o d i u m d i s u l p h i t e ( s o d i u m p y r o s u l p h i t e ) ,
4 . T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris N a S O , A . R.
2 2 s
5. H y d r o c h l o r i c a c i d , A . R . , 1 N 11. Inorganic p y r o p h o s p h a t a s e , P P a s e
6. M a g n e s i u m c h l o r i d e , M g C l - 6 H 0 , 2 2
from yeast, crystalline suspension in 3.2 M
A . R. a m m o n i u m sulphate solution: ^ 200 U / m g .
(25 °C). C o m m e r c i a l p r e p a r a t i o n , see p . 508.
Purity of Reagents
Reagents 2 to 11 must be free from inorganic p h o s p h a t e .
P r e p a r e all s o l u t i o n s w i t h fresh, d o u b l y distilled w a t e r . To a v o i d t h e g r o w t h o f m i c r o - o r g a n i s m s

sterilize the c o n t a i n e r s .
I. P h o s p h a t e s t a n d a r d (1 m M ) :
D i s s o l v e 136 m g . K H P 0 2 4 in d i s t i l l e d w a t e r in a v o l u m e t r i c flask a n d m a k e u p t o 1 1.
II. Tris buffer ( 5 0 m M ; p H 7 . 0 ) :
D i s s o l v e 0.6 g. tris in ca. 7 0 m l . d i s t i l l e d w a t e r , adjust t o p H 7.0 w i t h 1 N H Q a n d d i l u t e
III. M a g n e s i u m c h l o r i d e ( 1 0 0 m M ) :
D i s s o l v e 2 . 0 3 g. M g C l * 6 H 0 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l .
2 2
IV. A m m o n i u m m o l y b d a t e ( 2 . 5 % w / v ) :
D i s s o l v e 2.5 g. a m m o n i u m m o l y b d a t e in distilled w a t e r a n d m a k e u p t o 100 m l .
V. Reducing solution ( 1 % w/v 4-methylaminophenol sulphate; 2.7% sodium disulphite):
D i s s o l v e 1 g. 4 - m e t h y l a m i n o p h e n o l s u l p h a t e a n d 2.7 g. s o d i u m d i s u l p h i t e in d i s t i l l e d
water and m a k e u p to 100 ml.
VI. Phosphate reagent:
M i x 8 0 m l . distilled w a t e r , 2 0 m l . 5 N H S 0 , 2 0 ml. a m m o n i u m m o l y b d a t e s o l u t i o n
2 4
(IV) and 20 ml. reducing solution (V).

V I I . I n o r g a n i c p y r o p h o s p h a t a s e , P P a s e (1 m g . p r o t e i n / m l . ) :
VIII. Perchloric acid (5.8 N ) :
Mix 50 ml. cold water with 50 ml. perchloric acid.
Store solutions I and II, and enzyme suspension VII, stoppered at 4 °C; store all other solutions at r o o m
temperature. Prepare reagent VI for each series of measurements, solution V freshly each day and solution
II freshly every week. Enzyme suspension VII is stable for ca. 6 m o n t h s , a n d all other solutions are stable
indefinitely.
Procedure
Juice f r o m t i n n e d s a u s a g e s a n d o t h e r s a m p l e s l o w in p r o t e i n c a n b e u s e d directly for the a s s a y .
Deproteinization:
W h e r e n e c e s s a r y , a d d 0.1 v o l u m e p e r c h l o r i c a c i d ( V I I I ) t o b i o l o g i c a l fluids; homogenize

t i s s u e s , m e a t a n d s a u s a g e g o o d s w i t h 4 - t i m e s their w e i g h t ( w / v ) o f c o l d d i l u t e p e r c h l o r i c a c i d
(distilled w a t e r + p e r c h l o r i c a c i d ( V I I I ) = 10 + 1) for 5 m i n . in a m i x e r a n d c e n t r i f u g e off
Inorganic Pyrophosphate 2241
the r e s u l t i n g p r e c i p i t a t e . N e u t r a l i z e t h e s u p e r n a t a n t fluid w i t h K O H a n d after a l l o w i n g t o

s t a n d for 5 m i n . in ice filter off t h e p r e c i p i t a t e o f p o t a s s i u m p e r c h l o r a t e . U s e t h e filtrate for t h e
assay. The deproteinization with perchloric acid d o e s not cause any hydrolysis o f p y r o p h o s p h a t e .
I n o r g a n i c p y r o p h o s p h a t e in b i o l o g i c a l fluids a n d t i s s u e s is h y d r o l y s e d b y a n y P P a s e w h i c h
m a y b e p r e s e n t . A f t e r d e p r o t e i n i z a t i o n i n o r g a n i c p y r o p h o s p h a t e is r e a s o n a b l y s t a b l e in n e u t r a l
solution.
Assay System
W a v e l e n g t h : H g 578 n m ; light p a t h : 1 c m . ; i n c u b a t i o n v o l u m e : 3.0 m l . ; final v o l u m e : 10 m l . ;

r o o m temperature; read against blank containing distilled water instead o f sample.
1. D e t e r m i n a t i o n o f i n o r g a n i c p h o s p h a t e (P;):
P i p e t t e i n t o a test t u b e : C o n c e n t r a t i o n in a s s a y m i x t u r e

Sample (deproteinized and
neutralized) 0.50 ml. u p to ca. 0.2 m M P {

Phosphate reagent (VI) 7.00 ml.
M i x , a l l o w t o s t a n d f o r 10 m i n . , measure extinction
E t a n d r e a d off t h e c o r r e s p o n d i n g /rniole P f r o m t h e
f
standard curve.
2. D e t e r m i n a t i o n o f i n o r g a n i c p h o s p h a t e ( P ) p l u s i n o r g a n i c p y r o p h o s p h a t e
{ (PPi):

Sample (deproteinized and
neutralized) 0.50 ml. u p to ca. 0.2 m M P { + PPj
MgCl 2 solution (III) 0.04 ml. 1.3 m M
PPase suspension (VII) 0.01 m l . 3.3/xg./ml. = 660 m U / m l .
M i x a n d i n c u b a t e for c a . 5 m i n .
Phosphate reagent (VI) 7.00 ml.
M i x , a l l o w t o s t a n d for 10 m i n . , m e a s u r e e x t i n c t i o n
E a n d read off t h e c o r r e s p o n d i n g /rniole Pj f r o m t h e
2
standard curve.
To p r e p a r e the p h o s p h a t e s t a n d a r d c u r v e s d i l u t e 0 . 4 m l . , 0.6 m l . , 0.8 m l . p h o s p h a t e s t a n d a r d

s o l u t i o n (I), c o r r e s p o n d i n g t o 0 . 4 , 0 . 6 , 0.8 /rniole, t o 3.0 m l . w i t h d i s t i l l e d w a t e r . A f t e r a d d i t i o n
o f 7.0 m l . p h o s p h a t e r e a g e n t ( V I ) m i x , a l l o w t o s t a n d for 10 m i n . M e a s u r e t h e e x t i n c t i o n
(ordinate) against the blank (distilled water instead o f p h o s p h a t e standard) and plot against
/rniole p h o s p h a t e ( a b s c i s s a ) .
Calculations
U n d e r the above conditions the Pi concentratio n of the sample (/zmole/ml.) is obtained by reading off the
value for 2 E x from the s t a n d a r d curve, similarly the value for 2 E 2 gives the P P 4 + P } concentration
(/miole/ml.).
The PPj concentration (/imole/ml.) is o b t a i n e d by subtraction of 2 E from 2 E a n d division by 2 (2 mole
t 2
P formed per mole PPj). Multiplication by 173.96 gives the c o n c e n t r a t i o n in (/zg./ml.).

4
In the juice of tinned sausages a m e a n value of 8708 /miole Pj/1. (2 s = 691 /miole) a n d 326.2 /miole PPj/1.
(2 s = 5 9 . 3 /miole) was found. T h e c o r r e s p o n d i n g coefficient of variation for Pj = 4 % a n d for PPj = 9.1 %.
N o r m a l Values
N o n e available.
Insufficient purity of the reagents a n d the water (see p. 2240), especially with regard to the content of
inorganic p h o s p h a t e , can give false values.
Inorganic p y r o p h o s p h a t a s e is specific for inorganic p y r o p h o s p h a t e . O r g a n i c p y r o p h o s p h a t e esters, such

as A D P , A T P or T P P are not hydrolysed.
UV-Assay
Uridine-5'-diphosphate glucose p y r o p h o s p h o r y l a s e ( U T P : a - D - g l u c o s e - l - p h o s p h a t e uridylyltransferase,
E C 2.7.7.9), because of its specificity, is n o t only suitable for the d e t e r m i n a t i o n of uridine t r i p h o s p h a t e
a n d uridine d i p h o s p h a t e glucose (see p . 2172), but also allows the specific d e t e r m i n a t i o n of inorganic
pyrophosphate with the aid of p h o s p h o g l u c o m u t a s e (a-D-Glucose-l,6-bisphosphate:a-D-glucose-l-
p h o s p h a t e phosphotransferase, E C 2.7.5.1) a n d glucose-6-phosphate dehydrogenase (D-Glucose-6-
p h o s p h a t e : N A D P 1-oxidoreductase, E C 1.1.1.49) as auxiliary a n d indicator enzymes.
Principle
UDPG - pyrophosphorylas^ T j y p _|_ Q _ \ _ p

(1) PPi + U D P G
PGluM
(2) G-l-P G -1,6-P
G-6-P
2
G6P-DH
(3) G-6-P + N A D P +
Gluconate-6-P + N A D P H + H +
T h e increase in the c o ncentratio n of N A D P H , as measured by the change in extinction at 340 (334, 365)
n m , is p r o p o r t i o n a l to the q u a n t i t y of inorganic p y r o p h o s p h a t e .
Though the equilibrium constant of the U D P G P reaction is K = 0.3 and that of the P G l u M reaction is
K = 17.2, stoichiometric reaction of inorganic pyrophosphate is achieved, since the equilibrium of reaction
(3) lies almost completely on the right. Since the U D P G pyrophosphorylase from bovine liver is inhibited
by sulphate ions, the auxiliary and indicator enzymes must be dialysed to free them from ammonium
sulphate.
Apparatus
S p e c t r o p h o t o m e t e r o r s p e c t r u m - l i n e p h o t o m e t e r c a p a b l e o f m e a s u r e m e n t at 3 4 0 ( 3 3 4 o r 3 6 5 )
n m ; laboratory centrifuge.
Reagents
1. T r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e , tris 8. P h o s p h o g l u c o m u t a s e , P G l u M
2. U r i d i n e d i p h o s p h a t e g l u c o s e , U D P G from rabbit muscle, suspension in 3.2 M am
disodium salt U D P G - N a ; commercial prep
2 monium sulphate solution; ^ 200 U/mg. (25 °C),
arations, see p. 555. commercial preparations, see p. 499.
2 2 9. U r i d i n e d i p h o s p h a t e g l u c o s e p y r o p h o s
4. N i c o t i n a m i d e - a d e n i n e dinucleotide p h o s phorylase, U D P G P
phate, N A D P from bovine liver, solution in 50% (v/v) glycerol;
disodium salt N A D P - N a H , commercial prep
2 ^ 100 U/mg. (25 °C). Commercial preparations,
arations, see p. 546. see p. 519.
5. G l u c o s e - 1 , 6 - d i p h o s p h a t e , G - l , 6 - P 2 10. S o d i u m d i h y d r o g e n p h o s p h a t e ,
tetracyclohexylammonium salt G-l,6-P -2 NaH P0 H 0
2 4 2
( C H A ) - 4 H 0 ; commercial preparations, see

4 2 11. D i s o d i u m h y d r o g e n p h o s p h a t e ,
p. 537. Na HP0 -2H 0
2 4 2
6. H y d r o c h l o r i c a c i d A . R., 1 N 12. S o d i u m a c e t a t e , C H C O O N a - 3 H 0
3 2
7. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , 13. A c e t i c a c i d , A . R . , 0 . 0 5 M
G6P-DH
from yeast, suspension in 3.2 M ammonium
sulphate solution; ^ 1 4 0 U/mg. (25 °C). Com
mercial preparations, see p. 458.
The enzymes used must be substantially free from N A D P H oxidase.
M a k e u p all s o l u t i o n s w i t h f r e s h l y p r e p a r e d d o u b l y d i s t i l l e d w a t e r .
I. Tris buffer ( 5 0 m M ; p H 8 . 2 ) :
D i s s o l v e 0.6 g. t r i s - h y d r o x y m e t h y l - a m i n o m e t h a n e in 8 0 m l . w a t e r , a d j u s t t o p H 8.2
with 1 N HC1, and m a k e u p to 100 ml. with water.
II. U D P G s o l u t i o n ( 2 9 m M ) :
Dissolve 20 mg. U D P G - N a 2 in 1 m l . w a t e r .
III. M a g n e s i u m c h l o r i d e ( 0 . 2 M ) :
D i s s o l v e 4 . 0 6 g. M g C l * 6 H 0 in w a t e r a n d m a k e u p t o 100 m l .
2 2
I V . N A D P s o l u t i o n ( a p p r o x . 11 m M ) :
D i s s o l v e 10 m g . N A D P - N a H in 1 m l . w a t e r .
2
V. G - l , 6 - P 2 solution (approx. 1 m M ) :
D i s s o l v e 1 m g . G - 1 , 6 - P - ( C H A ) - 4 H 0 in 1 m l . w a t e r .
2 4 2
VI. G 6 P - D H (2 m g . / m l . ) :
D i a l y s e s u s p e n s i o n in a m m o n i u m s u l p h a t e s o l u t i o n a g a i n s t 0 . 0 5 M p h o s p h a t e buffer
p H 7.6 ( I X ) at 4 ° C . A d j u s t w i t h t h e s a m e buffer t o 2 m g . p r o t e i n / m l .
VII. P G l u M (2 m g . / m l . ) :
C e n t r i f u g e s u s p e n s i o n in a m m o n i u m s u l p h a t e s o l u t i o n . T a k e u p p r e c i p i t a t e w i t h 0 . 0 5 M
a c e t a t e buffer, p H 5.3 ( X ) , d i a l y s e a g a i n s t t h e s a m e buffer at 4 ° C , a n d adjust w i t h t h e
s a m e buffer t o 2 m g . p r o t e i n / m l .
V I I I . U D P G p y r o p h o s p h o r y l a s e (5 m g . / m l . ) :
U s e s o l u t i o n in 5 0 % g l y c e r o l w i t h o u t d i l u t i o n .
I X . P h o s p h a t e buffer ( 0 . 0 5 M ; p H 7 . 6 ) :
D i s s o l v e 6.9 g. N a H P 0 2 4 • H 0 in w a t e r a n d m a k e u p t o 1 litre; d i s s o l v e 8.9 g. N a H P 0 •
2 2 4
• 2 H 0 in w a t e r a n d m a k e u p t o 1 litre. M i x s o l u t i o n s t o g i v e p H 7.6.
2
X . A c e t a t e buffer ( 0 . 0 5 M ; p H 5 . 3 ) :
D i s s o l v e 6.8 g. C H C O O N a - 3 H 0 in w a t e r a n d m a k e u p t o 1 litre. A d j u s t t o p H 5.3
3 2
w i t h 0.05 M a c e t i c a c i d .
Keep solutions I, II, IV, a n d V in stoppered containers at 4 °C, a n d solution III at r o o m temperature. Store
enzyme solutions VI a n d VII frozen at - 20 °C, a n d solution VIII at 4 °C. E n z y m e solutions VI and VII
are stable for a b o u t 2 weeks, a n d solution VIII for a b o u t 6 m o n t h s . Solutions I, II, IV, a n d V must be
freshly prepared every week, whereas solution III keeps indefinitely.
Procedure
Collection, Treatment and Stability of Sample, see p . 2240.

Assay System
ature. M e a s u r e m e n t a g a i n s t air.
Pipette into cuvettes C o n c e n t r a t i o n in a s s a y m i x t u r e

UDPG (II) 0.20 ml. 1.9 m M
MgCl 2 (III) 0.10 ml. 6.7 m M
NADP (IV) 0.10 ml. 0.36 m M
G-l,6-P 2 (V) 0.05 ml. 17 fiM
G6P-DH (VI) 0.01 m l . 6.7 /xg./ml. = 9 3 2 m U / m l .
PGluM (VII) 0.02 ml. 1 3 . 3 /zg./ml. = 2.7 U / m l .
Sample 0.10 ml. u p to 0.2 m M P^
M i x , and w h e n reaction stops read extinction E . x
UDPGP (VIII) 0.02 ml. 3 3 / / g . / m l . = 3.3 U / m l .
Mix, and w h e n reaction stops read extinction E .

2
AE = E — E .2 l Take into a c c o u n t extinction due to

UDPGP.
Calculations
The reaction proceeds stoichiometrically u n d e r the conditions indicated. F o r m u l a (2), (3) on page 312 is
therefore valid. T h e result is obtained as pmole of PPj/ml. of sample. If the sample has been deproteinized,
neutralized, or otherwise diluted, this value must be multiplied by a c o r r e s p o n d i n g factor.

c = ZIE x 4.92 AE x 4.82 AE x 8.70 [//mole/ml.]
c = AE x 856 AE x 839 AE x 1513 [/ig./ml.]
Specificity, A c c u r a c y and P r e c i s i o n
The m e t h o d described has n o t yet been investigated for specificity a n d sources of error. M o r e o v e r , it has
not yet been used in routine work. N o d a t a regarding precision can therefore be given.
References
1 K. Bailey in H. U. Bergmeyer: M e t h o d s of enzymatic analysis. 1st. Edn., Verlag Chemie, Weinheim

a n d Academic Press, N e w York a n d L o n d o n 1963, p . 644.
2 J. Josse, J. biol. C h e m . 241, 1938 [1966].
3 M. Kunitz & P. W. Robbins in S. P. Colowick & N. O. Kaplan: T h e E n z y m e s . A c a d e m i c Press, N e w York
1961, Vol. 5, p . 169.
4 M. Kunitz, J. G e n . Physiol. 35, 423 [1952].
Inorganic Peroxides
Erich Bernt and H a n s Ulrich Bergmeyer
Inorganic peroxides are d e c o m p o s e d by peroxidase, P O D ( D o n o r : hydrogen peroxide oxidoreductase,

E C 1.11.1.7) a n d the oxygen liberated in this process oxidizes colourless hydrogen d o n o r s to coloured
c o m p o u n d s (e. g. o-dianisidine, o-, m - or p-toluidine). A r o m a t i c amines are the usual hydrogen d o n o r s ,
but amine derivatives, phenols, q u i n o n e s , etc., can be used.
Application of Method: In inorganic chemistry, biochemistry and foodstuff chemistry.
Principle
(1) H 0
2 2 + DH 2 2H 0 + D
2
o-Dianisidine which is used in this m e t h o d yields a red-brown dye with a b r o a d a b s o r p t i o n m a x i m u m

at 460 n m . T h e extinction coefficient d e p e n d s o n the experimental conditions, therefore the measured
extinction is c o m p a r e d to the extinction of an H 0 2 2 s t a n d a r d . T h e m e a s u r e m e n t s are m a d e at 436 n m or
an adjacent wavelength. T h e reaction is complete in 3 min. a n d the colour is stable for several h o u r s .
T h e equilibrium at p H 7 is completely o n the side of water a n d the dye. P O D shows m a x i m u m activity

between p H 4 a n d 8. T h e a m o u n t of o-dianisidine present in the assay mixture is sufficient for the con
centration range indicated below.
Equipment
P h o t o m e t e r s u i t a b l e for m e a s u r e m e n t s at 4 0 0 - 4 6 0 n m .
Reagents
1. S o d i u m d i h y d r o g e n p h o s p h a t e , 4. P e r o x i d a s e , P O D
NaH P0 -2H 0
2 4 2 from horse radish, lyophilized; ^36 U/mg.
2. D i s o d i u m h y d r o g e n p h o s p h a t e , (25 °C). C o m m e r c i a l p r e p a r a t i o n , see p . 494.
Na HP0 -2H 0
2 4 2 5. H y d r o g e n p e r o x i d e * , A . R . , c a . 35 % ( w / w )
3. o - D i a n i s i d i n e h y d r o c h l o r i d e 6. P e r c h l o r i c acid, A. R., sp. gr. 1.67;
ca. 7 0 %
Purity of Reagents
T h e P O D should have a K 4 value** of at least 35000. This is equivalent to an activity of ca. 36 U / m g .

with guaiacol as substrate. T h e enzyme m u s t n o t contain a m i n o oxidases.
* e. g. Perhydrol from E. Merck, D a r m s t a d t , G e r m a n y .

1 x
** According t o K 1
4 = x — (a G = initial concentration of guaiacol in the assay mixture,
a e t Q x
e = enzyme concentration, — = hydrogen peroxide decomposed per s e c ) .
I n o r g a n i c Peroxides 2247
I. B u f f e r - e n z y m e s o l u t i o n ( 0 . 1 2 M p h o s p h a t e buffer, p H 7 ; 4 0 pg. POD/ml.):

D i s s o l v e 2 . 0 7 g. N a H P 0 - 2 H 0 , 1.09 g. N a H P 0 - 2 H 0 a n d 4 m g . P O D in distilled
2 4 2 2 4 2
w a t e r a n d m a k e u p t o 100 m l .
II. C h r o m o g e n (5 m g . o - d i a n i s i d i n e h y d r o c h l o r i d e / m l . ) :
D i s s o l v e 10 m g . o - d i a n i s i d i n e h y d r o c h l o r i d e in 2 m l . distilled w a t e r .
III. P e r o x i d e r e a g e n t :
A d d 0.5 m l . c h r o m o g e n s o l u t i o n (II) t o 5 0 m l . b u f f e r - e n z y m e s o l u t i o n (I) w i t h v i g o r o u s
stirring. S t o r e t h e m i x t u r e in a d a r k b o t t l e . P r e v e n t t h e g r o w t h o f b a c t e r i a b y t h e a d d i t i o n
of a few drops of chloroform.
I V . H y d r o g e n p e r o x i d e s t a n d a r d s o l u t i o n ( 2 0 pg. H 0 / m l . ) :
2 2
a) D i l u t e 1.00 m l . c a . 3 5 % h y d r o g e n p e r o x i d e s o l u t i o n in a 2 5 0 m l . v o l u m e t r i c flask
to the mark with distilled water. C h e c k the H 0 2 2 concentration: dilute 20.00 ml. o f
t h i s s o l u t i o n w i t h 3 0 m l . d i s t i l l e d w a t e r a n d 5 m l . c a . 1 N H S 0 , a n d titrate w i t h
2 4
0.1 N K M n 0 4 s o l u t i o n t o a p e r m a n e n t p i n k c o l o u r . 1.00 m l . 0.1 N K M n 0 4 is e q u i v a l e n t

t o 1.70 m g . h y d r o g e n p e r o x i d e .
b ) A c c o r d i n g t o t h e r e s u l t s o f t h e t i t r a t i o n , d i l u t e t h e a p p r o p r i a t e v o l u m e ( b e t w e e n 10
a n d 2 0 m l . ) o f s o l u t i o n a) t o 1 0 0 0 m l . w i t h d i s t i l l e d w a t e r .
V. Perchloric acid (1.0 N ) :
Store all solutions, stoppered, in a refrigerator at 0 to 4 °C. P r e p a r e the peroxide reagent (solution III)
freshly each w e e k ; always p o u r solution (I) d o n o t pipette. If solution III becomes t u r b i d it must be
discarded. Always p r e p a r e the hydrogen peroxide s t a n d a r d solution just before use by dilution of the
ca. 3 5 % (w/w) solution which is stable.
Procedure
Collection:
Dilute solutions o f h y d r o g e n peroxide, s o d i u m peroxide, m a g n e s i u m peroxide, s o d i u m per

borate, percarbonate, urea peroxide, strontium peroxide, etc., to a concentration o f 5 - 5 0 pg.
H 0 / m l . Clarify t u r b i d s o l u t i o n s , f a t - c o n t a i n i n g f r a c t i o n s o r e x t r a c t s o f f o o d s t u f f s b y
2 2 filtra
t i o n ; t h e fat r e m a i n s o n t h e filter p a p e r . If t h e s a m p l e is c o l o u r e d b y p r o t e i n it m u s t b e d e p r o t e i n i
zed with perchloric acid.
Deproteinization :
P i p e t t e s u c c e s s i v e l y i n t o a 10 m l . c e n t r i f u g e t u b e 1 m l . p e r c h l o r i c a c i d ( s o l u t i o n V ) a n d 1 m l .
s a m p l e . M i x t h o r o u g h l y w i t h a t h i n g l a s s r o d , c e n t r i f u g e for 5 - 1 0 m i n . at c a . 3 0 0 0 g, p o u r
off t h e c l e a r s u p e r n a t a n t fluid i n t o a test t u b e a n d u s e 0.1 m l . f o r t h e a s s a y .
2248 M e t a b o l i t e s : Miscellanous Substrates a n d Effectors
B e c a u s e o f the i n s t a b i l i t y o f d i l u t e p e r o x i d e s o l u t i o n s s a m p l e s m u s t b e w o r k e d u p a n d a n a l y s e d
w i t h i n 2 - 3 hr.
Assay System
W a v e l e n g t h : H g 4 3 6 n m ( 4 2 0 t o 4 8 0 n m ) ; light p a t h : 1 c m . ; final v o l u m e : 2.6 m l . ; r o o m t e m p e r a

ture. P r e p a r e a r e a g e n t b l a n k c o n t a i n i n g w a t e r i n s t e a d o f s a m p l e a n d a p e r o x i d e s t a n d a r d
c o n t a i n i n g 0.1 m l . H 0 2 2 s t a n d a r d s o l u t i o n I V b i n s t e a d o f s a m p l e for e a c h series o f m e a s u r e
m e n t s . R e a d a g a i n s t t h e r e a g e n t b l a n k . E q u i l i b r a t e t h e p e r o x i d e r e a g e n t ( s o l u t i o n III) t o r o o m
temperature before use.
Peroxide reagent (III) 2.50 ml. 115 m M p h o s p h a t e , 0 . 0 0 5 %

o - d i a n i s i d i n e ; 4 0 pg. POD/ml.
= 1.4 U / m l .
Sample or standard solution (IV b) 0.10 ml. u p t o 6 0 pM
M i x , a l l o w t o s t a n d for 5 m i n . at r o o m t e m p e r a t u r e
a n d read e x t i n c t i o n s E s a m p l e and E s t a n d a r d against the
blank.
Calculations
S t a n d a r d curves are linear u p to ca. 10 pg. (ca. 0.3 pmole) H 0 / a s s a y mixture. W i t h extinctions over
2 2
0.600 dilute the sample with water a n d analyse again.

F o r the calculations the extinction of the sample is c o m p a r e d to that of the peroxide standard. This
contains 2 pg. H 0 / a s s a y mixture a n d therefore with a sample volume of 0.1 ml. the concentration is:
2 2
c = ^ S A M
P L E
x 20 = [/ig./ml.]
^standard
A n y preliminary dilution of the sample must be allowed for in the calculations.
T h e standard deviation (S. D.) of the m e t h o d for a mean value of 20 pg. H 0 / m l . sample was 0.05 pg. 2 2
H 0 / m l . The coefficient of variation is 2 . 5 % .

2 2
Specificity and S o u r c e s o f Error
Peroxidase is specific for inorganic peroxides. T h e analytical results are n o t reproducible for organic
peroxides and therefore they c a n n o t be determined with this m e t h o d .
References
1 P. George, J. biol. C h e m . 201, 413 [1953].

2 B. Chance & A. S. Maehly in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology, vol. II, p . 764.
Academic Press, N e w Y o r k 1955.
Organophosphorus and Carbamate Insecticides
Paul A. Giang
O r g a n o p h o s p h o r u s a n d c a r b a m a t e insecticides inhibit the cholinesterase C h E ( E C 3.1.1.8) of animals

and insects. This accounts b o t h for their toxicity to w a r m - b l o o d e d animals a n d to a large extent for their
effectiveness in the c o n t r o l of insects. M a n y of these insecticides, a n d also some of their oxidation a n d
metabolic p r o d u c t s , are such powerful inhibitors of cholinesterase t h a t very sensitive enzymatic m e t h o d s
for their micro-determination have been developed on the basis of this inhibitory p r o p e r t y .
T h e most widely used m e t h o d in which the inhibition of an enzyme is used for the analysis of i n s e c t i c i d e s 1,2
is described below. It is based on Michel's 21

simplified m e t h o d for the estimation of cholinesterase activity
in h u m a n red blood cells a n d plasma. T h e cholinesterase used in this m e t h o d m u s t be carefully s t a n d a r d
ized to avoid any interference from other related e s t e r a s e s 4 - 6
.
Application of Method: In agriculture a n d food chemistry.
Principle
(1) C h E + Inhibitor • Inhibited C h E + Free C h E
(2) Acetylcholine + H 0 2
f r e e C h E
> Acetic acid + Choline
The less cholinesterase is inhibited in reaction (1), the m o r e acetic acid will be formed in reaction (2)
during the specified time of hydrolysis.
The sample is extracted with an organic solvent, the solvent is evaporated off a n d the residue is incubated
for 30 min. with a k n o w n excess of cholinesterase ( C h E ) in a buffered solution. At the end of this "inhibit
i o n " period, a k n o w n excess of acetylcholine is a d d e d to the reaction mixture. After 60 min. the acetic
acid produced by the hydrolysis of acetylcholine is determined from the change in p H (measured with
a pH-meter).
Equipment
p H - m e t e r ; c o n s t a n t t e m p e r a t u r e b a t h ; c r y s t a l l i z a t i o n d i s h e s (9 c m . d i a m e t e r ) a s s m a l l c o n s t a n t
t e m p e r a t u r e b a t h s ; m a g n e t i c stirrer ( m a g n e t i c flea: i r o n wire s e a l e d in g l a s s ; 1 c m . l o n g , 2 m m .
o u t s i d e d i a m e t e r ) ; 10 m l . b e a k e r s , e a c h e n c l o s e d b y a l e a d - s o l d e r wire c o i l t o h o l d t h e b e a k e r
in p l a c e in t h e w a t e r b a t h ; s y r i n g e s (2 a n d 25 m l . ) ; s t o p w a t c h . F i g . 1 s h o w s t h e a r r a n g e m e n t
o f t h e w a t e r b a t h s , t h e r m o s t a t a n d m a g n e t i c stirrers.
Reagents
1. 5 , 5 - D i e t h y l b a r b i t u r i c a c i d 6. S o d i u m c h l o r i d e , N a C l
(Veronal, barbital) 7. D i c h l o r o m e t h a n e , r e d i s t i l l e d ,
2. S o d i u m h y d r o x i d e , 1 N CH C1 2 2
3. P o t a s s i u m c h l o r i d e , K C 1 8. A c e t y l c h o l i n e c h l o r i d e
4. P o t a s s i u m d i h y d r o g e n p h o s p h a t e , 9. Cholinesterase
KH P0 2 4 e. g. from Winthrop-Stearns Inc., from bovine
5. H y d r o c h l o r i c a c i d , 0.1 N erythrocytes; ;>46 U / m g . (37 °C).
T h e s u b s t r a t e a n d buffer c o n c e n t r a t i o n s d e s c r i b e d b e l o w are s u i t a b l e for t h e purified b o v i n e

and h u m a n serum cholinesterase that can be obtained from Winthrop-Stearns, Inc., N e w
Y o r k 18, N . Y . 1 0 0 1 8 . B y s u i t a b l e a l t e r a t i o n o f t h e s u b s t r a t e a n d buffer c o n c e n t r a t i o n s t h e
following preparations can be used: " L y o v a c " plasma (Sharpe & D o h m e , Philadelphia, Pa.
19100); lyophilized horse serum (Schwarz Bio-Research, Inc., Orangeburg, N . Y. 10962);
h u m a n p l a s m a ( o l d b l o o d f r o m b l o o d b a n k s o r h o s p i t a l s ) " . F o r o t h e r r e a g e n t s , see " P r o c e
7 9
dure".
Fig. 1. D i a g r a m of the c o n s t a n t t e m p e r a t u r e a p p a r a t u s :
A = cooling w a t e r ; B = t h e r m o m e t e r ; C = stirrer; D = heating
element with t h e r m o s t a t ; E = compressed air; F = small, in
sulated c o n s t a n t t e m p e r a t u r e b a t h s (crystallization dishes);
G = magnetic stirrer; H = c o p p e r p i p e ; I = large constant
t e m p e r a t u r e water b a t h .
I. V e r o n a l buffer ( 3 6 m M v e r o n a l ; 8 m M p h o s p h a t e ; 1.2 M K C 1 ; p H 8 . 1 ) :
A d d 6 . 6 4 7 g. v e r o n a l t o 8 0 0 m l . d i s t i l l e d w a t e r w i t h stirring, a n d a d d 36 m l . 1 N N a O H t o
d i s s o l v e the v e r o n a l . A d d 8 9 . 9 g. K C 1 a n d 1.089 g. K H P 0 2 4 t o this s o l u t i o n . A d j u s t t h e
p H o f t h e s o l u t i o n t o 8.1 w i t h c a . 7 m l . 0.1 N H C 1 , a n d d i l u t e t o 1 0 0 0 m l . w i t h d i s t i l l e d
w a t e r ( v o l u m e t r i c flask). A d d 2 d r o p s o f t o l u e n e .
II. S o d i u m c h l o r i d e ( 0 . 9 % w / v ) :
D i s s o l v e 0.9 g. N a C l in 1 0 0 m l . d i s t i l l e d w a t e r , sterilize t h e s o l u t i o n ( h e a t t o b o i l i n g ) a n d
p o u r i n t o sterile b o t t l e s .
III. A c e t y l c h o l i n e ( 0 . 2 2 M ) :
D i s s o l v e 4 g. a c e t y l c h o l i n e c h l o r i d e in 100 m l . distilled w a t e r a n d a d d 2 d r o p s o f t o l u e n e .
IV. Cholinesterase
a) S t o c k s o l u t i o n ( c a . 4 2 U / m l . ) :
I n t r o d u c e i n t o a vial c o n t a i n i n g 1 0 0 0 U o f dried c h o l i n e s t e r a s e 2 2 m l . o f i c e - c o l d , sterile
N a C l s o l u t i o n (II) w i t h a sterile 25 m l . s y r i n g e ( p u n c t u r e t h e s t o p p e r o f t h e vial) a n d m i x
by s h a k i n g . I m m e d i a t e l y p l a c e t h e s o l u t i o n in a refrigerator (0 t o 5 ° C ) . T h e activity o f
this s t o c k s o l u t i o n m u s t b e d e t e r m i n e d (see " D e t e r m i n a t i o n o f t h e A c t i v i t y o f t h e C h o
linesterase Stock Solution").
b) D i l u t e s o l u t i o n (ca. 1 U / m l . ) :
A d d t o 1.0 m l . o f t h e s t o c k s o l u t i o n t h e v o l u m e o f 0 . 9 % N a C l s o l u t i o n c a l c u l a t e d f r o m t h e
Organophosphorus and Carbamate Insecticides 2251
results o f t h e c h o l i n e s t e r a s e a c t i v i t y a s s a y (p. 2 2 5 3 ) . T h e d i l u t e s o l u t i o n s h o u l d d e c r e a s e
the p H o f t h e c o n t r o l m i x t u r e C 2 b y 2 u n i t s u n d e r t h e c o n d i t i o n s g i v e n u n d e r " A s s a y
S y s t e m " . A d d 2 d r o p s o f t o l u e n e t o t h e d i l u t e s o l u t i o n a n d s t o r e at 0 t o 5 ° C .
V . C h o l i n e s t e r a s e buffer m i x t u r e :
M i x equal parts of the cooled solutions I and IVb immediately before use.
F o r other solutions see " O x i d a t i o n o f Insecticides."
S t o r e t h e a c e t y l c h o l i n e a n d buffer s o l u t i o n s in a refrigerator. T h e c h o l i n e s t e r a s e s o l u t i o n s k e e p
for 6 m o n t h s at 0 t o 5 ° C w i t h o u t a n y a p p r e c i a b l e l o s s o f a c t i v i t y .
Procedure
Extract
Additional reagents:
Dichloromethane
Active charcoal (Nuchar)
S o d i u m sulphate, N a S 0 , anhydrous
2 4
S o d i u m chloride, 10% (w/v)
C u t t h e s a m p l e i n t o s m a l l p i e c e s a n d c h o p in a m i x e r (2 m i n . ) w i t h 2 m l . o f d i c h l o r o m e t h a n e p e r
g. o f s a m p l e . F i l t e r t h r o u g h a l a r g e f u n n e l w i t h a p l u g o f c o t t o n w o o l . If t h e e x t r a c t c o n t a i n s
p l a n t p i g m e n t s , s h a k e w i t h a f e w g r a m s o f a 1 : 1 m i x t u r e o f c h a r c o a l (Nuchar) and anhydrous
s o d i u m s u l p h a t e , a n d filter t h r o u g h a f o l d e d filter. M e a s u r e v o l u m e . C o n c e n t r a t e the e x t r a c t
t o a b o u t 5 0 m l . in a c u r r e n t o f air. W a s h in a 125 m l . s e p a r a t i n g f u n n e l w i t h 10 m l . p o r t i o n s o f
10% N a C l s o l u t i o n until t h e w a s h i n g s are n e u t r a l t o l i t m u s . F i l t e r t h e e x t r a c t t h r o u g h a Gooch
c r u c i b l e c o n t a i n i n g a b o u t 10 g. o f a n h y d r o u s s o d i u m s u l p h a t e i n t o a 100 m l . g r a d u a t e d flask.
M a k e u p t o a l m o s t 1 0 0 m l . w i t h d i c h l o r o m e t h a n e , p l a c e g r a d u a t e d flask in ice b a t h for 2 0 m i n . ,
m a k e u p t o 100 m l . w i t h i c e - c o l d d i c h l o r o m e t h a n e , a n d m i x .
D e t e r m i n e t h e a p p r o x i m a t e i n s e c t i c i d e c o n t e n t o f t h e s o l u t i o n a s f o l l o w s : p i p e t t e i n t o a 10 m l .
b e a k e r c o n t a i n i n g a m a g n e t i c flea
2 ml. ice-cold sample solution
a n d e v a p o r a t e t o d r y n e s s i n a c u r r e n t o f air. A d d
6 ml. cholinesterase-buffer mixture (V)
a n d stir f o r 3 0 m i n . i n a w a t e r b a t h at 2 5 ° C . M i x i n
0.6 ml. acetylcholine s o l u t i o n (III),
c o n t i n u e t o stir at 2 5 ° C a n d after e x a c t l y 10 m i n . , d e t e r m i n e t h e p H o f t h e s o l u t i o n . S u b t r a c t
t h e r e a d i n g f r o m p H 8.0 a n d m u l t i p l y t h e difference b y 6 ( 1 0 m i n . —• 6 0 m i n . ) . W i t h t h i s v a l u e
obtain the a p p r o x i m a t e insecticide content o f the sample solution from a standard curve
c o n s t r u c t e d for t h e p a r t i c u l a r i n s e c t i c i d e . D i l u t e t h e s o l u t i o n o f t h e s a m p l e s o t h a t it c o n t a i n s ,
e . g . a b o u t 0 . 0 1 5 fig. P a r a o x o n / m l . ( F i g . 3 ) .
2252 Metabolites: Miscellanous Substrates and Effectors
Oxidation of Insecticide
P r a c t i c a l l y all t h e k n o w n o r g a n o p h o s p h o r u s i n s e c t i c i d e s a r e t h i o - o r d i t h i o p h o s p h a t e s . T h e s e
c o m p o u n d s are n o r m a l l y o x i d i z e d in vivo t o their r e s p e c t i v e o x y g e n a n a l o g u e s o r t o their
s u l p h o x i d e s o r s u l p h o n e s , w h i c h a r e p o w e r f u l i n h i b i t o r s o f c h o l i n e s t e r a s e . T h e r e are f o u r
m e t h o d s f o r t h e in vitro o x i d a t i o n o f i n s e c t i c i d e s , w h i c h a r e s u i t a b l e f o r all t h e e n z y m a t i c
procedures.
Additional reagents:
Bromine water, saturated Nitric acid, cone, a n d fuming

N-Bromosuccinimide S o d i u m bicarbonate, 10% solution
Phenol m-Chloroperbenzoic acid
Chloroform FMC-Corp., Carteret, N . J. 07008
Benzene Sodium pyrosulphite, N a S 0 2 2 5
Hydrogen peroxide, 30% 0.5% solution
Glacial acetic acid S o d i u m chloride, saturated solution
L i q u i d paraffin (heat 40 g. NaCl with 100 ml. H 0 , allow to 2
cool, and decant)
1. O x i d a t i o n w i t h b r o m i n e o r N - b r o m o s u c c i n i m i d e 1 0
" 1 2
: A d d 0.4 ml. saturated bromine water
to 100 m l . distilled w a t e r . A d d 1 m l . o f this s o l u t i o n t o t h e d r y r e s i d u e f r o m t h e d i c h l o r o m e t h a n e
e x t r a c t o f t h e s a m p l e i m m e d i a t e l y b e f o r e t h e a d d i t i o n o f buffer a n d e n z y m e ( s e e u n d e r " A s s a y
S y s t e m " ) . T h e b r o m i n e c o n t e n t o f t h e b r o m i n e w a t e r is n o t critical, s i n c e t e n - f o l d v a r i a t i o n s
give t h e s a m e results.
B r o m i n e reacts i m m e d i a t e l y w i t h m o s t t h i o p h o s p h a t e s . C e r t a i n c o m p o u n d s , s u c h a s D e m e -
ton (0,0-diethyl-0-[2-(ethylthio)ethyl] thiophosphate) or Sulfotepp (tetra-ethyl dithiopyro-
p h o s p h a t e ) a r e e x c e p t i o n s , a s t h e y c a n n o t b e c o n v e r t e d t o c h o l i n e s t e r a s e i n h i b i t o r s either b y
b r o m i n e w a t e r o r b y N - b r o m o s u c c i n i m i d e i n a q u e o u s s o l u t i o n . H o w e v e r , t h e y are s l o w l y
o x i d i z e d b y N - b r o m o s u c c i n i m i d e in c h l o r o f o r m , c a r b o n t e t r a c h l o r i d e o r 1 . 1 . 1 - t r i c h l o r o e t h a n e .
In this c a s e , a d d 1 m l . o f a s o l u t i o n o f 2 5 m g . N - b r o m o s u c c i n i m i d e in 1 0 0 m l . o f o n e o f t h e
a b o v e - m e n t i o n e d solvents t o the dry residue o f t h e d i c h l o r o m e t h a n e extract. A l l o w t o stand
for 5 m i n . at r o o m t e m p e r a t u r e , a d d 1 m l . o f a 0 . 0 2 % s o l u t i o n o f p h e n o l in c h l o r o f o r m a n d
e v a p o r a t e off t h e s o l v e n t . Treat t h e r e s i d u e w i t h buffer a n d e n z y m e s o l u t i o n a s d e s c r i b e d u n d e r
"Assay System".
2. O x i d a t i o n w i t h H 0 / a c e t i c a c i d
2 2
1 3 - 1 6
: Extract the sample with benzene. A d d 3 ml. o f a
freshly p r e p a r e d m i x t u r e o f 3 0 % H 0 2 2 a n d g l a c i a l a c e t i c a c i d (1 : 5 v / v ) t o 5 m l . o f t h e e x t r a c t
in a test t u b e ( w i t h a g r o u n d - g l a s s s t o p p e r ) c o n t a i n i n g b o i l i n g c h i p s . S t o p p e r t h e t u b e , s h a k e
briefly, r e m o v e t h e s t o p p e r , h e a t f o r 2 0 m i n . at 75 ° C a n d c o o l in a n i c e b a t h . A d d 5 m l . d i s t i l l e d
water, stopper a n d shake t h o r o u g h l y . W h e n the phases have separated, pipette a portion o f
the b e n z e n e layer i n t o a 10 m l . b e a k e r a n d a d d a d r o p o f N u j o l ( l i q u i d paraffin). M i x t h o r o u g h
ly, e v a p o r a t e off t h e b e n z e n e a n d u s e t h e r e s i d u e f o r t h e a s s a y .
3. O x i d a t i o n w i t h nitric a c i d : E v a p o r a t e 5 m l . d i c h l o r o m e t h a n e e x t r a c t t o d r y n e s s in a 1 2 5 m l .
1
r o u n d - b o t t o m e d flask, c o o l t h e flask i n a n i c e b a t h a n d c a r e f u l l y a d d 1 0 m l . o f a m i x t u r e o f
cone. H N 0 3 and fuming H N 0 3 (1 : 1 v / v ) . Wet t h e w a l l s o f t h e flask w i t h t h e s o l u t i o n , r e m o v e
t h e flask f r o m t h e i c e b a t h a n d a l l o w t o s t a n d f o r 5 m i n . a t r o o m t e m p e r a t u r e . C a r e f u l l y a d d
Organophosphorus and Carbamate Insecticides 2253
25 m l . c o l d d i s t i l l e d w a t e r a n d p o u r t h e s o l u t i o n i n t o a 1 2 5 m l . s e p a r a t i n g f u n n e l . R i n s e t h e
flask w i t h t w o 2 5 m l . p o r t i o n s o f d i c h l o r o m e t h a n e a n d p o u r t h e w a s h i n g s i n t o t h e s e p a r a t i n g
f u n n e l . S h a k e t h o r o u g h l y , d r a w off t h e a q u e o u s l a y e r a n d d i s c a r d . W a s h t h e d i c h l o r o m e t h a n e
phase with 10 ml. portions o f 10% N a H C 0 3 solution until the w a s h i n g s give a n alkaline react
i o n t o l i t m u s . F i n a l l y , w a s h t w i c e w i t h s a t u r a t e d N a C l s o l u t i o n a n d filter t h e d i c h l o r o m e t h a n e
p h a s e t h r o u g h a s m a l l , d r y p l u g o f c o t t o n w o o l i n t o a 1 0 0 m l . v o l u m e t r i c flask. W a s h t h e
s e p a r a t i n g f u n n e l t w i c e w i t h 2 0 m l . p o r t i o n s o f d i c h l o r o m e t h a n e a n d filter t h e w a s h i n g s
t h r o u g h t h e s a m e c o t t o n w o o l p l u g i n t o t h e v o l u m e t r i c flask. D i l u t e w i t h d i c h l o r o m e t h a n e
to 100 ml. A n a l y s e a p o r t i o n o f this solution.
4. O x i d a t i o n w i t h m - c h l o r o p e r b e n z o i c a c i d 1 7 - 1 9
: Dissolve 100 m g . m-chloroperbenzoic acid
in 1 0 0 m l . o f a freshly p r e p a r e d m i x t u r e o f 9 0 m l . d i c h l o r o m e t h a n e a n d 1 0 m l . b e n z e n e . A d d
2 5 m l . o f this s o l u t i o n t o t h e d r y r e s i d u e f r o m t h e e x t r a c t , s h a k e w e l l , s t o p p e r t h e v e s s e l a n d
a l l o w t o s t a n d i n a n i c e b a t h f o r 3 0 m i n . I m m e d i a t e l y after t h i s t i m e , p o u r i n t o a 1 2 5 m l . s e p a
rating funnel. Wash o u t t h e o x i d a t i o n vessel with t w o 2 0 ml. p o r t i o n s o f d i c h l o r o m e t h a n e a n d
p o u r t h e w a s h i n g s i n t o t h e s e p a r a t i n g f u n n e l . W a s h t h e o x i d i z e d e x t r a c t w i t h 5 0 m l . o f a freshly
prepared 0.5% N a S 0 2 2 5 solution a n d with t w o 50 ml. portions o f saturated N a C l solution,
or u n t i l t h e w a s h i n g s a r e n e u t r a l t o l i t m u s . F i l t e r t h e d i c h l o r o m e t h a n e / b e n z e n e p h a s e t h r o u g h
a s m a l l p l u g o f c o t t o n w o o l a n d a little a n h y d r o u s s o d i u m s u l p h a t e i n t o a 2 0 0 m l . g r a d u a t e d
flask, m a k e u p t o 2 0 0 m l . w i t h d i c h l o r o m e t h a n e , a n d u s e a p o r t i o n o f t h i s s o l u t i o n f o r t h e a s s a y .
Determination of Activity of Cholinesterase Stock Solution (IVa)
Preliminary remarks: D u r i n g t h e a s s a y k e e p all t h e s o l u t i o n s in a n i c e b a t h . W h e n p i p e t t i n g

toxic solutions d o n o t use m o u t h . Clean glassware with h o t chromic acid, wash thoroughly
with water a n d dry at 100 ° C in a drying oven.
Method: W i t h a sterile a n d c h i l l e d 2 m l . s y r i n g e w i t h d r a w s l i g h t l y m o r e t h a n 1 m l . o f t h e i c e -
c o l d c h o l i n e s t e r a s e s t o c k s o l u t i o n ( I V a ) . P l u g t h e p o i n t o f t h e n e e d l e b y s t i c k i n g it u p r i g h t in a
c l e a n r u b b e r b u n g . R e m o v e t h e p i s t o n f r o m t h e s y r i n g e a n d w i t h a n a c c u r a t e p i p e t t e transfer
1.0 m l . o f t h e s o l u t i o n t o a 5 0 m l . v o l u m e t r i c flask. D i l u t e t o 5 0 m l . w i t h i c e - c o l d 0 . 9 % N a C l
solution (II) a n d m i x . This dilute cholinesterase solution c o n t a i n s ca. 0 . 8 4 U / m l .
I n t r o d u c e t h e i c e - c o l d s o l u t i o n s i n d i c a t e d i n t o t h r e e 10 m l . b e a k e r s a n d p l a c e at 2 m i n . i n t e r v a l s
in a w a t e r b a t h m a i n t a i n e d at 25 ± 0.5 ° C . Stir c o n t e n t s m a g n e t i c a l l y .
Control Assay mixtures C o n c e n t r a t i o n in

Beaker no. 1 2 3 assay mixture
Pipette into beakers:
0.9% NaCl solution (II) 3 ml. 770 m M

Buffer (I) 3 ml. 18 m M V e r o n a l
Cholinesterase-buffer mixture (V) 6 ml. 6 ml. c a . 2.7 U / m l .
c a . 15 m M buffer
A l l o w e a c h b e a k e r t o s t a n d in t h e w a t e r b a t h for e x a c t l y 3 0 m i n . A f t e r t h i s
t i m e t a k e first b e a k e r f r o m w a t e r b a t h a n d i m m e d i a t e l y m e a s u r e p H .
T h i s s h o u l d b e 8 . 0 0 ± 0.5 ("initial p H " ) . P i p e t t e i n t o b e a k e r s 2 a n d 3 :
Acetylcholine solution (III) — 0.6 ml. 0.6 ml. 20 m M
A l l o w t o s t a n d in w a t e r b a t h for e x a c t l y a further 6 0 m i n . A f t e r t h i s t i m e
measure p H values. These should agree to within 0.02 p H units. Take
average.
F i g . 2 s h o w s a p l o t o f arbitrary c h o l i n e s t e r a s e u n i t s / m l . a g a i n s t t h e p H . R e a d off f r o m t h i s
curve the cholinesterase activity o f the dilute cholinesterase s o l u t i o n corresponding to the
-60
e
CO
/
"1 50
ig 40
CD
f 30
"o
TO
QJ
% 20
Fig. 2. S t a n d a r d curve for the hydrolysis of acetyl-
^ | | | i choline chloride by cholinesterase (1 h o u r at 25 °C).
10 75 70 65 60 55 5.0 Reaction m i x t u r e : 3 ml. buffer (solution I ) ; 3 ml.
pH of the reaction mixture after incubation for cholinesterase solution; 0.6 ml. acetylcholine standard
one hour at 25°C and with an initial pH of 8.0. solution (III).
m e a s u r e d p H v a l u e . M u l t i p l y t h e n u m b e r o f arbitrary u n i t s / m l . b y 4 9 / 3 8 * . T h e r e s u l t i n g v a l u e
g i v e s t h e m l . o f 0 . 9 % N a C l s o l u t i o n (II) t h a t m u s t b e a d d e d t o 1 m l . c h o l i n e s t e r a s e s t o c k
s o l u t i o n ( I V a ) in o r d e r t o o b t a i n t h e d i l u t e c h o l i n e s t e r a s e s o l u t i o n ( I V b ) .
* T h e quotient is obtained as follows: 1 ml. cholinesterase stock solution (IVa) was mixed with 49 ml.
N a C l solution to obtain the dilute cholinesterase solution. Fig. 2. shows t h a t 38 arbitrary units choline-
sterase/ml. are required to lower the pH from 8.0 to 6.0.
O r g a n o p h o s p h o r u s a n d C a r b a m a t e Insecticides 2255
Standard Curve
To c a l c u l a t e t h e e x p e r i m e n t a l results a s t a n d a r d c u r v e is r e q u i r e d for e a c h i n s e c t i c i d e . T h e
p r o c e d u r e is a s s t a t e d u n d e r " A s s a y S y s t e m " , b u t different c o n c e n t r a t i o n s o f t h e p u r e i n s e c t i c i d e
in d i c h l o r o m e t h a n e are u s e d i n s t e a d o f t h e s a m p l e s o l u t i o n .
T h e % i n h i b i t i o n for e a c h s o l u t i o n is o b t a i n e d a s d e s c r i b e d u n d e r " C a l c u l a t i o n s " a n d this is
p l o t t e d ( a b s c i s s a , linear s c a l e ) a g a i n s t t h e fig. i n s e c t i c i d e / r e a c t i o n m i x t u r e ( o r d i n a t e , l o g a r i t h m i c
scale). F i g . 3 illustrates t w o s u c h s t a n d a r d c u r v e s .
0.2
0.15
|
0.1
/
B
2 0.08 u
3 0.07 A—
•| 0.06
§ 0.05 '/ 7-
-§
CD
CD
0.04
/
^ 0.03
0
/
Fig. 3. S t a n d a r d curves for (A) diethyl-p-nitrophenyl
0.02
/
/
/
p h o s p h a t e ( P a r a o x o n ) a n d (B) 0,0-diethyl-O-p-nitro-
phenyl t h i o p h o s p h a t e ( P a r a t h i o n ) , the latter after oxi 0.01
10 20 30 40 50 60 70 80 9 0 7 J 0 0
dation with a mixture of equal volumes of cone, and % Inhibition (measured under the condi
fuming H N 0 * . 3
tions described under "Assay System").
Assay System
Preliminary Remarks:
L a b e l the p i p e t t e s for m e a s u r i n g o u t t h e c h o l i n e s t e r a s e , buffer a n d a c e t y l c h o l i n e s o l u t i o n s .

W h e n p i p e t t i n g d o n o t t o u c h t h e w a l l s o f t h e b e a k e r . It is e s s e n t i a l t h a t t h e s t a t e d r e a c t i o n
t i m e s are a c c u r a t e l y a d h e r e d t o , s i n c e t h e p r i n c i p l e o f t h e a s s a y is b a s e d o n t h e m e a s u r e m e n t o f
r e a c t i o n rates. B e f o r e e a c h m e a s u r e m e n t , rinse t h e m i c r o e l e c t r o d e s o f t h e p H m e t e r w i t h
distilled w a t e r a n d s p o n g e w i t h a c l o t h o r t h i n a b s o r b e n t p a p e r . In a d d i t i o n , see t h e p r e l i m i n a r y
r e m a r k s o n p. 2 2 5 3 .
Method:
I n c u b a t i o n t e m p e r a t u r e : 2 5 + 0.5 ° C ; final v o l u m e : 6.6 m l . ; t w o c o n t r o l s ( C I a n d C 2 ) c o n t a i n

ing n o s a m p l e are r e q u i r e d for e a c h series o f m e a s u r e m e n t s . P l a c e t e n n u m b e r e d 10 m l . b e a k e r s
in a f u m e c u p b o a r d , a n d p l a c e a m a g n e t i c flea in e a c h b e a k e r .
* Oxidation with b r o m i n e water or H 0 / a c e t i c acid is equally suitable or m o r e s o

2 2
1 3
Control Assay Control

CI mixtures C2 C o n c e n t r a t i o n in
assay mixture
Beaker no. 1 2 to 9 10
Pipette into beakers:
S a m p l e (extract) 1 - 8 ml. up to 20 ng./ml.

CH C12 2 8 ml. 7 - 0 ml. 8 ml.
W i t h o u t h e a t i n g , e v a p o r a t e all s o l u t i o n s t o d r y n e s s in a g e n t l e c u r r e n t o f
air f r o m a n electric f a n , w i t h m a g n e t i c stirring. T h i s n o r m a l l y t a k e s
10 m i n . P i p e t t e in s u c c e s s i o n , at i n t e r v a l s o f e x a c t l y 2 m i n . , i n t o all t h e
beakers:
Cholinesterase-buffer ca. 2.7 U / m l .

mixture (V) 6 ml. 6 ml. 6 ml. ca. 15 m M buffer
P l a c e e a c h b e a k e r in a w a t e r b a t h (25 + 0.5 °C) a n d stir (cf. F i g . 1). A f t e r

e x a c t l y 30 m i n . r e m o v e t h e first b e a k e r ( C I ) f r o m t h e w a t e r b a t h a n d
m e a s u r e t h e p H o f t h e s o l u t i o n . T h i s s h o u l d b e 8 . 0 0 + 0 . 0 5 . It is d e s i g n a t
e d ( p H ) ^ , a n d m a y b e r e g a r d e d a s t h e initial p H o f all t h e o t h e r s o l u t i o n s .
A t intervals o f e x a c t l y 2 m i n . p i p e t t e :
Acetylcholine solution (III) — 0.6 m l . 0.6 m l . 20 m M
K e e p e a c h b e a k e r at 25 ± 0.5 ° C for e x a c t l y a further 6 0 m i n . , c a l c u l a t e d

f r o m t h e t i m e o f p i p e t t i n g , a n d stir w i t h a m a g n e t i c stirrer. A f t e r t h i s t i m e
m e a s u r e p H . T h e s e v a l u e s are d e s i g n a t e d ( p H ) g a n d ( p H ) f ° a m
Calculations
The inhibition of the cholinesterase is calculated from the p H values according to the f o r m u l a :
% inhibition = [(PHfe-(pH)g]-[(pH)S,-( H)fl,] P x m
KpH)^ - (pH)g]
where
(pH)ci = p H of the control CI (without acetylcholine) before the start of the incubation with acetyl
choline
(pH)c 2 = p H of the control C 2 at the end of the incubation with acetylcholine
(pH)fam. — p H of t n e
experimental mixtures at the end of the incubation with acetylcholine.
The insecticide concentration in /ig./reaction mixture corresponding to the calculated % inhibition is

obtained from a s t a n d a r d curve (see above and Fig. 3).
Sensitivity and P r e c i s i o n o f M e t h o d
The sensitivity of the m e t h o d d e p e n d s on the inhibitory action of the insecticide to be determined. F o r

example the sensitivity is 0.005 ^g- for P a r a t h i o n , 1.0 fig. for M e t h y l p a r a t h i o n , 0.01 fig. for Trithion,
5.0 fig. for Methyltrithion, 1.4 fig. for Di-Syston, and 0.5 fig. for Sevin.
T h e reproducibility of the m e t h o d d e p e n d s o n the accuracy with which the solutions are prepared, a n d
is limited by the p H meter. T h e accuracy of the m e t h o d d e p e n d s on the accuracy of the s t a n d a r d c u r v e ;
it is greatest when s t a n d a r d s are treated together with the samples.
T h o u g h interference due to plants or animal material (untreated with insecticide) has n o t been considered,
blanks of this n a t u r e are r e c o m m e n d e d .
T h e reagents a n d solutions must be of the highest purity, a n d all glassware must be washed with the
u t m o s t care. C o n t a c t of the solutions with t a p grease, rubber, cork, or detergents must be avoided, since
these substances m a y inhibit or even activate the cholinesterase.
This highly sensitive m e t h o d was developed for insecticides that inhibit cholinesterase. T h o u g h the m e t h o d
is not specific in the usual sense, it is possible to differentiate between insecticides with fairly large differen
ces in their inhibitory action, e. g. between P a r a t h i o n a n d M e t h y l p a r a t h i o n or between Trithion a n d
M e t h y l t r i t h i o n ; moreover, m a n y of the weakly inhibiting or non-inhibiting insecticides, such as p u r e
P a r a t h i o n , can be readily oxidized to P a r a o x o n (which has an extremely strong insecticidal action) and
be determined by this m e t h o d .
Enzymatic m e t h o d s for the d e t e r m i n a t i o n of o r g a n o p h o s p h o r u s a n d c a r b a m a t e insecticides can be

grouped as follows:
a) electrometric m e t h o d s 1
-3.9.11.13,14,20-24,63-68^ ^ titrimetric m e t h o d s ' ~ , c) m a n o m e t r i c m e t h o d s "
5 2 5 2 8 2 9
3 5
, d) p h o t o m e t r i c m e t h o d s ' 7 1 0 , 3 6
" ' "
3 9 6 9 7 4
[some p h o t o m e t r i c m e t h o d s use c h r o m o g e n i c substrates, which
form strongly coloured or fluorescent p r o d u c t s o n hydrolysis with cholinesterase or related esterases; with
a constant substrate concentration the colour intensity or fluorescence d e p e n d s on the activity of the
enzyme ' 8 4 0 - 4 4
] , e) m e t h o d s based o n p a p e r or thin layer c h r o m a t o g r a p h y " 4 5 4 9 , 7 5 - 7 8
, 0 a
g a r
diffusion
m e t h o d s " , and g) a u t o m a t e d chemical m e t h o d s "
5 0 5 3 5 4 5 8 , 7 9
" , h) gas liquid c h r o m a t o g r a p h i c m e t h o d s ,
8 4 8 5
i) N M R m e t h o d s . In addition, there are m e t h o d s t h a t allow the d e t e r m i n a t i o n of the loss of cholinesterase

86
activity in blood after the action of P a r a t h i o n or other o r g a n o p h o s p h o r u s i n s e c t i c i d e s 3 , 6 , 5 9

" . 6 2
References
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201 [1949].
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54 J. Byron Lovell, J. econ. E n t o m o l . 56, 310 [1963].
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56 Guenther Voss, J. econ. E n t o m o l . 59, 1288 [1966].
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58 G. D. Winter & Andres Ferrari in F. A. Gunther: Residues Reviews. Springer-Verlag N e w Y o r k Inc.,
New Y o r k , N . Y . 10010, 1964, Vol. 5, p . 139.
59 D. O. Hamblin & H. H. Golz: Cholinesterase Tests a n d Their Applicability in the Field. American
C y a n a m i d C o . , Stamford C o n n . 0 6 9 0 1 , 1963.
60 D. R. Davies & J. D. Nicholis, Brit. med. J., 1 373 [1955].
61 G. Limperos & K. E. Rauta, Science (Washington) 777, 453 [1953].
62 E. P. W. Winteringham & R. W. Disney, N a t u r e 795, 1 303 [1962].
63 F. D. Aldrich, A r c h Environ. H e a l t h 79, 617 [1969].
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65 P. E. Braid & M . Nix, C a n . J. Biochem. 47, 1 [1969].
66 H. Marchart, Microchim. A c t a p . 669 [1968].
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68 F. P. W. Winteringham & K. S. Fowler, Biochem. J. 101, 127 [1966].
69 H. 7. Colvin & A. T. Phillips, Bull. Environ. C o n t a m . Toxicol. 3, 106 [1968].
70 G. G. Guilbault & M. H. Sadar, Anal. C h e m . 41, 366 [1969].
71 G. G. Guilbault, S.S. Kuan & M.H. Sadar, J. Agri. F o o d C h e m . 18, 692 [1970].
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74 G. Zweig & J. M. Devine, Residue Reviews 26, 17 [1969].
75 H. Achermann, J. C h r o m a t o g r . 36, 309 [1968].
76 / . Crossley, J. Assoc. Off. Agric. Chemists 53, 1036 [1970].
77 P. Wales, H.A. McLeod & W.P. McKinley, Ibid. 51, 1239 [1968].
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86 G. Kato, M o l . P h a r m a c o l . 4, 640 [1968].
Nitrate
Fujio Egami and Shigehiko Taniguchi
Nitrates are c o m p o n e n t s of the so-called " n i t r o g e n c y c l e " ; they have a stable nitrogen a t o m of the highest
oxidation state. Nitrates are widely distributed t h r o u g h o u t the biosphere, e. g. in rain water, sea water, soil
and plant material. They have also been found in h e t e r o t r o p h i c organisms, e. g. in h u m a n urine a n d in horse
serum . 1
T h e existing colour reactions for nitrate are less sensitive and less specific t h a n the d e t e r m i n a t i o n of nitrite
2
via diazo c o m p o u n d s ' . Therefore m e t h o d s were developed in which nitrate was converted with a suitable
3 4
reducing agent to nitrite a n d the latter measured (see ). However, the reducing agents so far examined are n o t
2
sufficiently specific a n d it is normally difficult to reduce nitrate to nitrite quantitatively. T h e present m e t h o d

uses a specific particle-bound formate-nitrate reductase, F N R * obtained from certain strains of Escherichia
coli which have n o formate-nitrite reductase activity. F N R reduces nitrate quantitatively to nitrite with
formate as the specific hydrogen d o n o r , but does n o t cause any further reduction. F M N is a d d e d as the
hydrogen carrier. I m p r o v e m e n t s in the original m e t h o d are included here.
5
Application of Method: In biochemistry, agricultural chemistry a n d geochemistry.
Principle
(1) FMN + HCOO -
+ H 0 2 > FMNH 2 + C0 2 + OH -
(2) FMNH 2 + N(V > N0 ~ + FMN +

2 H 0
2
T h e nitrite formed in reaction (2) is converted in a diazo coupling reaction to a red azo dye which is measured
colorimetrically.
A l t h o u g h n a t u r a l electron carriers, such as c y t o c h r o m e b x

6
a n d u b i q u i n o n e are active endogenously with
7
the particulate F N R from E. coli, F M N * * is a d d e d in the assay so as to o b t a i n complete reduction within a

reasonable time. T h e F N R p r e p a r a t i o n also contains low activity of the electron t r a n s p o r t system from
formate to molecular oxygen, which has m u c h in c o m m o n with the system of formate t o nitrate a n d is
competitive with it. Therefore the reaction is carried out anaerobically in a Thunberg t u b e to obtain a
complete conversion.
F o r a routine test where the nitrate c o n c e n t r a t i o n of the sample is above 50 a n d an error of 1 0 - 1 5 % is
permissible, a cell suspension of E. coli (see A p p e n d i x , p . 2264) can be used if the cells are freed from nitrite
reductase activity and from nitrate a n d nitrite by t h o r o u g h washing.
Alternatively, the reduction of nitrate with the cell suspension can be carried o u t in test tubes w i t h o u t the
addition of F M N . D e t e r m i n a t i o n of nitrate by living cells of E. coli a n d of other bacteria has already been
described 8 - 1 0
.
F r o m d a t a obtained with the purified enzyme " r e d u c e d dye-nitrate r e d u c t a s e " the t u r n o v e r n u m b e r of
F N R is a b o u t 70000 mole nitrate per mole active g r o u p s per min. at 30 °C. T h e K
6
M
6
of F N R for nitrate
(130 uM) is unfortunately rather high, therefore excess of formate a n d relatively large a m o u n t s of enzyme
are required to bring the reaction to completion within several h o u r s .
* N o system n u m b e r has yet been assigned by the Enzyme C o m m i s s i o n .

* In contrast to the colour of methylene b l u e , the yellow colour of F M N or F A D does not interfere with the
5
m e a s u r e m e n t of the red azo dye. Therefore in the present m e t h o d F M N is a d d e d instead of methylene

blue, which simplifies the m e t h o d because of the omission of the decolourization step.
Nitrate 2261
Equipment
S p e c t r o p h o t o m e t e r o r s i m p l e filter p h o t o m e t e r s u i t a b l e for m e a s u r e m e n t s at c a . 5 4 0 n m ; bench

c e n t r i f u g e a n d T h u n b e r g t u b e s ( s i d e - a r m s are n o t n e c e s s a r y ) .
Reagents
1. D i s o d i u m h y d r o g e n p h o s p h a t e , 6. U r a n y l a c e t a t e , U 0 2 (CH COO) -2H 0,

3 2 2
N a H P 0 - 2 H 0 , A . R.
2 4 2 A . R.
2. S o d i u m d i h y d r o g e n p h o s p h a t e , 7. H y d r o c h l o r i c a c i d , A . R . 3 5 % ( w / w ) , s p .
N a H P 0 - 2 H 0 , A . R.
2 4 2
gr. 1.175
3. P o t a s s i u m n i t r a t e , K N 0 , A . R . 3 8. S u l p h a n i l a m i d e , A . R .
4. S o d i u m f o r m a t e , H C O O N a , A . R . 9. N - ( 1 - N a p h t h y l - ) e t h y l e n e d i a m m o n i u m -
5. F l a v i n e m o n o n u c l e o t i d e , F M N chloride, A . R.
m o n o s o d i u m salt, synthetic. C o m m e r c i a l p r e p 10. F o r m a t e - n i t r a t e r e d u c t a s e , F N R
a r a t i o n , see p . 533. from E. coli; ca. 0 . 5 - 1 U / m g . (25 °C). P r e p
Purity of Compounds
The F N R must be free from formate-nitrite reductase activity a n d from nitrate a n d nitrite. T h e other reagents
should n o t contain these c o m p o u n d s or heavy metals which inhibit F N R activity.
P r e p a r e all s o l u t i o n s w i t h fresh, g l a s s distilled w a t e r .

D i s s o l v e 6.38 g. N a H P 0 - 2 H 0 a n d 2 . 2 2 g. N a H P 0 - 2 H 0 in d i s t i l l e d w a t e r a n d m a k e
2 4 2 2 4 2
u p t o 100 m l .
II. P h o s p h a t e b u f f e r / f o r m a t e / F M N s o l u t i o n ( 0 . 5 M p h o s p h a t e , p H 7 . 2 ; 0 . 2 5 M f o r m a t e ;
0.1 m M F M N ) :
D i s s o l v e 1.7 g. H C O O N a H 0 a n d 5 m g . F M N - N a - 2 H 0 in 1 0 0 m l . p h o s p h a t e buffer (I).
2 2
III. N i t r a t e s t a n d a r d s o l u t i o n ( 2 5 0 fiM):
D i s s o l v e 126 m g . K N 0 3 in d i s t i l l e d w a t e r a n d m a k e u p t o 100 m l . ( c o r r e s p o n d i n g t o
12.5 m M ) ; d i l u t e 2.0 m l . o f this t o 100 m l . w i t h d i s t i l l e d w a t e r .
IV. Uranyl acetate solution (saturated):
A d d 1 0 . 0 g. U 0 ( C H C O O ) - 2 H 0 t o 100 m l . d i s t i l l e d w a t e r a n d w a r m t o 4 0 ° C . A l l o w t o
2 3 2 2
c o o l t o r o o m t e m p e r a t u r e , d e c a n t off t h e s u p e r n a t a n t fluid a n d u s e d i r e c t l y .
V. S u l p h a n i l a m i d e ' (58 m M ) :
3 4
M i x 75 m l . d i s t i l l e d w a t e r a n d 2 5 m l . c o n e . H C 1 a n d d i s s o l v e in t h i s m i x t u r e 1.0 g. s u l p h a
nilamide.
VI. N - ( l - N a p h t h y l - ) e t h y l e n e d i a m i n e s o l u t i o n ' (0.77 m M ) :
3 4
D i s s o l v e 2 0 m g . N - ( l - n a p h t h y l - ) e t h y l e n e d i a m i n e d i h y d r o c h l o r i d e in 100 m l . d i s t i l l e d
water.
V I I . F o r m a t e - n i t r a t e r e d u c t a s e (ca. 10 m g . p r o t e i n / m l . ) :
P r e p a r e t h e e n z y m e s u s p e n s i o n in p h o s p h a t e buffer (0.1 M ; p H 7.2) a c c o r d i n g t o t h e
p r o c e d u r e d e s c r i b e d in t h e A p p e n d i x ( p . 2 2 6 4 ) .
2262 Metabolites: Miscellanous Substrates and Effectors
Store all reagents in b r o w n bottles protected from the light. Store solutions II, V a n d VI in a refrigerator.
The freeze-dried enzyme F N R keeps its activity for at least 3 m o n t h s stored in evacuated a m p o u l e s at 0 - 5 ° C ;
as suspension VII is stable in t h e d a r k at 0 - 5 °C for 1-2 weeks. T h e o t h e r reagents are stable at r o o m
temperature.
Procedure
A l l s a m p l e s for d e t e r m i n a t i o n o f nitrate m u s t b e k e p t as sterile as p o s s i b l e , b e c a u s e t h e p r e s e n c e

o f n i t r a t e - r e d u c i n g b a c t e r i a o r m o u l d s c a n result in t h e r a p i d c o n v e r s i o n o f large a m o u n t s o f
nitrate t o o t h e r n i t r o g e n - c o n t a i n i n g c o m p o u n d s b e f o r e t h e start o f t h e a s s a y . N o r m a l l y d e
p r o t e i n i z a t i o n o f b i o l o g i c a l s a m p l e s is n o t n e c e s s a r y . S a m p l e s w i t h h i g h salt c o n c e n t r a t i o n
m u s t b e d i l u t e d t o a l l o w full a c t i v i t y o f t h e F N R . S a m p l e s w h i c h are s t r o n g l y a c i d o r a l k a l i n e
must be neutralized before the addition of the enzyme.
Stability of sample: S a m p l e s s h o u l d b e a n a l y s e d as s o o n a s p o s s i b l e . If s t o r a g e is u n a v o i d a b l e
this s h o u l d b e in t h e dark at 0 - 5 ° C u n d e r a s e p t i c c o n d i t i o n s .
Assay System
If t h e s a m p l e c o n t a i n s nitrate t o g e t h e r w i t h nitrite, t h e latter m u s t b e d e t e r m i n e d in a s e p a r a t e

a s s a y ( t u b e s 4 a n d 5) w i t h o u t a d d i t i o n o f e n z y m e a n d i n c u b a t i o n .
Fig. 1. R a t e of reaction with nitrate s t a n d a r d s

O r d i n a t e : pM nitrite formed by enzymatic
reduction of nitrate in the sample.
Abscissa: L e n g t h of enzymatic reaction (hr.)
C u r v e A : 2000 pM nitrate
C u r v e B : 1000 pM nitrate
C u r v e C : 500 pM nitrate
Curve D : 200 pM nitrate
0 1 2 Curve E : 100 pM nitrate
T h e nitrate c o n c e n t r a t i o n in t h e a s s a y s h o u l d b e 1 0 - 2 0 0 pM, so that within a few hours the

c o m p l e t e c o n v e r s i o n t o nitrite is a c h i e v e d . T h e a p p r o x i m a t e nitrate c o n c e n t r a t i o n o f t h e s a m p l e
s h o u l d b e d e t e r m i n e d w i t h t w o different a m o u n t s o f s a m p l e (e. g. 0 . 4 m l . a n d 2 . 0 m l . ) b y t h e
s t a n d a r d m e t h o d . S t o p t h e r e a c t i o n after 1 hr. a n d after 2 hr. (see F i g . 1). W i t h n i t r a t e c o n c e n t r a t
i o n s o v e r 2 0 0 pM d i l u t e the s a m p l e a c c o r d i n g l y .
Wavelength: 540 n m ( g r e e n filter); light p a t h : 1 c m . ; i n c u b a t i o n v o l u m e : 3.00 m l . ; 3 7 ° C
( c o n s t a n t t e m p e r a t u r e w a t e r b a t h ) ; a s s a y v o l u m e : 3.00 m l . R e a d a g a i n s t air at r o o m t e m p e r a
ture.
P i p e t t e s a m p l e s 1 - 3 in Thunberg test t u b e s , s a m p l e s 4 a n d 5 in test t u b e s . C o n c e n t r a t i o n s in
Nitrate 2263
i n c u b a t i o n m i x t u r e (after u r a n y l a c e t a t e a d d i t i o n ) : 6 . 7 - 1 3 3 pM nitrate or s a m p l e ; 100 m M

p h o s p h a t e ; 5 0 m M f o r m a t e ; 2 0 /xM F M N ; F N R c a . 0 . 6 7 m g . p r o t e i n / m l .
Thi mberg tul ) e s Test t u b e s

1 2 3 4 5
Pipette into tubes:
Nitrate Internal Nitrite Nitrite
Blank
test standard test blank
Sample 2.0 m l . 2.0 m l . 2.0 ml.

Phosphate/formate/FMN solution (II) 0.6 m l . 0.6 m l . 0.6 ml. 0.6 ml. 0.6 m l .
Nitrate standard solution (III) 0.2 m l .
Distilled water 0.2 ml. 2.2 ml. 0.4 ml. 2.4 ml.
F N R suspension (VII) 0.2 ml. 0.2 m l . 0.2 ml.
E v a c u a t e Thunberg t u b e s 1 - 3 , i n c u b a t e for 2 hr. A n a l y s e t u b e s 4 a n d 5 i m m e d i a t e l y .
Uranyl acetate solution (IV) 1.0 m l . 1.0 m l . 1.0 m l . 1.0 m l . 1.0 m l .
M i x a n d c e n t r i f u g e for 10 m i n . at 1 0 0 0 g. U s e t h e clear s u p e r n a t a n t fluid.
P i p e t t e i n t o test t u b e s :
Supernatant fluid 2.0 ml. 2.0 ml. 2.0 ml. 2.0 ml. 2.0 m l .
Sulphanilamide solution (V) 0.5 m l . 0.5 m l . 0.5 m l . 0.5 m l . 0.5 m l .
N-(l-Naphthyl-)ethylene diamine
solution (VI) 0.5 m l . 0.5 m l . 0.5 m l . 0.5 m l . 0.5 m l .
M i x , a l l o w t o s t a n d for 10 m i n . a n d t h e n read e x t i n c t i o n s .
Calculations
Analyse the nitrite s t a n d a r d s (from " s u p e r n a t a n t fluid") a n d p r e p a r e s t a n d a r d curve. O r d i n a t e : extinction,

abscissa: nitrite.
R e a d off the concentrations c o r r e s p o n d i n g to the m e a s u r e d extinctions from the s t a n d a r d curve a n d multi

ply by the a p p r o p r i a t e dilution factor (3 in this m e t h o d ) . This gives the nitrite c o n c e n t r a t i o n of the sample.
T h e nitrate concentration is calculated as follows:
C o n c e n t r a t i o n differences, _
expressed as nitrite in tubes C o r r e s p o n d i n g t o the c o n t e n t in t h e sample of
1-3 nitrate + nitrite

4-5 nitrite
(1-3)-(4-5) nitrate
2-1 nitrate as internal s t a n d a r d (should be 25 fiM)
T h e correct functioning of the nitrate assay should be checked for each sample by the recovery of the internal
s t a n d a r d (difference between t u b e 1 a n d 2). In the experimental material studied so far ( h u m a n urine, horse
serum, extracts of spinach leaves , culture m e d i u m of algae, etc.) 9 2 - 9 8 % of the a d d e d nitrate has been
5
recovered, which d e m o n s t r a t e s the applicability of the m e t h o d to these materials.

W i t h nitrate s t a n d a r d s solutions of 10, 25, 50 a n d 100 pM a m e a n recovery of 1 0 0 % (range 9 5 - 1 0 6 % ) was

found with 10 assays for each concentration. In the presence of equal a m o u n t s of nitrite the nitrate recovery
was 9 8 % (range 9 3 - 1 0 5 % ) . Using cell suspensions of E. coli a n d m o r e t h a n 50 pM nitrate in the assay, the
error is 1 0 - 1 5 % .
Normal Values
The nitrate content in agricultural or geochemical samples varies considerably, depending on the environ
mental a n d local conditions at the site of sampling. Too few analytical d a t a are available t o present n o r m a l
values. O n 5 samples of n o r m a l h u m a n urine the nitrate concentration was between 5 a n d 7 m M ; on 3
samples of horse serum it was between 20 a n d 50 pM.
It is particularly i m p o r t a n t that the enzyme is free from formate-nitrite reductase, otherwise t o o low values
will be obtained, especially with very low a m o u n t s of nitrate. Inhibitors of F N R cause a decrease in the rate
of nitrate reduction or prevent completion of the reaction. These inhibitors i n c l u d e , heavy metal chelating
1
agents, such as cyanide, azide, o-phenanthroline, a, a'-dipyridyl, t h i o u r e a , chlorate, b r o m a t e , i o d a t e a n d a

2 3
high salt concentration in the sample. C h l o r a t e , b r o m a t e a n d iodate are competitive i n h i b i t o r s ; the first
two react with the e n z y m e . Sea water which contains 3 % N a C l m u s t be diluted before assay with 2 parts
11
distilled water, otherwise the reaction does n o t p r o c e e d . In addition, all c o m p o u n d s which inhibit the
5
diazotization by nitrite interfere. This g r o u p includes reducing agents, such as cysteine, glutathione, ascorbic
acid, N A D H a n d N A D P H . T h e presence of these c o m p o u n d s can be determined by a recovery experiment
in which nitrite is a d d e d to the sample (as " i n t e r n a l s t a n d a r d " ) . This type of interference can be overcome by
specific oxidation of the interfering c o m p o u n d s . F o r example, interference by N A D H can be removed by
addition of acetaldehyde a n d a trace of yeast alcohol dehydrogenase (alcohol: N A D oxidoreductase,
E C 1.1.1.1) before the diazotization r e a c t i o n . 12
The m e t h o d is specific for nitrate, because a l t h o u g h chlorate a n d b r o m a t e 1 1

are also reduced by F N R , the
diazotization reaction is completely specific for nitrite. C r u d e F N R p r e p a r a t i o n s can contain a nitro
reductase, which reduces a r o m a t i c n i t r o c o m p o u n d s . This c o n t a m i n a n t , however, causes n o interference in
this m e t h o d .
Appendix
Preparation of Formate-nitrate Reductase
G r o w Escherichia coli, strain Yamagutchi ( I F O 12433) or strain B* anaerobically for 8 hr. in a p e p t o n e

m e d i u m containing 0 . 1 % K N 0 ; 0 . 1 % K H P 0 ; 0 . 1 % glucose, 0 . 2 5 % s o d i u m formate, 0 . 1 % casein
3 2 4
hydrolysate a n d 0 . 2 % p o w d e r e d yeast e x t r a c t ; p H 7.2. Transfer this subculture in a m o u n t s of 5 % (v/v) to
* Other strains are also suitable, provided they contain n o significant a m o u n t s of formate-nitrite reductase
activity. N o r m a l E. coli strains cultured u n d e r similar conditions contain only slight formate-nitrite
reductase a n d the major p o r t i o n of this is destroyed if the cells are frozen for several h o u r s at —15 °C. In
addition, in the cell fractionation, the major p o r t i o n of the activity is r e m o v e d from the particulate
fraction where it is located.
Nitrate 2265
the m e d i u m of the main culture (same composition, ca. 500 ml.) a n d incubate for a further 8 hr. Collect
the cells by centrifugation a n d wash with distilled water to remove the total nitrate a n d nitrite. Freeze the
washed cell paste (ca. 2 g. wet wt.) for several h o u r s at - 1 5 °C a n d then grind for 30 min. in a cold m o r t a r
with double its weight of A 1 0 2 3 powder. G r i n d for a further 10 min. with five times its weight of cold
p h o s p h a t e buffer (I). T h e extraction can also be carried out with a l a b o r a t o r y sonic disintegrator ( 1 0 - 2 0 k c ;
with cooling), suspend the frozen cells with five times their weight of buffer (I). Centrifuge the resulting brei
for 20 min. at 2000 g in the cold. Centrifuge the s u p e r n a t a n t fluid for 40 min. at 20000 g in the c o l d ; the
particle-bound F N R fraction sediments. Wash this sediment by twice re-suspending in p h o s p h a t e buffer (I)
a n d centrifugation. Suspend the washed precipitate (ca. 200 mg. protein) in p h o s p h a t e buffer (I) a n d dialyse
for 24 hr. in the cold against the same buffer. After adjustment of the protein content to ca. 10 mg./ml. the
dialysed material can be used directly as the F N R p r e p a r a t i o n for the assay. If the p r e p a r a t i o n is to be
stored for long periods in the freeze-dried state it should be dialysed against distilled water.
References
1 H. Takahashi, S. Taniguchi & F. Egami in M. Florkin & H. S. Mason: C o m p a r a t i v e Biochemistry.

A c a d e m i c Press N e w York 1963, Vol. 5, p . 93.
2 H. J. Taras: Colorimetric D e t e r m i n a t i o n of N o n m e t a l s . Interscience Publishers, N e w York 1958, p . 135.
3 F. D. SnellScC. T. Snell: Colorimetric M e t h o d s of Analysis. Van N o s t r a n d , N e w York 1949, 3rd edn.,
p . 804.
4 D. J. D. Nicholas & A. Nason in S. P. Colowick & N. O. Kaplan: M e t h o d s in Enzymology. A c a d e m i c
Press, New York 1957, Vol. / / / , p . 9 8 1 .
5 F. Egami, K. Iida, T. Doke & S. Taniguchi, Bull. C h e m . Soc. J a p a n 27, 619 [1954].
6 S. Taniguchi & E. Itagaki, Biochim. biophys. Acta 44, 263 [I960].
7 E. Itagaki, J. Biochem. (Tokyo) 55, 432 [1964].
8 R. Williams, J. L a b . clin. M e d . 41, 157 [1953].
9 G. B. Garner, J. S. Baumstark, M. L. Muhrer & W. H. Pfander, A n a l . C h e m . 28, 1589 [1956].
10 R. M. Hill, H. Pivnick, W. E. Engelhard & M. Bogard, Agr. F o o d C h e m . 7, 291 [1959].
WE. Itagaki & S. Taniguchi, unpublished results.
12 A. Medina & D. J. D. Nicholas, Biochim. biophys. A c t a 23, 440 [1957].
Concentrations of Metabolites in Animal Tissues
D . H . W i l l i a m s o n a n d J. T . B r o s n a n
Fourteen years have elapsed since publication o f the classic paper* entitled " U b e r Metabolit-
g e h a l t e u n d M e t a b o l i t - K o n z e n t r a t i o n e n in d e r L e b e r der R a t t e " a n d in this p e r i o d m e a s u r e m e n t
of metabolite concentrations in animal tissues h a s b e c o m e an important branch o f enzymatic
a n a l y s i s . M e a s u r e m e n t s o f c h a n g e s in m e t a b o l i t e p a t t e r n s in t i s s u e s in r e s p o n s e t o a l t e r a t i o n s
of substrate or h o r m o n a l balance have provided considerable information o n the regulation
of metabolic p a t h w a y s 1 0 5
" 1 0 8
.
T h e following Tables are m e a n t t o b e a guide t o the metabolite c o n c e n t r a t i o n s * * likely t o
o c c u r in vivo u n d e r a v a r i e t y o f different c o n d i t i o n s . O n l y v a l u e s w h i c h h a v e b e e n o b t a i n e d
w i t h a n enzymatic method on tissue which has been freeze-clamped or immersed in a refrigerant
(refer t o p . 4 0 0 ) are i n c l u d e d . C e r t a i n v a l u e s h a v e b e e n d e l i b e r a t e l y e x c l u d e d b e c a u s e t h e
tissue m a y h a v e b e e n a n o x i c o r b e c a u s e t h e a s s a y t e c h n i q u e w a s n o t c o n s i d e r e d reliable. T h e
v a l u e s are e x p r e s s e d in ^ m o l e / g . fresh w e i g h t o f t i s s u e o r / / m o l e / m l . w h o l e b l o o d e x c e p t
where otherwise stated.
R e c e n t l y there h a s b e e n a d e t a i l e d i n v e s t i g a t i o n o f t h e p r o b l e m s a s s o c i a t e d w i t h t i s s u e s a m p l i n g
f r o m e x p e r i m e n t a l a n i m a l s e s p e c i a l l y w i t h r e f e r e n c e t o t h e f r e e z i n g t e c h n i q u e , a n o x i a , stress
and n a r c o s i s 1 0 9
.
A n i m p o r t a n t a d v a n c e in t h e s t u d y o f b r a i n m e t a b o l i t e s is t h e d e v e l o p m e n t o f a n a p p a r a t u s
w h i c h r e m o v e s a n d freezes t h e b r a i n o f t h e c o n s c i o u s rat w i t h i n a s e c o n d , b y b l o w i n g it o u t o f
t h e cranial v a u l t i n t o a t h i n l a y e r b e t w e e n t w o m e t a l d i s k s p r e v i o u s l y c o o l e d in l i q u i d n i t r o g e n 1 1 6
.
T h e c o m p i l e r s h a v e s u r v e y e d t h e literature f o r t h e p e r i o d 1 9 5 9 - 1 9 7 3 , b u t n o c l a i m is m a d e
that t h e T a b l e s are in a n y w a y c o m p l e t e . F o r a c o m p i l a t i o n o f m e t a b o l i t e c o n c e n t r a t i o n s in
t i s s u e s f o r t h e p e r i o d p r i o r t o 1 9 6 0 refer t o B i o c h e m i s t s ' H a n d b o o k , E d . b y C . L o n g , S p o n
L t d . , L o n d o n , b u t n o t e t h a t f e w o f t h e s e v a l u e s c a n b e c o n s i d e r e d t o r e p r e s e n t in vivo c o n
centrations.
T h e Tables are a r r a n g e d in the following Sections: Page
1. Glycogen, Glucose and Glycolytic Glyceraldehyde-3-phosphate 2274

Intermediates Page Glycerol-3-phosphate 2274
3-Phosphoglycerate 2275
Glycogen 2267
2-Phosphoglycerate 2276
Uridine d i p h o s p h o g l u c o s e 2268
Phosphoenol pyruvate 2276
Glucose 2268
Pyruvate 2276
Glucose-1 - p h o s p h a t e 2269
Lactate 2278
Glucose-6-phosphate 2269
Glucose-1,6-diphosphate 2271
2. Tricarboxylic Acid Cycle
Fructose-1-phosphate 2271
Intermediates
Fructose-6-phosphate 2271
Fructose-l,6-diphosphate 2272 Oxaloacetate 2280
Dihydroxyacetone phosphate 2273 Citrate 2280
* H. J. Hohorst, F. H. Kreutz & Th. Bucher, Biochem. Z . 332, 18 [1959].

** Strictly the values given here a r e " c o n t e n t " n o t " c o n c e n t r a t i o n " because they have n o t been corrected
for the blood content of t h e tissue o r for the extracellular space.
Glycolytic Intermediates 2267
Page Page
Isocitrate 2281 Acetoacetate 2291
Oxoglutarate 2281 3-Hydroxybutyrate 2292
Succinate 2282 Acetate 2293
Fumarate 2282 Malonyl-CoA 2293
Malate 2282 Propionyl-CoA 2293
3. Amino Acids and Ammonia 6. Adenosine and other Nucleotides

Adenosine-5'-triphosphate 2293
Alanine 2284
Adenosine-5'-diphosphate 2295
Aspartate 2284
Adenosine-5'-monophosphate 2296
N-Acetylaspartate 2285
Adenosine-3': 5 '-monophosphate 2296
Glutamate 2285
Uridine-5'-triphosphate 2297
Glutamine 2285
Ammonia 2286 Guanosine-3': 5'-monophosphate 2297
4. Pentose Phosphate Pathway 7. Nicotinamide-Adenine Dinucleotides

Intermediates N i c o t i n a m i d e - a d e n i n e dinu61eotide 2298
N i c o t i n a m i d e - a d e n i n e dinucleotide,
6-Phosphogluconate 2286 reduced 2298
Pentose p h o s p h a t e s 2287 N i c o t i n a m i d e - a d e n i n e dinucleotide
Ribulose-5-phosphate 2287 phosphate 2298
Xylulose-5-phosphate 2287
N i c o t i n a m i d e - a d e n i n e dinucleotide
Sedoheptulose-7-phosphate 2287
p h o s p h a t e , reduced 2299
5. Coenzyme A and Intermediates of

8 . Creatine Phosphate, Creatine, Inorganic
Lipid Metabolism
Pyrophosphate and Phosphate
Coenzyme A 2287 Creatine p h o s p h a t e 2299
Acetyl-CoA 2288 Creatine 2300
Acetyl-carnitine 2288 P y r o p h o s p h a t e , inorganic 2300
Acyl-carnitine 2289 P h o s p h a t e , inorganic 2300
Carnitine 2290
Hydroxymethylglutaryl-CoA 2291 References 2300
1. Glycogen, Glucose and Glycolytic Intermediates
Metabolite Species Tissue State of Anaesthesia Tissue Refer- Notes

animal used content ences
(/xmole/g.
fresh wt.)
Glycogen Rat Liver Fed Ether 304 5

(as glucose) Fed Ether-air 218 3
Fed Nembutal 229 47
Starved (24 hr.) Phenobarbital 141 21
Starved (48 hr.) Ether-air 4.2 3
Alloxan-diabetes Ether 66 5
Alloxan-diabetes Ether-air 12.6 3
AIS*-diabetes Ether 173 5
Diabetes after administration of anti-insulin serum.

2268 Tables. C o n c e n t r a t i o n s of Metabolites in A n i m a l Tissues
Metabolite Species Tissue State of Anaesthesia Tissue Refer Notes

(/imole/g.
fresh wt.)
Glycogen Rat Kidney

(as glucose) (cortex) Fed Phenobarbital 1.6 16 N o change in
(medulla) Fed Phenobarbital 2.9 16 starvation . 16
Brain Fed None 3.8 12

(cortex)
Muscle Fed None 14 13
Fed Ether 24 13
Fed Pentobarbital 25 13
Mouse Liver Fed None 390 8
Fed None 176 56
Brain Fed None 5.5 8 10 day old
Fed None 1.9 26 mice . 10
Fed None 2.2 10

Fed Phenobarbital 2.7 10
Muscle Fed None 18 8
Adipose Fed None 4.2 8
tissue
(epididymal)
Frog Brain Fed None 19.3 55 F o r values for
fish a n d turtle,
see .
55
Uridine di Rat Liver Fed None 0.23 30 Decreases after

phospho Starved (24 hr.) Phenobarbital 0.23 21 injection of
glucose, fructose o r di-
UDPG hydroxy-
acetone . 71
Fed Pentothal 0.32 84 Decreases in

galactosamine-
hepatitis . 84
Mouse Liver Fed Phenobarbital 0.277 69

Starved (24 hr.) Phenobarbital 0.177 69
Kidney Fed Phenobarbital 0.144 69
Brain Fed 0.12 31
Fed 0.17 31
Fed None 0.083 60
Muscle Fed Phenobarbital 0.033 69 Decreases o n
stimulation . 69
G u i n e a pig Brain Fed 0.23 31

Hamster Brain Fed 0.20 31
Rabbit Brain Fed 0.115 31
Glucose Rat Liver Fed Ether 7.1 5
Fed Nembutal 6.85 47
Alloxan-diabetes Ether 35.4 5
AIS*-diabetes Ether 16.7 5

(/imole/g.
fresh wt.)
Glucose Rat Heart Fed Chloralose + 0.59 7 Estimated

urethane intracellular
Starved (24 hr.) Chloralose -f 0.45 7 glucose
urethane (/zmole/ml.) 7
Kidney Fed Phenobarbital 3.7 16 N o change in

(cortex) starvation . 16
(medulla) Fed Phenobarbital 2.6 16

Brain Fed None 1.54 12 F o r effects of
(cortex) Fed None 0.99 52 different
m e t h o d s of
Fed None 1.39 117 freezing . 52
Muscle Fed Inactin 1.84 68

Adipose Fed Ether 0.38 72
Blood Fed Ether 5.40 72
Fed Chloralose + 5.72 7
urethane
Starved (24 hr.) Chloralose + 4.10 7
urethane
Starved (72 hr.) Ether 4.36 72
Mouse Liver Fed None 25 8

Fed None 8.6 56
Brain Fed None 1.05 26 F o r effects of
Fed None 1.54 10 different an
Fed None 2.61 49 aesthetics,
Fed Phenobarbital 2.56 10 see .
51
10 day old
mice . 10
Blood Fed None 7.7 8

G u i n e a pig Heart Fed Ether 2.70 68
Frog Heart Fed 1.31 45
Brain Fed 0.36 55 F o r values for
fish a n d turtle,
see .
55
Glucose-1 - Rat Liver Starved (18 hr.) Ether 0.011 50 F o r values in

phosphate, cirrhotic,
G-l-P undernouris
hed a n d older
rats, s e e . 50
Muscle Fed Ether 0.05 24

Fed Inactin 0.0055 68
Glucoses- Rat Liver Fed None 0.160 28 Decrease o n
phosphate, Fed None 0.266 19 fructose o r
G-6-P Fed Ether-air 0.283 3 glucose
Fed Ether 0.370 2 feeding . 19
Fed Ether 0.195 5 Increases in

Fed Nembutal 0.322 47 anoxia . 28

(/imole/g.
fresh wt.)
Glucoses- Rat Liver Starved (24 hr.) Phenobarbital 0.082 21 F o r values in

phosphate, Starved (48 hr.) None 0.192 28 cirrhotic,
G-6-P Starved (48 hr.) Ether-air 0.088 3 undernourish
Alloxan-diabetes Ether-air 0.118 3 ed and older
Alloxan-diabetes Ether 0.209 5 rats, s e e . 50
AIS*-diabetes Ether 0.329 5 F o r effects of

stress o r nar
cosis, s e e . 1 0 9
Heart Fed Chloralose + 0.32 7

urethane
urethane
A c u t e alloxan- Chloralose -f 0.66 7
diabetes urethane
Kidney Fed None 0.039 28 N o change o n
(cortex) Fed Phenobarbital 0.041 16 starvation . 16
(medulla) Fed Phenobarbital 0.049 16

Starved (48 hr.) None 0.059 28 Decreases in
anoxia . 28
Brain None 0.146 117 Freeze-blown

brain 1 1 6

Adipose Fed Nembutal 0.006 74
Tissue
Mammary Fed Nembutal 0.034 75 See a l s o 1 3 2
gland
Mouse Liver Fed None 0.18 8

Fed None 0.38 56
Fed Ether 0.30 22
Brain Fed None 0.080 10 Decreases in
anoxia . 10
Fed Phenobarbital 0.224 10 10 day old

mice .10
Blood Fed Ether 0.138 22 per ml.

erythrocytes

Brain Fed None 0.11 55 F o r values for
fish a n d turtle,
* Diabetes after administration of anti-insulin serum


(/imole/g.
fresh wt.)
Glucose-1, Mouse Liver Fed Phenobarbital 0.014 69

6-diphosphate,
G-l,6-P2
Heart Fed Phenobarbital 0.012 69

Kidney Fed Phenobarbital 0.014 69
ischaemia . 69
Muscle Fed Phenobarbital 0.045 69 N o change o n

stimulation . 69
Erythrocytes F e d None 0.080 69
Fructose-1- Rat Liver Fed None 0.27 58 H i g h values

phosphate, Fed Nembutal 0-0.19 57 after feeding
F-l-P Fed Urethane 0.7 59 sucrose or 58
Starved (24 hr,) Phenobarbital 0.15 71 fructose . 59
Starved (48 hr.) None 0-0.03 58 H i g h values

after injection
of fruct
ose, ' .
5 7 7 1
Fructoses- Rat Liver Fed None 0.052 28 Decreases o n

phosphate, Fed None 0.070 19 feeding glucose
F-6-P Fed Ether 0.060 3 or f r u c t o s e . 19
Fed Ether 0.112 2 Increases in

Starved (48 hr.) None 0.046 28 F o r values in

Starved (48 hr.) Ether 0.036 3 cirrhotic,
Starved (18 hr.) Ether 0.027 50 undernourish
Alloxan-diabetes Ether 0.018 3 ed a n d older
rats, s e e . 50

urethane
urethane
Acute Chloralose + 0.165 7
Alloxan-diabetes u r e t h a n e
Kidney Fed None 0.013 28
Starved (48 hr.) None 0.013 28
tissue
Mammary Fed Nembutal 0.0075 75
gland
Mouse Liver Fed Ether 0.070 22

Fed Phenobarbital 0.050 10 anoxia . 10
10 d a y old
mice . 10

(/imole/g.
fresh wt.)
Fructose-6- Mouse Blood Fed Ether 0.042 22 per ml.

phosphate, erythrocytes.
F-6-P
Fructose-1,6- Rat Liver Fed None 0.016 35 F o r values in
diphosphate, Fed None 0.027 19 cirrhotic,
F-l,6-P2 Fed Ether 0.022 2 undernourish
Fed Ether 0.038 5 ed a n d older
Fed Nembutal 0.023 47 rats, s e e . 50

urethane
urethane
Alloxan-diabetes u r e t h a n e
Kidney Fed None 0.010 28 Increase in
Fed Ether 0.017 35 anoxia 28

Fed Ether 0.075 35
gland

Fed Ether 0.024 22
Brain Fed None 0.060 26 Rises in
Fed None 0.120 10 anoxia 10
Fed Phenobarbital 0.027 10 10 day old

mice 10
Blood Fed 0.051 22 per ml.

erythrocytes
Cat Brain Fed Phenobarbital 0.023 11
fish and turtle,
see .
55
* Diabetes after administration of anti-insulin serum.


0*mole/g.
fresh wt.)
Fructose-1,6- Cow Liver Non-lactating Xylotox local 0.062 9

diphosphate, anaesthesia
F-l,6-P2 Lactating Xylotox local 0.039 9
anaesthesia
Ketotic Xylotox local 0.026 9
anaesthesia
Dihydroxy- Rat Liver Fed None 0.027 19 N o change o n
acetone Fed None 0.035 35 feeding fruct
phosphate, Fed Ether 0.038 2 ose or glu
DAP Fed Ether 0.044 5 cose .
19

Starved (24 hr.) Phenobarbital 0.010 21 F o r values in
Starved (24 hr.) Phenobarbital 0.013 71 cirrhotic,
Alloxan-diabetes Ether 0.020 5 undernourish
AIS*-diabetes Ether 0.026 5 ed and older
rats, s e e . 50
Kidney Fed Ether 0.025 35

Fed Ether 0.024 35
Muscle Fed Ether 0.031 14 N o change
Fed Ether 0.036 35 during con
Fed Phenobarbital 0.040 25 tractions . 35
or ether
tissue
gland
erythrocytes.
Fed Ether 0.043 22
Brain Fed None 0.046 10 Increase in

Fed Phenobarbital 0.013 10 anoxia . 10
10 day old
mice .10

erythrocytes.

fish a n d turtle,


(/imole/g.
fresh wt.)
Glyceralde- Rat Liver Fed 0.011 67 F o r values in

hyde-3-phos- Fed Ether 0.012 27 cirrhotic,
phate, G A P Fed Nembutal 0.0014 47 undernourish
Starved (18 hr.) Ether 0.017 50 ed a n d older
Starved (72 hr.) 0.017 67 rats, s e e . 50
Alloxan-diabetes 0.010 67
Fed Inactin < 0.0005 68
gland
Mouse Brain Fed Phenobarbital 0.0009 10 10 day old
mice 10
Increases in
anoxia 10
G u i n e a Pig Heart Fed Ether 0.0027 68

Glycerol-3- Rat Liver Fed None 0.700 19 Decreases on
phosphate Fed None 0.140 28 feeding
Fed Ether 0.253 2 fructose or
Fed Ether 0.404 5 glucose . 19

Starved (24 hr.) Phenobarbital 0.142 21 Increases in
Starved (24 hr.) Phenobarbital 0.164 71 ischaemia 28
Starved (48 hr.) None 0.230 28 and after ethanol

Alloxan-diabetes Ether 0.303 5 injection . 118
AIS*-diabetes Ether 0.246 5 F o r values in

cirrhotic,
undernourish
ed and older
rats, s e e . 50

urethane
urethane
Alloxan-diabetes u r e t h a n e 0.028 7
Kidney Fed None 0.115 28 Increases in
Fed Nembutal 0.131 28 ischaemia . 28

Muscle Fed Ether 0.130 14 Increase
Fed Phenobarbital 0.120 25 during con
tractions . 25
(abdomen) Fed Inactin 0.0055 68

tissue
gland


(/zmole/g.
fresh wt.)
Glycerol-3- Rat Blood Fed Ether Not 5

phosphate detectable

Fed Ether 0.30 22
Brain Fed Phenobarbital 0.048 10 10 day old
mice . 10
G u i n e a Pig Heart Fed Ether 0.076 68
Cat Brain Fed Phenobarbital 0.038 11 Increases in

ischaemia . 11

fish a n d turtle,
see .
55
Cow Liver Non- Xylotox local 0.68 9

Lactating anaesthesia
Lactating X y l o t o x local 0.73 9
anaesthesia
anaesthesia
3-Phospho Rat Liver Fed None 0.284 28 Decreases in

glycerate, Fed Ether 0.380 2 anoxia . 28
3-PG Fed Ether 0.296 5 F o r values in

Starved (18 hr.) Ether 0.572 50 cirrhotic,
Starved (24 hr.) Phenobarbital 0.076 21 undernourish
Starved (48 nr.) None 0.292 28 ed a n d older

Kidney Fed None 0.085 28 Decreases in

gland
Blood Fed 0.041 2

Brain Fed Phenobarbital 0.025 10 10 day old
mice . 10
Increases in
anoxia . 10
Blood Fed 0.225 22 per ml.

erythrocytes.


(//mole/g.
fresh wt.)
2-Phospho- Rat Liver Fed None 0.034 28 Decreases in

glycerate, Starved (18 hr.) Ether 0.150 50 anoxia . 28
2-PG Starved (48 hr.) None 0.028 28 F o r values in

cirrhotic,
undernourish
ed a n d older
rats, s e e . 50

gland
Mouse Brain Fed Phenobarbital 0.0028 10 10 day old
mice . 10
Increases in
anoxia . 10

Phosphoenol Rat Liver Fed None 0.099 28 Decreases in
pyruvate, P E P Fed Ether 0.117 2 anoxia . 28
Fed Ether 0.113 5 F o r values in

Starved None 0.130 28 cirrhotic,
Starved (18 hr.) Ether 0.397 50 undernourish
Starved (24 hr.) Phenobarbital 0.037 21 ed a n d older

Kidney Fed None 0.035 28 Increases in
Fed Pentobarbital 0.097 15 acidosis . 15
Fed Nembutal 0.047 28 Decreases in

Starved None 0.051 28 anoxia . 28

Fed None 0.0048 117
gland
Brain Fed Phenobarbital 0.0035 10 10 days old
mice . 10
Increases in
anoxia . 10

Pyruvate Rat Liver Fed None 0.130 1 Increases after
Fed Ether 0.154 2 administration
Fed Ether 0.252 5 of fructose, di
Fed Ether-air 0.212 3 hydroxyacet
Fed Nembutal 0.138 47 one or glyce
Starved (24 hr.) Phenobarbital 0.080 21 rol ' .
7 0 7 1


0«nole/g.
fresh wt.)
Pyruvate Rat Liver Starved (24 hr.) Phenobarbital 0.040 71

Starved (48 hr.) Ether-air 0.073 3
Alloxan-diabetes None 0.054 1
Heart Fed Ether 0.030 16
Fed Chloralose + 0.040 7
urethane
urethane
Alloxan-diabetes Chloralose + 0.038 7
urethane
Kidney Fed Ether 0.043 46 Decreases in
Fed Ether 0.091 35 anoxia .28
Fed Nembutal 0.089 6 N o significant

Fed Pentobarbital 0.120 15 change in
Starved (48 hr.) None 0.038 6 acidosis . 15
Starved (48 hr.) Nembutal 0.056 6

(cortex) Fed None 0.086 117
Fed None 0.12 35
Fed Ether 0.12 35
(base) Fed None 0.158 12
(thigh) Fed Pentobarbital 0.078 13
(skel.) Fed Ether 0.097 14
Fed Ether 0.080 25
(abd.) Fed Inactin 0.116 68
tissue Fed Nembutal 0.178 74
gland
Whole Fed Ether 0.085 72
blood Fed Ether 0.190 2
Fed Ether 0.210 46
Plasma Fed Ether 0.234 2
Fed None 0.23 8
Fed Ether 0.17 22
Brain Fed None 0.091 17 F o r effect of
Fed None 0.144 56 thiamine de
Fed 0.164 49 ficiency, s e e . 56
F e d (10 d a y Phenobarbital 0.039 10

old mice)

Oumole/g.
fresh wt.)
Pyruvate G u i n e a pig Heart Fed Ether 0.022 68

Dog Blood Starved (18 hr.) Chloralose + 0.094 48
(art.) Urethane
A c u t e alloxan- Chloralose + 0.042 48
diabetes Urethane
Cat Brain Fed Phenobarbital 0.065 11 Little change
Fed Pentobarbital 0.089 11 during
anoxia . 11
Cow Liver Fed Local 0.033 73

Non-lactating Local 0.065 9
Lactating Local 0.045 9
Ketotic Local 0.026 9
Lactate Rat Liver Fed None 1.62 1 Increases rapid

Fed Ether 1.54 2 ly during isch
Fed Ether 2.64 5 aemia ' - . 4 6 79
Fed Ether-air 1.53 3 D r i n k i n g water

Fed Nembutal 1.50 47 contained 1 0 %
Starved (24 hr.) Phenobarbital 0.95 21 sucrose . 5
Starved (24 hr.) Phenobarbital 0.45 71 Increases after

Starved (48 hr.) None 0.78 1 administration
None 0.54 70 of glycerol, di
Starved (48 hr.) Ether-air 0.66 3 hydroxyacet
Alloxan-diabetes N o n e 2.37 1 one o r fruc
Alloxan-diabetes Ether 1.52 4 tose ' 7 0 7 1
Alloxan-diabetes Ether 3.01 5 Considerably

AIS*-diabetes Ether 1.80 5 lower values
for alloxan-
diabetes have
been reported . 3

Fed Chloralose -f 0.57 7
urethane
urethane
Alloxan-dia Chloralose + 1.00 7
betes urethane
Kidney Fed None 0.47 28 Increases dur
Fed Ether 0.65 14 ing isch
Fed Nembutal 1.17 28 aemia * . 16 28
Starved (48 hr.) None 0.42 28 F o r distribut

Starved (48 hr.) Nembutal 0.83 28 ion of lactate
cortex Fed Phenobarbital 0.65 16 in renal cortex
medulla Fed Phenobarbital 1.1 16 and medulla,
see .
16


(/imole/g.
fresh wt.)
Lactate Rat Brain Fed None 1.7 51 Decreases if

cortex Fed None 1.29 117 phenobarbital
Fed None 1.94 12 chloroform,
base Fed Ether 1.26 14 ether o r
Fed None 2.98 12 chlorpromazi-
ne are used for
anaesthesia . 51
F o r a study o n
the effects of
different tech
niques of freez
ing brains,
see ' .
5 2 1 1 6

skel. Fed Ether 4.65 13
Fed Pentobarbital 1.44 13
o r ether
abdm. Fed Inactin 0.90 68
gland
Fed Ether 0.95 72
Fed Ether 2.23 2
plasma Fed Ether 2.96 2
Fed None 2.8 8
Fed None 2.6 56
Brain Fed None 2.26 10 Increases ra
Fed None 1.20 26 pidly d u r i n g
Fed None 1.86 49 anoxia ' . 1 0 2 6
F e d (10 days Phenobarbital 0.77 10

old)
Dog Blood Fed Chloralose + 1.42 48
(art.) urethane
Alloxan-dia Chloralose -f 1.12 48
betes urethane
Cat Brain Fed Phenobarbital 0.83 11 Increases ra
Fed Pentobarbital 1.83 11 pidly during
anoxia . 11
Frog Heart Fed None 4.41 45

Non-lactating Local 1.64 9
Lactating Local 1.37 9
2. Tricarboxylic Acid Cycle Intermediates

(/miole/g.
fresh wt.)
Oxaloacetate Rat Liver Fed Ether 0.007 4

Fed Ether 0.0037 36
Fed Ether 0.0062 2
Alloxan-dia Ether 0.0022 36
betes
betes
betes
(leg)
(abd.) Fed Ether 0.002 24
Mouse Brain Fed None 0.004 17
G u i n e a pig Liver Fed Ether 0.0012 38
Ketotic Ether 0.0008 38
Ketotic Ether 0.0014 38
Cow Liver Fed Local 0.012 73 M e a s u r e d with
Non-lactating Local 0.0022 9 a colorimetric
Lactating Local 0.0019 9 assay . 73

Citrate Rat Liver Fed Ether 0.17 54 Increases after

Fed Ether-air 0.26 3 administration
Fed Ether 0.21 34 of l a c t a t e .
21
Fed Ether 0.19 5 Decreases with

Fed Nembutal 0.23 47 a high fat
Starved (24 hr.) Ether 0.21 34 diet .
34
Starved (24 hr.) Phenobarbital 0.15 21 N o change in

Starved (27 hr.) Ether-air 0.15 3 riboflavin de
Starved (48 hr.) Ether-air 0.13 3 ficiency . 111

Alloxan-dia Ether-air 0.18 3
betes
betes
betes
betes
* Diabetes after administration of anti-insulin serum

Tricarboxylic Acid Cycle Intermediates 2281

(/xmole/g.
fresh wt.)
Citrate Rat Heart Fed Chloralose + 0.30 7 Corrected

urethane from dry
Fed Pentobarbital 0.44 20 weight
Starved (24 hr.) Chloralose + 0.80 7 values . 20
urethane
Alloxan-diabetes Chloralose + 0.97 7 Values f o r 7
urethane o b t a i n e d by
Alloxan-diabetes P e n t o b a r b i t a l 1.26 20 colorimetric
method.
Kidney Fed Nembutal 0.39 28 Decreases in
Fed Pentobarbital 0.43 15 acidosis ' . 15 28
Starved (48 hr.) N o n e 0.37 28

Mouse Brain Fed None 0.32 17 Decreases in

Fed None 0.22 26 anoxia . 17
N o change
d u r i n g con
vulsions . 26
Cow Liver Fed Local 0.21 73 Increases after

Ketotic Local 0.07 73 glucocorticoid
Starved (6 days) Local 0.075 115 treatment 90

Starved (6 days) N o n e 0.025 115
Isocitrate Rat Liver Fed None 0.020 39

Fed Ether 0.021 2
Heart Fed Pentobarbital 0.026 20
Alloxan-diabetes P e n t o b a r b i t a l 0.066 20
Oxoglutarate Rat Liver Fed None 0.14 1 Decreases in

Fed Nembutal 0.14 47 anoxia ' . 6 7 9
Starved (48 hr.) None 0.108 70 Increases after

Alloxan-diabetes None 0.045 1 dihydroxy-
acetone ad
ministration . 70
N o change in
riboflavin
deficiency . 111
Heart Fed Pentobarbital 0.054 20 Corrected to

Fed Chloralose + 0.078 7 wet wt. b a s i s . 20
urethane
urethane

(jumole/g.
fresh wt.)
Oxoglutarate Rat Kidney Fed Pentobarbital 0.11 15 Decreases in

Fed Pentobarbital 0.11 37 acidosis - . 1 5 2 8 , 3 7

(abd.) Fed Inactin 0.018 68
Mouse Liver Fed None 0.052 56 F o r effects of
of thiamine
deficiency,
see .56

Fed None 0.11 56 anoxia . 17

Cow Liver Non- Xylotox-local 0.11 9 Increases after
Lactating anaesthesia glucorticoid
Lactating Xylotox-local 0.092 9 treatment . 90
anaesthesia
Ketotic Xylotox-local 0.044 9
anaesthesia
Succinate Rat Liver Fed Ether 0.74 111 Increases in
riboflavin
deficiency . 111
Mouse Brain Fed None 0.69 17 Rises in

anoxia . 17
Fumarate Rat Liver Fed Ether 0.075 111 N o change in

riboflavin
deficiency . 111

anoxia . 17
Malate Rat Liver Fed None 0.34 19 Increases on

Fed Ether 0.50 4 feeding
Fed Ether 0.38 5 glucose or
Fed Ether 0.44 2 fructose . 19
Fed Ether 0.44 2

Fed Ether 0.28 36 Increases after
Fed Nembutal 0.42 47 lactate ad
Starved (24 hr.) Phenobarbital 0.22 21 ministration . 21

Alloxan-diabetes Ether 0.42 36 Increases on
Alloxan-diabetes Ether 0.55 4 ethanol
Alloxan-diabetes Ether 0.61 5 infusion 47
AIS*-diabetes Ether 0.40 5 or i n j e c t i o n . 118

Tricarboxylic Acid Cycle Intermediates 2283

(^mole/g.
fresh wt.)
Malate Rat Heart Fed Chloralose + 0.16 7 Corrected

urethane from dry
Fed Pentobarbital 0.12 20 weight b a s i s . 20

urethane
urethane
Kidney Fed Pentobarbital 0.45 15 Decreases in
Fed Nembutal 0.37 28 acidosis .1 5 , 2 8
Starved (48 hr.) None 0.21 28 Rises in

anoxia .28

Muscle Fed Pentobarbital 0.10 25 Increases
or ether during
(abd.) Fed Inactin 0.038 68 contraction . 25

gland
Fed Ether 0.031 2
Plasma Fed Ether not 2
detect
able
Mouse Brain Fed None 0.44 17 N o change in
anoxia .17

Cat Brain Fed Phenobarbital 0.091 11 Increases in
ischaemia . 11

Cow Liver Non- Xylotox-local 0.51 9 Increases after
lactating anaesthesia glucocorticoid
Lactating Xylotox-local 0.53 9 treatment . 90
anaesthesia
anaesthesia
3. Amino Acids and Ammonia

(jumole/g.
fresh wt.)
Alanine Rat Liver Fed None 1.23 78 Rises in

Fed None 0.87 79 ischaemia ' . 78 79
Starved (48 hr.) None 0.46 78 Increases

Starved (48 hr.) None 0.38 6 after 130
Alloxan-diabetes None 0.47 78 a m m o n i a load.

Brain Fed None 0.57 6 Rises in
ischaemia . 6

ischaemia . 89
Cow Liver Normal Xylotox-local 1.57 9

lactating anaesthesia
Non- Xylotox-local 1.64 9
anaesthesia
Aspartate Rat Liver Fed None 1.19 6 N o change in

Fed None 0.74 6 ischaemia . 79
Fed Nembutal 0.70 47 Increases

Starved (48 hr.) None 0.63 6 after 130
Starved (48 hr.) None 0.76 78 ammonia

Alloxan-diabetes None 0.35 78 load.
Kidney Fed None 0.39 6 Rises in

Fed Pentobarbital 1.18 15 ischaemia and
N o change in
acidosis . 15
Heart Fed None 2.72 6

Brain Fed None 2.34 6 N o change in
Starved (72 hr.) N o n e 2.42 119 ischaemia . 6

Starved (72 hr.) E t h e r 0.024 72
Fed Ether 0.029 72
Fed None 2.20 26 ischaemia;
slight rise in
hypo
glycemia . 88
Cow Liver Normal Xylotox-local 0.80 9

Non- Xylotox-local 0.46 9
anaesthesia
A m i n o Acids a n d A m m o n i a 2285

(/miole/g.
fresh wt.)
N-Acetylasp- Mouse Brain Fed None 6.00 88 N o change in

artate ischaemia or in
hypo
glycemia . 88
Glutamate Rat Liver Fed None 2.41 1 N o change in

Fed Nembutal 2.39 47 ischaemia ' . 6 79

Kidney Fed Pentobarbital 7.30 37 Decreases in

Fed Nembutal 3.01 28 acidosis . 37
Starved (48 hr.) Nembutal 2.70 28 Increases in

ischaemia . 28

Starved (72 hr.) N o n e 8.6 119 ischaemia 6
Muscle Fed None 1.6 80

(leg) Starved (48 hr.) None 1.0 80

tissue
Fed Ether 0.091 6
Mouse Liver Fed None 3.68 56 N o change in

thiamine
deficiency 56
Brain Fed None 12.7 56 N o c h a n g e in

cortex Fed None 11.8 77 thiamine
cerebellum Fed None 8.7 77 deficiency . 56
Cow Liver Normal non- Xylotox local 3.47 9

Lactating Xylotox local 3.06 9
anaesthesia
anaesthesia
Glutamine Rat Liver Fed None 3.54 80 Increase o n

Fed None 5.03 6 high C H O
Fed None 4.35 78 diet a n d high
Starved (48 hr.) None 5.10 80 protein d i e t 80

Starved (48 nr.) None 4.43 78 Only slight
Alloxan-diabetes None 2.15 78 decrease in
short term
ischaemia 79
Kidney Fed None 1.72 6 Falls in

Fed Pentobarbital 1.60 37 ischaemia a n d


(/imole/g.
fresh wt.)
Glutamine Cow Liver Normal Xylotox-local 2.56 9

Non-lactating Xylotox-local 3.28 9
anaesthesia
anaesthesia
Ammonia Rat Liver Fed None 0.47 1 N o change
Fed None 0.36 114 during 2 min.

Kidney Fed Nembutal 0.88 6 Increases in
Starved (48 hr.) Nembutal 1.71 6 acidosis 28
Brain Fed None 0.34 6 Increases in

Starved (72 hr.) N o n e 0.39 119 ischaemia 6
Muscle Fed None 0.27 64 Increases in

(leg) Fed Ether 0.25 6 tetany 64

(art.)
Brain Fed None 0.36 65 Increases in
Fed None 0.24 77 ischaemia . 77
4. Pentose Phosphate Pathway Intermediates

(/imole/g.
fresh wt.)
6-Phospho Rat Liver Fed None 0.019 67 Decrease on

gluconate Starved (24 hr.) N o n e 0.020 67 refeeding a
Alloxan-diabetes N o n e 0.033 67 high fat d i e t . 67
Brain Fed None 0.012 96 100-fold

increase on
administration
of 6-amino-
nicotin-
amide - .
9 6 1 1 0
Adipose Fed None 0.0013 74

tissue
Mouse Liver Fed None 0.021 56 N o change in
thiamine
deficiency . 56
C o e n z y m e A a n d Intermediates of Lipid M e t a b o l i s m 2287
Metabolite Species Tissue State of Anaesthesia Tissue Refer- N o t e s

(/imole/g.
fresh wt.)
6 Phospho- Mouse Brain Fed None 0.008 56 Rises in

gluconate Fed None 0.010 81 thiamine
deficiency . 56
Falls in
ischaemia . 81
Pentose Rat Liver Fed None 0.429 67 Decreases o n

phosphates Starved (72 hr.) N o n e 0.236 67 refeeding high
Alloxan-diabetes N o n e 0.345 67 fat d i e t .
67
Ribulose-5- Rat Liver See pentose

phosphate phosphates
aa nnHd 67
Mouse Brain Fed None 0.007 81 Rises d u r i n g

nembutal
anaesthesia . 81
Xylulose-5- Rat Liver See pentose

phosphate phosphate
and 6 7

Fed None 0.014 81 thiamine
deficiency . 56
Falls in
ischaemia;
rises in
nembutal
anaesthesia . 81
Sedoheptulose Rat Liver Fed None 0.026 67

-7-phosphate Starved (72 hr.) N o n e 0.017 67
Alloxan-diabetes N o n e 0.046 67
5. Coenzyme A and Intermediates of Lipid Metabolism

(/zmole/g.
fresh wt.)
Coenzyme A, Rat Liver Fed None 0.135 97

CoA* Fed None 0.125 70
tissue
F o r values of total acid soluble C o A , see 1


(/xmole/g.
fresh wt.)
C o e n z y m e A, Mouse Brain Fed None 0.077 49

CoA*
Sheep Liver Fed None 0.050 112 F o r values in
alloxan-
diabetes, s e e 1 1 2

Cow Liver Lactating Local 0.012 120
Acetyl-CoA** Raatt
R Liver Fed None 0.027 70 Decrease in
Fed Ether-air 0.029 3 ischemia 41
Fed Ether 0.022 34 Decreases

Fed Nembutal 0.041 41 after e t h a n o l
Fed Nembutal 0.040 47 infusion . 47

Starved (48 hr.) None 0.076 70 For a com
Starved (48 hr.) Ether 0.120 41 parative study
Starved (48 hr.) Ether-air 0.082 3 of techniques
Starved (48 hr.) Nembutal 0.102 3 of sampling,
Starved (48 h r . ) Nembutal 0.120 41 see " .
Alloxan-diabetes Ether-air 0.080 3
Starved (24 hr.) None 0.0054101 hypoxia . 101

alloxan-dia
betes, s e e . 1 1 2

Acetyl Rat Liver Fed None 0.041 41 Decreases dur
carnitine Fed Nembutal 0.074 41 ing a n o x i a . 41
Starved None 0.095 41

( 3 6 - 4 8 hr.)
Starved Ether 0.33 41
( 3 6 - 4 8 hr.)
Starved Nembutal 0.29 41
( 3 6 - 4 8 hr.)
( 3 6 - 4 8 hr.)
( 3 6 - 4 8 hr.)
Alloxan-dia- None 0.20 41
betes
* F o r values of total acid soluble C o A , s e e . 1 0 2
** Values in the literature prior to 1965 are liable t o be in error 1


(/miole/g.
fresh wt.)
Acetyl Rat Kidney Fed None 0.093 41

carnitine Fed Nembutal 0.12 41
( 3 6 - 4 8 hr.)
( 3 6 - 4 8 hr.)
Brain Fed None <0.01 41
Muscle Fed None <0.05 41
Diaphragm Fed None 0.21 41
Adrenal Fed None <0.02 41
Testis Fed None 0.047 41
Epididymis Fed None 0.54 41

Epididymal Fed None < 0.002 41
fat p a d
alloxan-dia
betes, s e e . 1 1 2

Rat Liver Fed None 0.011 41

(acid insoluble) Fed None 0.009 43*
Fed Nembutal < 0.010 41
( 3 6 - 4 8 hr.)
( 3 6 - 4 8 hr.)
Starved Nembutal < 0.010 41
( 3 6 - 4 8 hr.)
Heart Fed None 0.12 43* see a l s o .
4 1

Fed None 0.026 43*
Fed Nembutal <0.01 41
( 3 6 - 4 8 hr.)
Starved Nembutal <0.01 41
( 3 6 - 4 8 hr.)
Brain Fed None 0.00 43*
Muscle Fed None 0.081 43*
Adrenal Fed None 0.36 43*
Testis Fed None 0.046 43*
* Recalculated from g. dry weight basis.


(/imole/g.
fresh wt.)
Liver Fed None 0.17 41 Increases dur

Fed None 0.26 42* ing a n o x i a .
41
Fed None 0.19 43*

( 3 6 - 4 8 hr.)
( 3 6 - 4 8 hr.)
( 3 6 - 4 8 hr.)
Fed None 0.18 42*
Fed None 0.13 43*
Fed None 0.25 44*
( 3 6 - 4 8 hr.)
( 3 6 - 4 8 hr.)
Heart Fed None 0.30 41 Lower values
Fed None 1.23 42* in hearts of
Fed None 0.50 43* neonatal
Fed None 0.97 44* rats .
1 0 4

( 3 6 - 4 8 hr.)
Alloxan- None 0.17 41
diabetes
Fed None 0.65 42*
Fed None 0.33 43*
Fed None 0.54 44*
Fed None 0.22 42*
Fed None 0.075 43*
Fed None 0.11 44*
Diaphragm Fed None 0.28 41
Adrenal Fed None 0.18 41

Fed None 7.86 42*
Fed None 7.75 43*
Testis Fed None 0.091 41
Fed None 0.82 42*
Fed None 2.06 43*
Fed None 0.35 44*
Epididymis Fed None 6.20 41


(//mole/g.
fresh wt.)
Carnitine Rat Epididymal Fed None 0.01 41

(cont.) fat p a d .
Pancreas Fed None 0.43 42*
Lung Fed None 0.16 42*
Brown Fed None 0.63 44*
adipose
Sheep Liver Fed None 0.074 112
Hydroxy- Rat Liver Fed Ether 0.023' 53
methyl- Starved (24 hr.) Ether 0.016 53
glutaryl-CoA Starved (48 hr.) Ether 0.017 53
Alloxan-dia- Ether 0.026 53
betic
Acetoacetate Rat Liver Fed None 0.055 1 Increases o n

Fed None 0.057 62 fat f e e d i n g . 66
Fed Ether 0.156 66

Starved (48 hr.) None 0.69 62 Falls d u r i n g
Starved (24 hr.) None 0.63 62 anoxia and
6 , 7 9
Alloxan-dia None 3.75 1 o n injection

betes of e t h a n o l . 1 1 8
Kidney Fed Nembutal 0.032 6

Heart Fed Chloralose- 0.020 7
urethane
Starved (24 hr.) Chloralose- 0.11 7
urethane
Alloxan-dia Chloralose- 0.74 7
betes urethane
Blood Fed None 0.076 61 ^mole/ml.
Fed None 0.20 62 blood. High
Starved (12 hr.) None 0.29 63 values in suck
Starved (24 hr.) None 0.80 62 ling r a t s .1 0 3

Cow Liver Non-lactating X y l o t o x (local) 0.01 9
Lactating X y l o t o x (local) 0.01 9
Ketotic Xylotox (local) 0.53 9
Normal 0.068 73
Starved 0.130 73
Ketotic 0.280 73

(/miole/g.
fresh wt.)
Acetoacetate Cow Blood Non-lactating Xylotox (local) 0.011 9 /miole/ml.

Lactating Xylotox (local) 0.019 9 blood
Starved (6 days) None 0.80 115
Sheep Blood Fed None 0.026 113
(10 days)
Dog Blood Fed Chloralose- 0.026 48 /miole/ml.
(arterial) urethane blood
betes urethane
3-Hydroxy- Rat Liver Fed None 0.144 1 Increases o n

butyrate Fed Ether 0.288 66 fat f e e d i n g . 66
Fed Nembutal 0.144 47 Increases

Starved (48 hr.) None 1.79 1 after ethanol
Alloxan-dia None 7.73 1 infusion . 47
betes
Kidney Fed Nembutal 0.088 6
Heart Fed Chloralose- 0.11 7
urethane
Starved (24 hr.) Chloralose- 0.50 7
urethane
betes urethane
Blood Fed None 0.082 61 /miole/ml.
Fed None 0.08 62 blood. High
Starved (12 hr.) None 0.59 63 values in suck
Starved (24 hr.) None 2.10 62 ling r a t s .
1 0 3

Cow Liver Non-lactating Xylotox (local) 0.50 9
Lactating Xylotox (local) 0.51 9
Normal 0.41 73
Fasted 0.67 73
Ketotic 1.83 73
Blood Non-lactating Xylotox (local) 0.27 9 /miole/ml.

Lactating Xylotox (local) 0.34 9 blood
Starved (6 days) None 2.86 115
Sheep Blood Fed None 0.252 113 /miole/ml.

Starved None 0.723 113 blood
(10 days)
Dog Blood Fed Chloralose- 0.028 48 ^mole/ml.

(arterial) urethane blood
Alloxan-diabetes Chloralose- 3.46 48
urethane
Adenosine a n d other Nucleotides 2293

Gumole/g.
fresh wt.)
Acetate Rat Liver Fed 0.46 40

Starved (20 hr.) 0.27 40
Heart Fed 0.11 40
Kidney Fed 0.24 40
Starved (20 hr.) 0.13 40
Muscle Fed 0.03 40
Spleen Fed 0.16 40
Malonyl-CoA Rat Liver Fed None 0.013 121

Fat-fed None 0.004 121
Meal-fed None 0.025 121
Propionyl-CoA Rat Liver Fed Pentobarbital 0.0088 122

Starved (48 hr.) 0.011 122
6. Adenosine and other Nucleotides

(/imole/g.
fresh wt.)
Adenosine Rat Liver Fed None 2.45 6 R a p i d de

s'-triphos Fed Ether 2.10 2 crease in
phate., Fed Ether 2.8 27 ischaemia 6,79
ATP* Fed Ether 2.45 3 Decreases on

Fed Ether 3.65 34 fat f e e d i n g 34
Fed 2.2 32 Decreases

Fed Ether 2.85 54 after ad
Fed 2.6 33 ministration of
Fed Nembutal 3.02 47 fructose ; no
71
Starved (24 hr.) Phenobarbital 2.81 71 c h a n g e after

Starved (48 hr.) None 2.31 70 dihydroxy
Starved (48 hr.) Ether 1.86 3 acetone .
7 0 , 7 1
Starved (48 hr.) Ether 3.50 34 Decreases o n

Alloxan-diabetes Ether 2.33 3 fat-feeding a n d
Alloxan-diabetes Ether 3.24 34 in alloxan-
diabetes . 54
F o r values in
n e w b o r n rat
liver, see, . 8 2
F o r effects of
stress o r
109
narcosis, see
* These values may be 1 0 - 1 5 % t o o high d u e to interference in the assay m e t h o d s from o t h e r nucleotides. F o r

results by a m o r e specific m e t h o d , see p . 2078 a n d .
8 3

(/miole/g.
fresh wt.)
Adenosine- Rat Heart Fed Chloralose + 4.34 7 F o r other

5'-triphos urethane values,
phate., Starved Chloralose + 4.45 7 see p . 2086.
ATP* urethane
urethane
Kidney Fed None 1.43 6 Rapid
Fed Nembutal 1.71 6 decrease in
Starved (48 hr.) None 1.38 6 ischaemia ' . 6 16
Starved (48 hr.) Nembutal 1.99 6 F o r content of

cortex a n d
medulla, s e e . 16
N o change in
acidosis . 6
Muscle Fed Ether 6.00 24 N o change

(abd.) Fed Inactin 5.78 68 in t e t a n y .24
(thigh) Fed None 2.38 13 F o r other

Fed Ether 3.62 13 values,
Fed Pentobarbital 7.25 13 see p . 2086
tissue
gland
Submaxillary F e d Ether 1.24 123
gland Fed Nembutal 1.30 131
Mouse Liver Fed None 1.66 56 F o r effects of

Fed Ether 3.30 22 pyrithiamine,
see 5 6
Brain Fed None 2.92 49 F o r effects

Fed None 3.27 56 of anaesthesia,
Fed None 2.95 17 see 1 7

fish a n d turtle,
see .5 5
These values m a y be 1 0 - 1 5 % t o o high due to interference in the assay m e t h o d s from other nucleotides.
F o r results by a m o r e specific m e t h o d , see p. x x and . 8 3
Adenosine and other Nucleotides 2295

Oimole/g.
fresh wt.)
Adenosine-5'- Rat Liver Fed None 1.17 6 No large

diphosphate, Fed Ether 0.90 2 change in
ADP* Fed Ether 0.49 27 ischaemia ' . 6 79
Fed Ether 0.76 3 For values in

Fed Ether 0.67 34 newborn rat
Fed Ether 1.08 54 liver, see . 8 2
Fed 1.8 32
Fed 1.0 33 For effects
Starved (48 hr.) None 1.45 70 of stress or
Starved (48 hr.) Ether 0.82 3 narcosis, s e e 109
Starved (48 hr.) Ether 0.91 34 For other

Starved (48 hr.) Ether 1.46 54 values,
Alloxan-diabetes Ether 0.89 3 see p. 2086.
Kidney Fed None 1.34 28 No large
Fed Nembutal 0.96 28 change in
Starved (48 hr.) None 1.27 28 ischaemia or
Starved (48 hr.) Nembutal 0.91 28 acidosis . 28

Muscle Fed Ether 0.70 24 For effects of
(abdomen) Fed Inactin 0.64 68 anaesthesia,
(thigh) Fed None 0.82 13 see . 1 3
Ether 0.60 13
Pentobarbital 0.48 13
tissue
Mammary Fed Nembutal 0.170 75 See a l s o 132
gland
Submaxillary Fed Ether 0.74 123
gland Fed Nembutal 0.26 131
Brain Fed None 0.33 17 For the effects
Fed None 0.41 26 of anaesthesia,
c e k t 3 t 17
Guinea pig Heart Fed Ether 0.67 68

These values may be 10-15% too high due to interference in the assay methods from other nucleotides. For
results by a more specific method, see p. x x and . 8 3

(/imole/g.
fresh wt.)
Adenosine-5 '- Rat Liver Fed None 0.30 6 Large increase

mono Fed 1.0 32 in
phosphate, Fed 0.33 33 ischaemia . 6,79
AMP Fed Ether 0.20 27 F o r values in

Fed Ether 0.13 3 n e w b o r n rat
Fed Ether 0.21 34 liver, see . 8 2
Starved (48 hr.) None 0.33 70 F o r other values,

Starved (48 hr.) Ether 0.18 3 see p . 2086.
Starved (48 hr.) Ether 0.31 34 F o r effects
Alloxan-diabetes Ether 0.23 3 of stress or
Alloxan-diabetes Ether 0.27 34 narcosis, s e e 1 0 9
Kidney Fed None 0.42 28 Increases

Fed Nembutal 0.25 28 rapidly in
Starved (48 hr.) None 0.43 28 ischaemia . 28
Starved (48 hr.) Nembutal 0.21 28 N o change in

acidosis . 28
Muscle Fed Ether 0.3 24 F o r effects of

(abdomen) Fed Inactin 0.17 68 anaesthesia,
(thigh) Fed None 0.072 13 see .
Fed Ether 0.041 13
tissue
gland
Submaxillary F e d Ether 0.58 123
gland
Mouse Brain Fed None 0.050 17 F o r the effects

of anaesthesia,
see 17

Adenosine- Rat Liver Fed None 1.4** 124 Decreases 30
3':5'- min. after
monophosphate, insulin
A-3:5-MP* injection 93
Fed Pentobarbital 0.49** 93 N o change

Fed Pentobarbital 1.2** 125 on adrena
Fed Inactin 0.44** 126 lectomy 98
Starved None 1.2** 98

Alloxan- Pentobarbital 0.96** 93
diabetes
* F o r relative a m o u n t s of A-3 : 5 - M P a n d G-3 : 5-MP in various tissues, s e e . 87
* nmole/g. fresh weight!

A d e n o s i n e a n d other Nucleotides 2297

Ozmole/g.
fresh wt.)
Adenosine-3': Rat Kidney Fed None 1.5** 124

5'-mono Fed Inactin 0.8-1.8** 126
phosphate, Brain Fed None 5** 124
A-3:5-MP* Fed None 2** 98
Skeletal Fed Pentobarbital 1.0** 128
muscle Fed 0.66** 127
Starved None 0.3** 98
Adipose Fed Pentobarbital 0.51** 128
tissue
Mouse Brain Fed Pentobarbital 0.9** 91 Increases

in
ischaemia 91
Uridine-5'- Rat Liver Fed Ether 0.23 71 Decreases

triphosphate, Starved (24 hr.) Phenobarbital 0.23 71 after ad
UTP ministration
of fructose,
but no change
after di-
hydroxy-
acetone . 71
F o r s u m of
UTP + UDP
see .
8 4
Guanosine-3'- Rat Liver Fed Pentobarbital 8.5*** 87

5-monophos-
phate,
G-3:5-MP*
Heart Fed Pentobarbital 44*** 87
Brain Fed Pentobarbital 68*** 87
Skeletal Fed Pentobarbital 41*** 87
muscle
* F o r relative a m o u n t s of A - 3 : 5 - M P a n d G - 3 : 5 - M P in various rat tissues, s e e .

87
E
* nmole/g. fresh weight!
'* pmole/g. fresh weight!
7. Nicotinamide-Adenine Dinucleotides
(/imole/g.
fresh wt.)
Nicotinamide- Rat Liver Fed None 0.76 86 Falls in

adenine, Fed Ether 0.73 66 ischaemia 27,79
.
dinucleotide, Fed Ether 0.84 5
NAD Fed Ether 0.67 85
Fed Ether 0.67 85
(abdominal)
Brain Fed Ether 0.30 85
Fasted Local 0.21 73
Nicotinamide- Rat Liver Fed None 0.140 86 Rises in
adenine Fed Ether 0.090 27 ischaemia 27,79
.
dinucleotide Fed Ether 0.078 5
reduced, Fed Ether 0.041 85
NADH Starved (48 hr.) None 0.160 86
Cow Liver Fed local 0.24 73
anaesthesia
Fasted local 0.33 73
anaesthesia
Ketotic local 0.19 73
anaesthesia
Nicotinamide- Rat Liver Fed None 0.067 86 Falls in
adenine Fed Ether 0.066 85 ischaemia 85
dinucleotide Fed Ether 0.070 27 Higher values

phosphate, Fed Ether 0.059 95 using weak
NADP Starved (48 hr.) None 0.080 86 acid ( p H 2.3)
for extrac
tion .
85

Fed 0.027 95

Creatine P h o s p h a t e , Creatine, I n o r g a n i c P y r o p h o s p h a t e a n d P h o s p h a t e 2299

(/zmole/g.
fresh wt.)
Nicotinamide- Rat Brain Fed Ether 0.0053 94

adenine
Blood Fed Ether 0.005 94 jumole/ml.
dinucleotide
phosphate, blood
NADP Cow Liver Fed Local 0.071 73
anaesthesia
Starved Local 0.078 73
anaesthesia
anaesthesia
Nicotinamide- Rat Liver Fed None 0.30 86 Little change

adenine Fed Ether 0.39 85 in i s c h a e m i a .
85
dinucleotide Fed Ether 0.25 27

phosphate, Fed Ether 0.49 94
reduced, Fed Ether 0.36 95
NADPH Starved (48 hr.) None 0.36 86
Fed 0.108 95
Brain Fed Ether 0.026 94
Blood Fed Ether 0.015 94 jumole/ml.
blood
Starved Local 0.28 73
8. Creatine Phosphate, Creatine, Inorganic Pyrophosphate and Phosphate

Gumole/g.
fresh wt.
Creatine Rat Kidney Fed Phenobarbital 0.60 16 Fordistribu-

phosphate tion between
cortex a n d
medulla, s e e . 16

Fed None 1.0 12
Fed None 3.55 117
Fed None 3.51 119
Fed Ether 8.28 13
Fed Inactin 23.0 68

(/imole/g.
fresh wt.)
Creatine Mouse Brain Fed None 3.44 49 Rapid

phosphate Fed None 4.44 77 decrease in
ischaemia . 10

Creatine Rat Muscle Fed Ether 10 24 Rises in
Fed Inactin 6.98 68 tetany 24

Pyrophosphate Rat Liver Fed None 0.01-0.015 92
Inorganic Mouse Liver Fed None 3.37 ;129 G o o d agree
p h o s p h a t e , Pi. Fed Phenobarbital 2.99 29 m e n t with
chemical assay
Heart Fed Phenobarbital 3.29 29
Brain Fed None 2.91 :129
Muscle Fed Phenobarbital 4.23 29
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54 O. Wieland, A d v . in M e t a b o l i c Disorders, 3, 1, [1968].
55 D. B. McDougal, J. Holowack, J. Howe, E. M. Jones & C A. Thomas, J. N e u r o c h e m . 75, 577 [1968].
56 / . Holowack, F. Kauffman, M. G. Ikossi, C Thomas & D. B. McDougal, J. N e u r o c h e m . 75, 621 [1968].
57 F. Heinz, Z . physiol. C h e m i e , 349, 859 [1968].
58 L. V. Eggleston, unpublished experiments
59 G. Michal, H. Mollering & W. Gruber, Enz. biol. clin. 9, 154 [1968].
60 S. R. Nelson, D. W. Schulz, J. V. Passonneau & O. H. Lowry, J. N e u r o c h e m . 75, 1271 [1968].
62 M. N. Berry, D. H. Williamson & M. B. Wilson, Biochem. J. 94, 17 C [1964].
63 G. Stein & K. H. Bassler, Z. fur Klin. C h e m i e u n d Klin. Biochemie, 6, 27 [1968].
64 C. Gerez & R. Kirsten, Biochem. Zeit. 341, 534 [1965].
65 K. L. Reichelt, E. Kvamme & B. Tveit, Scand. J. clin. & Lab.-Invest. 16, 433 [1964].
66 H. D. Soling, R. Kattermann, H. Schmidt & P. Kneer, Biochem. b i o p h y s . Acta, 775, 1 [1965].
67 K. A. Gumaa & P. McLean, F E B S Letters, 7, 227 [1968].
68 P. Arese, A. Bosia & G. Pescarmona, unpublished results.
69 / . V. Passonneau, O. H. Lowry, D. W. Schulz & J. G. Brown, J. biol. C h e m . 244, 902 [1969].
70 D. H. Williamson, D. Veloso, E. V. Ellington & H. A. Krebs, Biochem. J. 774, 575 [1969].
71 H. B. Burch, P. Max, K. Chyu & O. H. Lowry, Biochem. biophys. R e s . C o m m u n . 34, 619 [1969].
72 F. J. Ballard & R. W. Hanson, Biochem. J. 772, 195 [1969].
73 F. J. Ballard, R. W. Hanson, D. S. Kronfeld & F. Raggi, J. N u t r . 95, 160 [1968].
74 A. L. Greenbaum & E. D. Sagger son, unpublished results.
75 A. L. Greenbaum & A. Reilly, unpublished results.
76 S. van den Noort, R. E. Eckel, K. Brine & J. T. Hrdlicka, J. clin. Invest. 47, 2133 [1968].
77 J. Folbergrova, J. V. Passonneau, O. H. Lowry & D. W. Schulz, J. N e u r o c h e m . 16, 191 [1969].
79 J. T. Brosnan, H. A. Krebs & D. H. Williamson, Biochem. J. 777, 91 [1970].
80 P. Lund, unpublished results.

81 F. C. Kauffman, J. G. Brown, J. V. Passonneau & O. H. Lowry, J. biol. C h e m . 244, 3647 [1969].
82 F. J. Ballard, Biochem. J. 117, 231 [1970].
83 W. Gruber, H. Mollering & H. U. Bergmeyer, Enzym. biol. clin. 7, 115 [1966].
84 D. Keppler & K Decker, E u r o p . J. Biochem. 10, 219 [1969].
86 D. H. Williamson, F. Mayor & D. Veloso, 9th Conference of the G e r m a n Society for Biological
Chemistry on " R e g u l a t i o n of Gluconeogenesis", Ed. by H.-D. Soling & B. Willms, p . 92 [1971].
87 E. Ishikawa, S. Ishikawa, J. W. Davis & E. W. Sutherland, J. biol. C h e m . 244, 6371 [1969].
88 M. C. Fleming & O. H. Lowry, J. N e u r o c h e m . 13, 779 [1966].
89 R. L. Young & O. H. Lowry, J. N e u r o c h e m . 13, 785 [1966].
90 G. D. Baird 8c R. J. Heitzman, Biochem. J. 116, 865 [1970].
91 B. M. Breckenridge, Proc. N a t . Acad. Sci. 52, 1580 [1964].
92 H. Flodgaard, Abstr. C o m m u n . Meet. F e d . Eur. Biochem. Soc. 8, 1192 [1972].
93 L. S. Jefferson, J. H. Exton, R. W. Butcher, E. W. Sutherland & C. R. Park, J. biol. Chem.X43, 1031.
94 H. B. Burch, M. E. Bradley & O. H. Lowry, J. biol. C h e m . 242, 4546 [1967].
95 V. Neuhoff&c E. Desselberger, Arch. Exp. P a t h o l . P h a r m a k o l . 252, 43 [1965].
96 H. Herken, K. Lange & H. Kolbe, Biochim. biophys. Res. C o m m u n . 36, 93 [1969].
97 7 . B. Allred8cD. G. Guy, A n a l . Biochem. 29, 293 [1969].
98 N. D. Goldberg, J. Lamer, H. Sasko & A. G. O'Toole, A n a l . Biochem. 28, 523 [1969].
99 J. Lumbers, C. J. Threlfall & H. B. Stoner, A n a l . Biochem. 31, 21 [1969].
100 D. J. Pearson, Biochem. J. 95, 23 C [1965].
101 J. Schoberth, J. Sollenberg, A. Sundwall & B. Sorbo, J. N e u r o c h e m . 13, 819 [1966].
102 P. J. Tubbs & P. B. Garland, Biochem. J. 93, 559 [1964].
103 M. A. Page, H. A. Krebs & D. H. Williamson, Biochem. J. 121, 49 [1971].
104 B. Wittels & R. Bressler, J. Clin. Invest. 44, 1639 [1965].
105 K. A. Gumaa, P. McLean & A. L. Greenbaum in Essays in Biochemistry, Vol. 7, p . 39 [1971].
106 R. L. Veech, L. Raijman & H. A. Krebs, Biochem. J. 117, 499 [1970].
107 J. R. Williamson in T h e Energy Level a n d M e t a b o l i c C o n t r o l in M i t o c h o n d r i a , p . 385 [1969].
108 A. L. Greenbaum, K A. Gumaa & P. McLean, Arch. Biochem. Biophys. 143, 617 [1971].
109 R. P. Faupel, H. J. Seitz, W. Tarnowski, V. Thiemann & Ch. Weiss, A r c h . Biochem. Biophys. 148, 509
[1972].
110 K Lange, H. Kolbe, K Keller & H. Herken, Z. Physiol. C h e m . 351, 1241 [1970].
111 H.B. Burch, O. H. Lowry, M. E. Bradley & P. F. Max, A m e r . J. Physiol. 219, 409 [1970].
112 A. M. Snoswell8cP. P. Koundakjin, Biochem. J. 127, 133 [1972].
113 P. P. Koundakjin & A. M. Snoswell, Biochem. J. 119, 149 [1970].
114 K. A. Gumaa, P. McLean & A. L. Greenbaum, F E B S Letters 13, 5 [1971].
115 G.D. Baird, R.J. Heitzman & KG. Hibbit, Biochem. J. 128, 1311 [1972].
116 R. L. Veech, R. L. Harris, D. Veloso & E. H. Veech, J. N e u r o c h e m . 20, 183 [1973].
117 A. L. Miller, R. A. Hawkins & R. L. Veech, Science 173, 904 [1973].
118 R. L. Veech, R. Guynn & D. Veloso, Biochem. J. 127, 387 [1972].
119 D. Veloso, J. V. Passonneau & R. L. Veech, J. N e u r o c h e m . 19, 2679 [1972].
120 G.D. Baird, R.J. Heitzman & A.M. Snoswell, E u r o p . J. Biochem. 29, 104 [1972].
121 R. W. Guynn, D. Veloso & R. L. Veech, J. biol. C h e m . 247, 7325 [ 1972].
122 H. D. Soling & B. Volkmann, Analyt. Biochem. 52, 305 [1973].
123 R. H. Dreisbach & E. Gerlach, Proc. Soc. Exp. Biol. M e d . 126, 281 [1967].
124 G. L. Pauk & W. J. Reddy, Analyt. Biochem. 21, 298 [1967].
125 G. M. Walton & L. D. Garren, Biochemistry, 9, 4223 [1970].
126 G. D. Aurbach & B. A. Houston, J. biol. C h e m . 243, 5935 [1968].
127 / . B. Posner, K. E. Hammermeister, G. E. Bratvold & E. G. Krebs, Biochemistry 3, 1040 [1964].
128 J. R. Turtle & D. M. Kipnis, Biochemistry 6, 3970 [1967].
129 R. W. Guynn, D. Veloso & R. L. Veech, Analyt. Biochem. 45, 277 [1972].
130 / . T. Brosnan & D. H. Williamson, Biochem. J. 138, 453 [1974].
131 M. P. Thompson, unpublished results.
132 G. Murphy, A. D. Ariyanayagam & N. J. Kuhn, Biochem. J. 136, 1105 [1973].
Index
Index
The key w o r d s are a r r a n g e d alphabetically without regard to prefixes such as D , L, ( + ) , ( — ) , or, Where
necessary reference to other s y n o n y m s is given.
A - determination
- - other methods 2004
Abbreviations - with H O A D H 2001
- biochemical reagents 417 - p r e p a r a t i o n with diketene from C o A S H 1970
- l i s t of XXXIV N-Acetylaspartate
A b s o r p t i o n coefficient - c o n c e n t r a t i o n in animal tissues 2285
- Bunsen's 253 Acetylcarnitine
A b s o r p t i o n curve - concentration in a n i m a l tissues 2288
- dinitrophenylhydrazones - determination 1764
- - of pyruvate a n d 2-oxoglutarate 184 Acetylcholine
-general 182 - determination 1819
- N A D and N A D H 104, 184 - electrophoretic separation from choline 1821
Absorption photometry 180 -hydrolysis 1822
ABTS 1212, 1215 - n o r m a l values 1823
Acceptor activity of t R N A Acetylcholinesterase, see cholinesterase
- determination of 1894 Acetyl-CoA
Acetaldehyde - characteristics a n d commercial p r e p a r a t i o n s
- determination with 524
- alcohol d e h y d r o g e n a s e 1506 - concentration in animal tissues 2288
- - aldehyde de hydrogenase 1509 - determination
- n o r m a l values in b l o o d 1508 - catalytic assay with P T A 1975
Acetate - - fluorimetric with C S a n d M D H 1993
- concentration in a n i m a l tissues 2293 - - fluorimetric with o x o g l u t a r a t e dehydrogenase
- determination with and PTA 1994
- acetate kinase a n d h y d r o x y l a m i n e 1528 - - o t h e r methods 1992
- - acetate kinase, P T A , C S a n d M D H 1520 - radiochemical 1994
- - acetyl-CoA synthetase a n d sulphanilamide --UV-assay 1988
1532 - n o r m a l values 1991, 1994
- preceding indicator r e a c t i o n ; N-Acetyl-D-glucosamine
principle a n d t h e o r y 113 - characteristics a n d commercial p r e p a r a t i o n s
- distillation according to Bartley 1525 524
- n o r m a l values 1527 Acetyl g r o u p s
Acetate kinase - d e t e r m i n a t i o n in proteins 1640
- assay of activity 425 Acetylhydroxamic acid
- characteristics a n d commercial p r e p a r a t i o n s - a b s o r p t i o n curve of ferric complex 1529
425 Acetyl p h o s p h a t e
- relative rates of reaction with nucleoside - characteristics a n d commercial p r e p a r a t i o n s
triphosphates 2081 524
Acetoacetate -determination 1538
- c o n c e n t r a t i o n in a n i m a l tissues 2291 Acetylpyridine-adenine dinucleotide
-determination 1840 - characteristics a n d commercial p r e p a r a t i o n s
- n o r m a l values 1843 525
- stability in sample 1842 - reduced, extinction coefficient 159£
Acetoacetyl-CoA S-Acetyl-N-succinyl-cysteamine
Ace-Ade Index XLIV
S-Acetyl-N-succinyl-cysteamine - skeletal muscle 2295

- extinction coefficient 1972 - determination
Acid p h o s p h a t a s e - - with M K , P G K a n d G A P D H 2078
- see p h o s p h a t a s e , acid - with P K a n d L D H 2127
Aconitase - n o r m a l values in b l o o d 2131
- in progressive muscular dystrophy 38 - stability in b l o o d 166
Acrylyl-CoA Adenosine-5 '-diphosphoglucose
- determination 2005 - determination 2204
Acylcarnitines - see also A D P - g l u c o s e
- concentration in a n i m a l tissues 2289 A d e n o s i n e - 3 ' : 5 ' - m o n o p h o s p h a t e (cyclic)
- determination - characteristics a n d commercial p r e p a r a t i o n s
- acetylcarnitine 1764 526
- long-chain ( C > 12) 1769 - c o n c e n t r a t i o n in a n i m a l tissues 2296
- other m e t h o d s 1770 - d e t e r m i n a t i o n with
- - short-chain ( C - C ) 3 1 0 1767 - - F D P a s e by A M P inhibition 2144
Acyl-CoA - - other m e t h o d s 2136
- determination - - protein binding test 2137
- colorimetric assay 2010 - n o r m a l values 2142
- fluorimetric 2015 A - 3 : 5 - M P - b i n d i n g protein
Acyl-CoA dehydrogenase -isolation 2143
- isolation 2014 Adenosine-5'-monophosphate
A c y l p h o s p h a t e : D-glucose-6-phosphotransferase - characteristics a n d commercial p r e p a r a t i o n s
- for determination of glucose 1222 526
- Michaelis constants 1223 - c o n c e n t r a t i o n in
Additional (side) activities - - a n i m a l tissues 2086, 2296
-definition 108 - - rat heart 2086
Additive models 343 - - rat liver 2086, 2296
Adenine - - rat skeletal muscle 2086, 2296
-determination 1909 - determination 2088, 2127
- n o r m a l values 1915 - n o r m a l values in b l o o d 2131
Adenosine - stability in b l o o d 166
- cleavage by nucleoside phosphorylase 1934 Adenosine-5'-nucleotides 2088
- determination 1919 Adenosine phosphates
- extinction coefficient 1921 - determination 2132
Adenosine deaminase Adenosine-5' -triphosphate
- assay of activity - characteristics a n d commercial p r e p a r a t i o n s
- colorimetric 1092 527
- - UV-assay 426 - concentration in
- characteristics a n d commercial p r e p a r a t i o n s - a n i m a l tissues 2293
426 - liver in viral hepatitis 8
- i m p o r t a n c e in clinical chemistry 1092 - - rat heart 2294
- in serum of patients with t y p h u s 1097 - - rat liver 2293
- n o r m a l values 1098 - - rat skeletal muscle 2294
Adenosine-5' -diphosphate - d e t e r m i n a t i o n with
- characteristics a n d commercial p r e p a r a t i o n s - AK, A G T and G6P-DH 2080
525 - formyltetrahydrofolate synthetase 2110
- content in - HK & G6P-DH 2101
- animal tissues 2295 - luciferase 2112
- - rat heart 2295 - - o t h e r methods 2109
- - rat liver 2295 - - PGK & GAPDH 2097
T h e key words are arranged alphabetically without regard t o prefixes such

XLV Index Ade-Ami
Adenosine-5 ' - t r i p h o s p h a t e - in b l o o d diseases 54

- n o r m a l values 2100 - in m u s c u l a r d y s t r o p h y
- stability in b l o o d 166 - - in muscle 38
Adenosine triphosphatase (ATPase) - in serum 38
- in b l o o d diseases 52 - in s e r u m of rats after exercise 8
- in m u s c u l a r d y s t r o p h y - in various m y o p a t h i e s 43
- - in muscle 38 - stability in s e r u m 168
Adenylate deaminase Aldolase, from liver
- in progressive m u s c u l a r d y s t r o p h y 38 - for d e t e r m i n a t i o n of F - l - P 1308
Adenylate kinase -isolation 1312
- assay of activity 486 Allitol
- characteristics a n d c o m m e r c i a l p r e p a r a t i o n s - reaction with sorbitol d e h y d r o g e n a s e 572
486 Allowable limits of error, A . L. E. 378
- see also m y o k i n a s e Alkaline p h o s p h a t a s e
2'-Adenylic acid - see p h o s p h a t a s e , alkaline n o r m a l values
- hydrolysis by myo-inositol-1 - p h o s p h a t a s e 1340 Altronate dehydrogenase
ADP-glucose - isolation 1302
- see adenosine-5'-diphosphoglucose Altrose
ADP-glucose pyrophosphorylase - oxidation by G O D 1212
- isolation 2207 A m i n e oxidase
Age dependence -isolation 1747
- of n o r m a l range 388, 731, 756 A m i n o acid a r y l a m i d a s e
- of experimental values 384, 731, 756 - for the diagnosis of kidney diseases 62
D-Alanine - in diseases of kidney a n d u r i n a r y tract 62
-determination 1686 - in h u m a n urine 67
L-Alanine - in structures of h u m a n kidneys 65
- c o n c e n t r a t i o n in a n i m a l tissues 2284 - stability in s e r u m 169
- d e t e r m i n a t i o n with A m i n o acid arylamidases
- alanine d e h y d r o g e n a s e 1679 - assay of activity 958
- - G P T and L D H 1682 - n o r m a l values 962
- n o r m a l values 1681 A m i n o acid decarboxylases
Alanine d e h y d r o g e n a s e -preparation 1668
- assay of activity 427 - reaction p r o d u c t s 1663
- characteristics a n d commercial p r e p a r a t i o n 427 D - A m i n o acid oxidase
Alcohol - apoenzyme, preparation 2184
- d e t e r m i n a t i o n with - assay of activity 431
- - A D H and A P A D 1502 - characteristics a n d commercial p r e p a r a t i o n s
- - A D H and N A D 1499 431
Alcohol d e h y d r o g e n a s e -isolation 1654
- assay of activity 429 - rate of oxidation of D - a m i n o acids 1648
- characteristics a n d c o m m e r c i a l p r e p a r a t i o n s A m i n o acids
428 - c o n c e n t r a t i o n s in a n i m a l tissues 2284
- equilibrium c o n s t a n t s 428 D - A m i n o acids
- in acute hepatitis 15 - determination
- specificity 429 - - manometric 1648
Aldehyde d e h y d r o g e n a s e - micro methods 1653
- isolation from yeast 1513 L - A m i n o acids
Aldolase - determination
- assay of activity 430 - manometric 1662
- characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 430 - colorimetric with fluorodinitrobenzene 1669
as D , L , ( + ) , ( — ) , a , p. Where necessary reference to other s y n o n y m s is given.

Ami-Aspa Index XLVI
L-Amino acids, d e t e r m i n a t i o n - t r a n s p o r t in a u t o m a t i c analysers 207

- - t R N A loading test, isotope dilution 1656 Aneurin pyrophosphate
>>-Aminobutyric acid - see thiamine p y r o p h o s p h a t e
-determination 1690 Angiotensinase
- n o r m a l values 1694 - assay of activity 964
Aminopeptidases a n d a m i n o acid arylamidases - n o r m a l values in serum 966
- activity o p t i m a 952, 953 Anti-oxidants
- classification 952, 953 - enzymatic determination in foodstuff chemistry
- general information 950 75
- occurrence 952, 953 Apoaminotransferase
- purification 952, 953 -preparation 2198
- substrates 952, 953 Apparatus
Aminotransferase - for a u t o m a t i o n of analysis 205 et seq.
- apoenzyme, isolation 2198 - for electrophoresis 262 et seq.
Ammonia - for luminescence m e a s u r e m e n t s 2114
- concentration in animal tissues 2286 - for microtechniques 230 et seq.
-determination 1802 - for p h o t o m e t r y 184 et seq.
A m p o u l e divers 243 - for r a d i o m e t r y 283 et seq.
Amylase L-Arabinose
- in honey 74 - characteristics a n d commercial p r e p a r a t i o n s
- in milk 74 - determination with
- in p r o d u c t i o n of grain 84 - L-arabinose isomerase 1350
a-Amylase - oxidation by G a l - D H 453
- assay of activity 432 L-Arabinose isomerase
- characteristics a n d commercial p r e p a r a t i o n s -isolation 1352
432 L-Arginine
- for the quality control of plant p r o d u c t s 84 - determination
- general information 885 - colorimetric assay with fluorodinitrobenzene
- measurements 1669
- after electrophoretic separation 909 - manometric 1662
- by end-point determination o n p a p e r 903 Arylesterases
- of degradation p r o d u c t s , maltose a n d glucose - assay with
890 - /?-naphthylpropionate as substrate 810
- of iodine-starch-complex 898 - phenylacetate as substrate 807
- of reducing g r o u p s 885 - general information 860
- with coloured insoluble substrates 894 - n o r m a l values 809, 813
- n o r m a l values 888, 897, 902, 908 Arylsulphatase
- stability in serum 168 - assay of activity 435
^-Amylase - characteristics a n d commercial p r e p a r a t i o n s
- assay of activity 433 435
- characteristics a n d commercial p r e p a r a t i o n s - in muscular d y s t r o p h y 38
433 - in serum in pregnancy 58
Amylo-1,6-glucosidase L-Asparagine
- assay of activity 434 -determination 1696
- characteristics a n d commercial p r e p a r a t i o n s - n o r m a l values 1700
434 L-Asparaginase
- for determination of glycogen 1127 - assay of activity 435
Amylose - characteristics a n d commercial p r e p a r a t i o n s 435
- preparation 902 D-Aspartate
Analytical mixture - determination, possible m e t h o d of 1700
The key words are arranged alphabetically without regard to prefixes such
XLVII Index Aspa-Car
L-Aspartate Butyleneglycol
- concentration in animal tissues 2284 - reaction with sorbitol dehydrogenase 1323
- determination Butyraldehyde
- manometric 1662 - oxidation by a l d e h y d e dehydrogenase 1513
--UV-assay 1696 - reduction by A D H 1508
- electrophoretic separation from citrate 1998 Butyryl-CoA
- n o r m a l values 1700 - determination
L-Aspartate decarboxylase 1663 - colorimetric 2010
Assessment - fluorimetric 2015
- longitudinal 383
- transverse 391
A s y m m e t r y (in statistics) 326 C
A u t o m a t i c analysers
- commercial 221 Caeruloplasmin
- fast analysers 213 - isoenzymes 11
- technique 205 Calculation of experimental results 308 et seq.
Automation Capillary pipettes 232
- continual flow w o r k i n g 203 C a r b a m a t e s with insecticide activity
- discontinuous w o r k i n g 203 - determination 2249, 2257
- of analysis 202 Carbamoyl phosphate
- serial a n d parallel analysis 203 - characteristics a n d commercial p r e p a r a t i o n s
Auxiliary enzyme 109 528
Auxiliary reaction 109 - determination 1749
Azocasein Carbonic anhydrase
- preparation 1005 - in n e p h r o n of h u m a n kidneys 64
- substrate for proteinases 1000 Carboxypeptidase A
- assay of activity 437, 989, 993
B - characteristics a n d commercial p r e p a r a t i o n s
436, 437
Bartley, distillation flask 1525 - n o r m a l values 992, 996
Benzoyl-CoA Carboxypeptidase B
- determination 2008 - assay of activity 996
Bile acids - n o r m a l values 998
-determination 1886 Carboxypeptidases
- n o r m a l values 1889 - effectors 987, 988
Biochemical reagents - general information 986
- control of 158 et seq. - occurrence 987, 988
- manufacturers a n d suppliers 559 - pH-optima 987, 988
- s t a b i l i t y of 164 et seq. - purification 987, 988
- storage of 158 et seq. - substrate o p t i m a 987, 988
Biological m a n o m e t r y 248 Carnitine
Biuret m e t h o d for protein 174 - c o n c e n t r a t i o n in a n i m a l tissues 2290
Blood - determination
- calculation of p r o p o r t i o n in tissue 2107 - - D T N B method 1762
- mailing of samples o n p a p e r 167 - thiokinase m e t h o d 1758
Brodie solution 253 C a r n i t i n e acetyltransferase
Buffer capacity 257 - assay of activity 438
Bunsen, a b s o r p t i o n coefficient 253 - characteristics a n d commercial p r e p a r a t i o n s
Burettes 438
- for micro-analysis 232 Cartesian diver 243
as D , L , ( + ) , (—), a, /?. Where necessary reference to other s y n o n y m s is given.

Cata-Chymo Index XLVIII
Catalase - n o r m a l values 1823

- assay of activity Cholinesterase
- other m e t h o d s 680 - inhibition by insecticides 2249
- titrimetric 678 - in liver diseases
--UV-assay 439,674 - acute hepatitis 15, 26
- characteristics a n d commercial p r e p a r a t i o n s - chronic hepatitis a n d cirrhosis 27
438, 439 - - hepatic o b s t r u c t i o n 26
- general information 673 - in serum 26, 27
- in cereal p r o d u c t s 74 - - liver t u m o u r s 23, 27
- in milk 73 - - obstructive j a u n d i c e 22
- kinetics 674 - toxic liver d a m a g e 20
- n o r m a l values in b l o o d 677 - isoenzymes 11
- units 681 - stability in serum 169
Catalytic assays 132 Cholinesterases
CDP-glucose - assay m e t h o d s
- see cytidine-5'-diphosphoglucose - colorimetric 840
CDP-glucose d e h y d r a t a s e - - electrometric 838
- isolation 2212 - manometric 835
Cell disintegration 396 - assay of activity of A C h E
Cell fractionation 407 - in erythrocytes 844
Cell suspensions, heterogeneous 399 - - in serum 845
Cellular enzymes 7 - in whole b l o o d 843
Cellulase, crude enzyme - assay of activity of P C h E
-preparation 1142 - in serum 846
Cellulose - d e t e r m i n a t i o n of dibuccaine n u m b e r a n d
- determination by m e a s u r e m e n t s of fluoride n u m b e r in serum 846
- of glucose formed 1139 - general i n f o r m a t i o n 831
- - of insoluble residue 1132 - i m p o r t a n c e in clinical chemistry 833
- of soluble p r o d u c t s 1137 - m e a s u r e m e n t s with a u t o m a t i c analysers, in blood
- n o r m a l values 1136 851
Chitosamine, - nomenclature 83
- see D-glucosamine - n o r m a l values
Choice of p r o b a n d s - - dibuccaine n u m b e r a n d fluoride n u m b e r 849
- for d e t e r m i n a t i o n of n o r m a l r a n g e 386 - - in erythrocytes 837, 840, 845
/?-Chlorolactate - in serum 845
- oxidation by L D H 1468 - in whole b l o o d 843
Chloroplasts - substrates 832
- fraction of total activity 412 - trivial n a m e s 831
- isolation 411 C h o n d r o i t i n sulphate
Cholesterol - a b s o r p t i o n spectrum of dissacharide
- determination 1890 - after enzymatic d e g r a d a t i o n 1166
-e ste rifie d 1890 - determination 1165
- n o r m a l values 1893 - isolation from biological samples 1168
Cholesterol oxidase -occurrence 1165
- assay of activity 440 Chymotrypsin
- characteristics a n d commercial p r e p a r a t i o n s 440 - assay of activity
Choline - with N-acetyl-L-tyrosine-ethylester as substrate
- determination 1819 441
- electrophoretic separation from acetyl choline - with N-benzoyl-L-tyrosine-ethylester as
1821 substrate 1009
IL Index Chymo-Crea
C h y m o t r y p s i n , assay of activity - higher saturated fatty acids

- with casein as substrate 1007 - - d e t e r m i n a t i o n , colorimetric 2010
- different forms, formation 1006 C o e n z y m e A thioesters
-general 1006 - determination 2008
- isoenzymes 11 Coenzyme B 1 2
Chymotrypsin A - determination 2200

- assay of activity 4 4 1 , 442 Coenzymes
- characteristics a n d c o m m e r c i a l p r e p a r a t i o n s - as biochemical reagents 523
440, 441 Collagenases
C h y m o t r y p s i n inhibitors - assay of activity 1059
- d e t e r m i n a t i o n of activity 1074 - general information 1058
Citrate Colorimeter 190
- concentration in a n i m a l tissues 2280 Competitive inhibition 151, 153, 154, 155
- determination Concentration
- fluorimetric 1565 -calculation 312
- UV-assay 1562 - definition 310
- electrophoretic separation from a s p a r t a t e 1998 - of b l o o d constituents, diurnal variations 390
- n o r m a l values 1569 - of metabolites in a n i m a l tissues 2266 et seq.
Citrate lyase Constriction pipettes 323
- assay of activity 442 C o n t a m i n a t i n g activity, definition 108
- characteristics a n d commercial p r e p a r a t i o n s C o n t e n t , definition 310
442 Control
Citrate synthase - of experimental results 374
- assay of activity 443 - of quantitative chemical analysis 370
- characteristics a n d commercial p r e p a r a t i o n s C o n t r o l chart 371, 372, 376
443 Conway diffusion a n d disher 1535
Cleavage of proteins C o u p l e d reaction 109
- to peptides with Creatine
- chymotrypsin 1629 - concentration in animal tissues 2300
- other proteases 1631 -determination 1772
- pepsin 1630 - n o r m a l values 1776
- trypsin 1625 Creatine kinase
Clostridiopeptidase A - assay of activity with
- assay of activity 1059 - a u t o m a t i c analysers 793
Cocarboxylase - creatine as substrate 785
- see thiamine p y r o p h o s p h a t e - - creatine p h o s p h a t e as substrate 789
Coefficient of variation 312, 314, 327 - characteristics a n d commercial p r e p a r a t i o n s
Coenzyme A 444
- characteristics a n d commercial p r e p a r a t i o n s - general information 784
528 - in heart diseases
- concentration in animal tissues 2287 - differential diagnosis 36
- determination - myocardial infarct 31
- fluorimetric with oxoglutarate dehydrogenase - in muscle, striated 38
1981 - in m u s c u l a r d y s t r o p h y 38
- - m e t h o d of choice 1967 - in m y o p a t h i e s , various 43
- - w i t h HOADH 1968 - n o r m a l values 788, 791, 797
- - with P T A , catalytic m e t h o d 1975 - relative rates of reaction with nucleoside
- - with P T A , end-point m e t h o d 1972 diphosphates 2079
- n o r m a l values 1967 - stability in serum 169
Coenzyme A derivatives - stabilization a n d reactivation 171
as D , L , ( + ) , ( - ) , a , /?. Where necessary reference t o other s y n o n y m s is given.

Crea-DeRitis Index L
Creatine p h o s p h a t e Cytosine
- characteristics a n d commercial p r e p a r a t i o n s 529 - determination 1916
- concentration in a n i m a l tissues 2299 Cytosine d e a m i n a s e
- determination -isolation 1918
- - o t h e r methods 1785
- with C K , H K a n d G 6 P - D H 1777 D
- - with C K , P G K a n d G A P D H 1781
- with luciferase 2112 D a t a , theoretical t r e a t m e n t 332
Creatininase D a t a processing 4
- assay of activity 446 D a y - t o - d a y reproducibility 378
- characteristics a n d commercial p r e p a r a t i o n s 445 Decision 347
Creatinine Decision m e t h o d , statistical 347
- determination 1786 Dehydrogenases
- n o r m a l values 1790 - assay after electrophoretic separation 273
"Creep" 308 Deoxy-CDP-glucose
Crotonase - reaction with C D P - g l u c o s e dehydratase 2212
-isolation 2021 Deoxyeytidine
Crotonyl-CoA -determination 1923
- determination - n o r m a l values 1926
- - other m e t h o d s 2020 Deoxy-GDP-mannose
--UV-assay 2017 - reduction with G D P - m a n n o s e dehydrogenase
C T P effect 1899 2215
Cuvettes 191 Deoxyribonuclease I
Cyclodeaminase - assay of activity 447
-isolation 1561 - characteristics a n d commercial p r e p a r a t i o n s 447
Cytidine Deoxythymidine
-determination 1923 -determination 1935
- n o r m a l values 1926 - extinction coefficient 1935
Cytidine d e a m i n a s e - n o r m a l values 1939
- isolation 1927 Deoxythymidine-5'-diphosphoglucose
Cytidine-5' - d i p h o s p h a t e - characteristics a n d commercial p r e p a r a t i o n s 530
Cytidine-5 '-diphosphoglucose Deoxythymidine phosphorylase
- characteristics a n d commercial p r e p a r a t i o n s 529 -isolation 1939
- determination 2209 Deoxyuridine
Cytidine-5 ' - m o n o p h o s p h a t e -determination 1935
- determination 2153 - extinction coefficient 1935
Cytidine-5 '-triphosphate - n o r m a l values 1939
- determination Deproteinization
- - other m e t h o d s 2148 -general 177
--withF-6-PK 2145 - volume displacement effect 177
Cytochrome c - with barium-zinc 1253
- in muscular d y s t r o p h y 38 - with perchloric acid 1448, 2104
C y t o c h r o m e c-reductases - with uranyl acetate 1208
- in muscular d y s t r o p h y DeRitis quotient
- in muscle 38 - in liver diseases
C y t o c h r o m e oxidase - acute hepatitis 14
- in plant diseases 83 - chronic hepatitis a n d cirrhosis 16
Cytolysis - liver obstruction 20
- of blood elements 639, 640 - liver t u m o u r s 24
LI Index DeRitis-Electro
DeRitis quotient, in liver diseases - see D-glycerate-2,3-diphosphate

- obstructive j a u n d i c e 21 D i p h o s p h o p y r i d i n e nucleotide
- toxic liver d a m a g e 19 - see n i c o t i n a m i d e - a d e n i n e dinucleotide
- in myocardial infarct 31 Disaccharides
Dermatan sulphate - assay of activity 916
- determination 1165 - general information 916
- occurrence 1165 -nomenclature 916
- n o r m a l values 1170 - n o r m a l values 921
Detergents - specificity 921
- effects o n enzyme activities 168 Disc electrophoresis 268
D e t e r m i n a t i o n of n o r m a l range 385 Disintegration
D e t e r m i n a t i o n of substrates - general 396
- with radiobiochemicals 283 - of cells a n d tissues 399, 401
D i a m i n e oxidase - of plant tissues 410
- assay of activity 660 - biological-enzymatic 405
- n o r m a l values in h u m a n serum 664 - chemical 405
Diaphorase - thermal 404
- assay of activity 448 Dispensing
- characteristics a n d commercial p r e p a r a t i o n s 448 - of samples a n d reagents 206
Diastase in honey 74 Dispensing units
Dibuccaine n u m b e r 846 - for micro-analysis 232
Dihydrofolate reductase Distillation vessel a c c o r d i n g t o Bartley 1525
- assay of activity, UV-assay 666 Distribution
- n o r m a l values in various diseases of blood a n d - empirical 322
bone marrow 669 - frequency 322
- other m e t h o d s 670 - probability 336
Dihydrofolic acid D i u r n a l variations
- preparation 671 - of n o r m a l range 390
D i h y d r o o r o t i c acid d e h y d r o g e n a s e - of the concentrations of b l o o d constituents 384
- isolation 1966 Documentation
Dihydroxyacetone - in a u t o m a t i o n 209
-determination 1442 dTDP-glucose
- values in serum 1445 - see deoxythymidine-5'-diphosphoglucose
Dihydroxyacetone phosphate d T D P - g l u c o s e dehydrase
- characteristics a n d commercial p r e p a r a t i o n s 531 - isolation 2220
- c o n c e n t r a t i o n in a n i m a l tissues 2273 Duplicate analysis
- determination 1314
Diketene E
- for conversion of C o A - S H to A c A c - C o A 1970
D i k e t o acid hydrolase Effect of t r e a t m e n t (t-test) 353
-isolation 1847 Elastase
Di- a n d polypeptidases - assay of activity with orceinelastin 1041
- activity o p t i m a 979, 980 - general information 1041
- classification 979, 980 Electrochemical m e t h o d s
- general information 978 - in microtechniques 245
- occurrence 979, 980 Electron-transferring flavoprotein ( E T F )
- o p t i m u m c o n d i t i o n s for m e a s u r e m e n t s 981 - isolation 2014
- purification 979, 980 Electrophoresis
- substrates 979, 980 - p r e p a r a t i o n of plates 265
2,3-Diphosphoglycerate Electrophoretic separation 262
as D , L , ( + ) , ( — ) , a , /?. Where necessary reference t o other s y n o n y m s is given.

Emi-Enzy Index LII
Emission spectra - in liver diseases

- usual sources of radiation 184 - acute hepatitis 14
Empirical distribution - chronic hepatitis and cirrhosis 16
- function 322 - liver obstruction 20
End-point m e t h o d - liver t u m o u r s 23
-schematic 105 - obstructive j a u n d i c e 21
End-point, n o n - c o n s t a n t 308 - in muscle diseases
Enolase - in striated muscle 37, 45
- assay of activity 449 - neural myopathies 43
- characteristics a n d commercial p r e p a r a t i o n s 449 - progressive muscular dystrophies 37
- in muscular dystrophy - various myopathies 43
- in muscle 38 - in serum
- in serum 38 - in rats after exercise 8
Enzymatic cycling - over a decade 9
-examples 133 - in soil 85
- for determination of nicotinamide-adenine - in urine
nucleotides 2059 - in diseases of kidney a n d urinary tract
- for determination of prostaglandins 1877 - n o r m a l values 67
-kinetics 135 - of kidney structures 65
- sensitivity of the assays 134 Enzyme activity assays
Enzymatic analysis - reproducibility from day to day 379
- basis 94 et seq. Enzyme distribution patterns
- definition 94 - in n e p h r o n s of h u m a n kidney 64
- for control of growth of micro-oroganisms 88 Enzyme elimination
- for control of microbiological metabolism 90 - immediate effect 10
- in beverages 76 - i n humans 10
- in biochemistry 3 Enzyme induction
- in b o t a n y a n d agricultural chemistry 82 - in micro-organisms 87
- in foodstuff chemistry 71 Enzyme inhibitors
- in medicine 6 - in urine 63
- in microbiology 87 Enzyme level
- of confectionery, chocolate, sugar a n d sugar - in serum of healthy subjects 9
products 76 Enzyme patterns
- of sugars a n d sugar p h o s p h a t e s (schematic) 111 -distortion 13
- with radiobiochemicals 283 - in h u m a n tissues 12
Enzymatic b r o w n i n g of plant p r o d u c t s 84 - in infectious mononucleosis 18
Enzymatic isotope dilution principle 296 - in serum in liver diseases 26, 27
Enzyme activities Enzyme reactions
- assay after electrophoresis 272 - p H dependence 129
- determination with radiobiochemicals 301 Enzyme repression
- for diagnosis, control of progress a n d therapy - in micro-organisms 88
14 et seq. Enzymes
- for differential diagnosis 25, 36 - as biochemical reagents 425
- for evaluation of plant p r o d u c t s 83 - in blood plasma 7
- in blood diseases 50 - in liver d a m g e , toxic 19
- in fruit a n d berries 84 - in milk 71
- in gynaecology 56 - of glycolysis
- in heart diseases 31 et seq. - in muscle 45
- differential diagnosis 36 - of respiratory chain
- myocardial infarct 31 - in muscle 46
LIII Index Enzy-For
Enzymes F
- of tissue metabolism 7
- organ-specific 7 Fast analyser 213
- plasma-specific 7 F a t t y acid synthetase
- release from cells 7 - d e t e r m i n a t i o n of activity in isotopic assay 305
- stability in sample 168 - isolation from b a k e r ' s yeast 2037
E n z y m e units 121, 311, 421 F a t t y acids, u n s a t u r a t e d
Equilibrium c o n s t a n t s - determination 1807
- of enzymatic reactions 107 F I G L U transferase
Errors -isolation 1561
- type I 371 Filter p h o t o m e t e r 190
- type II 372 Findings
- in collection a n d t r a n s p o r t of biological samples - not in agreement with clinical picture 393
369 - transmission of 212
- random 363, 366- Fixing the n o r m a l range 385
- systematic 363, 368 Flavin-adenine dinucleotide
- theory 363 et seq. - characteristics a n d commercial p r e p a r a t i o n s 532
D-Erythrose-4-phosphate - determination 2182
- characteristics a n d commercial p r e p a r a t i o n s Flavin m o n o n u c l e o t i d e
531 - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 533
L-Erythrulose Fluorescence, decrease of b a c k g r o u n d
- d e t e r m i n a t i o n with polyol dehydrogenase 1394 - of tissue extracts 1575
- reduction by N A D - x y l i t o l dehydrogenase 1369 Fluorimeter
Esterases - for micro-analysis 231
- after electrophoretic separation 275 Fluorimetry
- in b l o o d diseases 54 - in m i c r o t e c h n i q u e s 239 - 2 4 2
E t h a n o l , see alcohol - principle 240
Evaluation of - procedure 240
- electropherograms 271 et seq. - sensitivity 241
- experimental results 308 Folic acid
Experimental d a t a , evaluation 308 - characteristics a n d commercial p r e p a r a t i o n s 533
Experimental results Formaldehyde
- calculation of 312 - reduction by A D H 1508
- diurnal rhythms 384, 390 Formate
- evaluation, c o n t r o l , calculations a n d inter - d e t e r m i n a t i o n with
pretation 308 - - FH -synthetase
4 1546
- racial differences 317 - - formate d e h y d r o g e n a s e 1551
- relation to b o d y weight 317, 756 - values in urine i 549
- seasonal differences 317 Formate dehydrogenase
- sex-specific differences 317, 756 - isolation 1554
Experimental techniques 158 et seq. F o r m a t e - n i t r a t e reductase
Extinction 181 - isolation 2264
Extinction coefficient Formiminoglutamate
- for N A D H ( N A D P H ) 184, 546 - determination 1556
Extrapolation Formulae
- with n o n - c o n s t a n t end-points 308 - for calculation of experimental results 312, 313
as D , L , ( + ), ( - ) , a, p. Where necessary reference to other s y n o n y m s is given.

Frac-Galac Index LIV
Fractionation — in muscle 38, 46

- of tissues a n d cells 407 — in serum 38
Freeze-stop m e t h o d 400 Fumarate
/?-Fructofuranosidase - content in a n i m a l tissues 2282
- assay of activity 450 - determination
- characteristics a n d commercial p r e p a r a t i o n s 450 — fluorimetric 1600
D-Fructose - n o r m a l values 1603
- determination 1304 Fumarylacetoacetate
Fructose-1,6-diphosphatase -determination 1844
- activities in rabbit tissues 710 F u n c t i o n a l state of tissues 317, 397
- determination of activity 881
- n o r m a l values 884
D-Fructose-1,6-diphosphate G
- characteristics a n d commercial p r e p a r a t i o n s 534
- concentration in a n i m a l tissues 2272 Galactinol
- determination 1314 - cleavage by a-galactosidase 1174
- example of a d e t e r m i n a t i o n in liver 315 Galactocerebroside
Fructose-1,6-diphosphate aldolase - oxidation by G a l - O D 1286
- activity in rabbit tissues 710 D-Galactosamine
- activity in rat tissues 1100 - determination
- activity in serum 1100 - - s u b m i c r o m e t h o d with ATP-y- [ P ] 32
1232
- assay of activity - oxidation by G a l - O D 1286
- in tissues 1105 D-Galactose
- - UV-assay, m a n u a l m e t h o d 1100 - determination
- - UV-assay, a u t o m a t e d m e t h o d 1106 - colorimetric assay with galactose oxidase
- n o r m a l values in h u m a n serum 1104, 1108 1282
D-Fructose-1 - p h o s p h a t e - - s u b m i c r o m e t h o d with A T P - y - [ P ]
32
1232
- characteristics a n d commercial p r e p a r a t i o n s 534 - - UV-assay with G a l - D H 1279
- concentration in animal tissues 2271 - oxidation with G O D 1212
- determination with /^-Galactose d e h y d r o g e n a s e
- liver aldolase 1308 - assay of activity 453
- values in rat tissues 1311 - characteristics a n d commercial p r e p a r a t i o n s 453
F r u c t o s e - 1 - p h o s p h a t e aldolase - in d e t e r m i n a t i o n of lactose 1180
- see phosphofructoaldolase Galactose oxidase
D-Fruc tose -6-phos phate - assay of activity 454
- characteristics a n d commercial p r e p a r a t i o n s 535 - characteristics a n d commercial p r e p a r a t i o n s 454
- concentration in a n i m a l tissues 2271 D-Galactose-1 - p h o s p h a t e
- determination 1238 - characteristics a n d commercial p r e p a r a t i o n s 535
- reaction with m a n n i t o l - l - P dehydrogenase 1278 - determination
F r u c t o s e - 6 - p h o s p h a t e kinase - as galactose after cleavage of p h o s p h a t e 1201
- assay of activity 126, 451 - with uridyltransferase 1288
- characteristics a n d commercial p r e p a r a t i o n s 451 D-Galactose-6-phosphate
- relative rates with nucleoside triphosphates 2081 -determination 1296
- see also p h o s p h o f r u c t o k i n a s e a-Galactosidase
j9-D-Fucose - assay of activity 455
- oxidation by G a l - D H 1281 - characteristics a n d commercial p r e p a r a t i o n s 455
Fumarase /?-Galactosidase
- assay of activity 452 - assay of activity 456
- in muscular d y s t r o p h y - in determination of lactose 1180
LV Index Galac-Gluco
/?-D-Galactoside - - with G O D a n d P O D , rapid assay 1211

- determination 1180 - - with G O D , P O D a n d A B T S 1212
D-Galacturonate - - with G O D , P O D a n d o-dianisidine 1206
-determination 1299 - - with H K a n d G 6 P - D H 1196
- n o r m a l values 1302 - with test p a p e r , semiquantitative 1211
- oxidation by G a l - O D 1286 - n o r m a l values in blood, serum a n d p l a s m a 1210
G a l a c t u r o n a t e isomerase - stability in b l o o d 166
-isolation 1302 D-Glucose-1,6-diphosphate
Gastricsin - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 537
- characteristics 1055 - c o n c e n t r a t i o n in a n i m a l tissues 2271
GDP-mannose Glucose oxidase
- see g u a n o s i n e - 5 ' - d i p h o s p h a t e m a n n o s e - assay of activity 457
GDP-mannose dehydrogenase - characteristics a n d commercial p r e p a r a t i o n s 457
-isolation 2216 - rate of reaction with various sugars 458
Gentianose D-Glucose-1 - p h o s p h a t e
- cleavage by saccharase 1178 - characteristics a n d commercial p r e p a r a t i o n s 537
Glass electrode - concentration in a n i m a l tissues 2269
- m e t h o d s with aid of 261 -determination 1233
D-Gluconate - hydrolysis c o n s t a n t 1235
-determination 1243 - n o r m a l values 1236
- values in wine 1246 Glucose-6-phosphatase
G l u c o n a t e kinase - assay of activity 876
- assay of activity 457 D-Glucose-6-phosphate
- characteristics a n d commercial p r e p a r a t i o n s 457 - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 538
- relative rates of reaction with nucleoside - c o n c e n t r a t i o n in a n i m a l tissues 2269
triphosphates 2081 -determination 1238
D-Gluconate-6-phosphate Glucose-6-phosphate d e h y d r o g e n a s e
- c o n c e n t r a t i o n in a n i m a l tissues 2286 --UV-assay 459,636
-determination 1248 - characteristics a n d commercial p r e p a r a t i o n s 458
D-Glucosamine - in blood diseases 52
- characteristics a n d commercial p r e p a r a t i o n s 536 - in m u s c u l a r d y s t r o p h y
- determination - in muscle 38, 45
- - s u b m i c r o m e t h o d with ATP-y- [ P ] 32
1228 - in serum 38
- with hexokinase 1251 - in n e p h r o n s of h u m a n kidneys 64
D-Glucosamine-6-phosphate - Michaelis c o n s t a n t s 458, 637
- characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 536 - n o r m a l values in
-determination 1257 - b l o o d elements 643
D-glucosamine-6-phosphate-N-acetylase - liver tissue 643
-isolation 1262 Glucose p h o s p h a t e isomerase
D-Glucose - assay of activity, colorimetric assay 1113
- concentration in a n i m a l tissues 2268 - characteristics a n d commercial p r e p a r a t i o n s 501
- destruction in experimental material 1177 - n o r m a l values 1115
- determination a-Glucosidase
- in haemolysed blood, UV-assay 1199 - assay of activity 459
--withAGT 1222 - characteristics a n d commercial p r e p a r a t i o n s 459
- - with A T P - y - [ P ] , s u b m i c r o m e t h o d
32
1228 - for d e t e r m i n a t i o n of m a l t o s e 1185
- with a u t o m a t i c analysers, fluorimetric 1201 -specificity 459, 1188
- with a u t o m a t i c analysers, with G O D , P O D , 2-O-a-D-Glucosido-D-erythrose
andABTS 1215 - cleavage by a-glucosidase 1188
as D , L , ( + ) , ( — ) , v, /?. Where necessary reference to other s y n o n y m s is given.

Gluco-Gluta Index LVI
Glucotest® 1198 — in serum

^-Glucuronidase — n o r m a l values 654, 659
- assay of activity 460, 929 --stability 169
- characteristics a n d commercial p r e p a r a t i o n s 460 — n o r m a l values in organ extracts of various
- general information 929 species 654
- in diagnosis of kidney diseases 62 Glutamate-oxaloacetate transaminase
- in muscular dystrophy 38 — apoenzyme, isolation 2198
- measurements in — assay of activity
- - bile 940 — colorimetric assay according to Reitman and
- - cerebrospinal fluid 939 Frankel 735
- gastric juice 941 — colorimetric assay according to Tonhazy 739
- - serum 930, 932 — - colorimetric assay with B M T D 742
- - serum in pregnancy 58 — UV-assay with M D H , a u t o m a t e d 733
- - tissues 934, 937 — - UV-assay with M D H , m a n u a l 727
- - urine 938 — with a u t o m a t i c analysers 733, 768, 771
- - vaginal fluid 941 — characteristics a n d commercial p r e p a r a t i o n s 462
- n o r m a l values in serum 932 — dependence
L-Glutamate — on age and b o d y weight 731
- concentration in animal tissues 2285 — on sex 731
- determination — determination after electrophoretic separation
- manometric 1662 745
- - with a regenerating system 108 — in blood diseases 51
- - with G I D H a n d A P A D 1713 — in heart diseases
- - with G I D H a n d N A D 1704 — differential diagnosis 36
- - with G I D H a n d tetrazolium salt 1708 — myocardial infarct 31
- n o r m a l values 1707, 1712 — in liver diseases
G l u t a m a t e decarboxylase — acute hepatitis 14
- assay of activity by radiochemical m e t h o d 305 — chronic hepatitis a n d cirrhosis 16
L-Glutamate decarboxylase 1663 — in serum 26, 27
L-Glutamate d e h y d r o g e n a s e — liver t u m o u r s 23
- assay conditions for liver from various species — obstructive j a u n d i c e 21
655 — in meat 75
- assay of activity — in mononucleosis, infectious 18
- colorimetric assay 656 — in muscle diseases
- - UV-assay 650 — muscular d y s t r o p h y 39
- characteristics a n d commercial p r e p a r a t i o n s 461 — myopathies, various 44
- equilibrium constants 461 — in n e p h r o n s of h u m a n kidneys 64
- in liver diseases — in serum
- acute hepatitis 15 — healthy subjects 9
- chronic hepatitis a n d cirrhosis 16 — n o r m a l values 731
- in serum 26, 27 — of rats after exercise 8
- liver obstruction 20 --stability 169
- liver t u m o u r s 23 — in urine 66
- obstructive j a u n d i c e 21 -isoenzymes 10
- toxic liver d a m a g e 20 Glutamate-pyruvate transaminase
- in mononucleosis, infectious 18 — assay of activity
- in muscular d y s t r o p h y — colorimetric assay according to Reitman and
- in muscle 38, 46 Frankel 760
- in serum 38 — colorimetric assay according to Tonhazy 764
- in n e p h r o n s of h u m a n kidney 64 — - UV-assay L D H , a u t o m a t e d 758
LVII Index Gluta-Glyce
Glutamate-pyruvate transaminase - obstructive j a u n d i c e 21

— assay of activity - toxic liver d a m a g e 20
— UV-assay with L D H , m a n u a l 752 - n o r m a l values in s e r u m 718
— with a u t o m a t i c analysers 758, 768, 771 Glutathione (GSH + GSSG)
— characteristics a n d commercial p r e p a r a t i o n s 463 - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s
— dependence 538, 539
— on age a n d weight 756 -determination 1643
— on sex 756 - n o r m a l values 1647
— DeRitis quotie nt 14, 16, 17, 20, 2 1 , 24 G l u t a t h i o n e reductase
— in heart diseases - assay of activity 465
— differential diagnosis 36 - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 465
— m y o c a r d i a l infarct 31 G l u t a t h i o n e reductases
— in h u m a n liver 12 - in b l o o d diseases 52
— in liver diseases - in m u s c u l a r d y s t r o p h y 39
— acute hepatitis 14 G l u t a t h i o n e synthetase
— chronic hepatitis a n d cirrhosis 16 - in b l o o d diseases 52
— in serum 26, 27 Glyceraldehyde
— liver t u m o u r s 22 - oxidation by a l d e h y d e d e h y d r o g e n a s e 1508
— obstructive j a u n d i c e 21 D-Glyceraldehyde-3-phosphate
— toxic liver d a m a g e 20 - c o n c e n t r a t i o n in a n i m a l tissues 2274
— viral hepatitis in mice 8 - determination 1314
— in mononucleosis, infectious 18 DL-Glyceraldehyde-3-phosphate
— in muscle diseases - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 539
— muscular d y s t r o p h y 39 L-Glyceraldehyde-3-phosphate
— m y o p a t h i e s , various 44 -determination 1439
— in n e p h r o n s of h u m a n kidney 64 Glyceraldehyde-3-phosphate dehydrogenase
— in serum - assay of activity 466
— healthy subjects 9 - characteristics a n d commercial p r e p a r a t i o n s 466
— n o r m a l values 756 - in blood diseases 54
— of rats after exercise 8 - in m u s c u l a r d y s t r o p h y
--stability 169 - in muscle 39, 45
Glutaminase - in serum 39
— assay of activity 465 D-Glycerate
— characteristics a n d commercial p r e p a r a t i o n s 465 - d e t e r m i n a t i o n 1419
L-Glutamine - n o r m a l values 1422
— c o n c e n t r a t i o n in a n i m a l tissues 2285 D-Glycerate dehydrogenase
— d e t e r m i n a t i o n with - see glyoxylate reductase
— - glutaminase a n d G 1 D H 1719 D-Glycerate-1,3-diphosphate
— glutamine synthetase 1716 -characteristics 1430
— n o r m a l values 1718, 1722 - determination 1429
G l u t a m i n e synthetase D-Glycerate-2,3-diphosphate
y-Glutamyl t r a n s p e p t i d a s e -determination 1433
— activity in b l o o d elements a n d tissues of various - m e t h o d of choice 1433
species 718 - n o r m a l values in erythrocytes 1438
— assay of activity, colorimetric assay 715 D-Glycerate-2-phosphate
— in liver diseases - c o n c e n t r a t i o n in a n i m a l tissues 2276
— acute hepatitis 15 - determination 1446
— in serum 26, 27 D-Glycerate-3-phosphate
— liver t u m o u r s 23 - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 540
as D , L , ( + ) , ( — ) , a, (i. Where necessary reference to other s y n o n y m s is given.

Glyce-Hemi Index LVIII
D-Glycerate-3-phosphate - assay of activity 470

- concentration in a n i m a l tissues 2275 - characteristics a n d commercial p r e p a r a t i o n s 470
-determination 1424 Guanase
Glycerokinase - assay of activity, UV-assay 1086
- assay of activity 469 - characteristics a n d commercial p r e p a r a t i o n s 471
- characteristics a n d commercial p r e p a r a t i o n s - i m p o r t a n c e in clinical chemistry 1086
468, 469 - n o r m a l values 1090
- relative rate of reaction with nucleoside tri Guanine
phosphates 2081 - characteristics a n d commercial p r e p a r a t i o n s 541
Glycerol - d e t e r m i n a t i o n 1909
- determination - extinction coefficient 1913
- radiochemical 1409 - n o r m a l values 1915
- - UV-assay with G K a n d G D H 1404 Guanosine
- - UV-assay with G K , P K a n d L D H 1825 - characteristics a n d commercial p r e p a r a t i o n s 542
- n o r m a l values in serum 1408, 1830 - cleavage by nucleoside p h o s p h o r y l a s e 1934
- reaction with sorbitol d e h y d r o g e n a s e 1326 - determination 1928
L-Glycerol-3-phosphate Guanosine-5 '-diphosphate
- concentration in a n i m a l tissues 2274 - determination 2149
- determination 1415 Guanosine-5 '-diphosphomannose
Glycerol-3-phosphate dehydrogenase - determination 2213
- assay of activity 468 - see also G D P - m a n n o s e
- characteristics a n d commercial p r e p a r a t i o n s 468 G u a n o s i n e - 3 ' : 5 ' - m o n o p h o s p h a t e (cyclic)
- in muscular d y s t r o p h y - concentration in animal tissues 2297
- in muscle 39, 45 -determination 2166
- in serum 39 Guanosine-5 '-monophosphate
Glycocyamine - determination
- reaction with C K 1776 - other m e t h o d s 2165
Glycogen - - with G - 5 - M P - k i n a s e 2162
- characteristics a n d commercial p r e p a r a t i o n s 540 G u a n o s i n e - 5 ' - m o n o p h o s p h a t e kinase
- concentration in a n i m a l tissues 2267 - assay of activity 472
- determination with - characteristics a n d commercial p r e p a r a t i o n s 472
- amyloglucosidase 1127 Guanosine-5 '-triphosphate
-hydrolysis 1127 - characteristics a n d commercial p r e p a r a t i o n s 542
- n o r m a l values 1131 - concentration in
Glycolaldehyde - - r a t heart 2081
- determination 1514 - - r a t liver 2081
Glycollate-2-phosphate - - rat skeletal muscle 2081
- characteristics a n d commercial p r e p a r a t i o n s 541 - determination
- in determination of glycerate-2,3-diphosphate - other m e t h o d s 2161
1433 - - together with I T P 2078
Glycylglycine dipeptidase - - w i t h PGK 2158
- assay of activity 982
Glyoxalase I H
- characteristics a n d commercial p r e p a r a t i o n s 469 Hemicellulase
Glyoxylate - p r e p a r a t i o n of crude p r o d u c t 1147
- determination 1517 Hemicelluloses
- reduction by L D H 1451 - determination 1143
Glyoxylic acid reductase - n o r m a l values 1147
LIX Index Hep-Hyd
Heparin H y d r o g e n peroxide
- determination - d e t e r m i n a t i o n with peroxidase 2246
- spectrophotometric 1151 3-Hydroxyacyl-CoA dehydrogenase
- titrimetric 1154 - assay of activity 474
Hexokina se - characteristics a n d commercial p r e p a r a t i o n s 474
- assay of activity 473 3-Hydroxyanthranilic acid
- characteristics a n d commercial p r e p a r a t i o n s 473 -determination 1736
- in b l o o d diseases 52 - n o r m a l values in urine 1739
- in m u s c u l a r d y s t r o p h y 3-Hydroxyanthranilic acid oxidase
- in muscle 39, 45 -isolation 1739
- relative rates with nucleoside t r i p h o s p h a t e s 2081 2-Hydroxybutyrate
H g L a m p , lines 189 - oxidation by L D H 1468
Hill coefficient 2 - H y d r o x y b u t y r a t e dehydrogenase
- in P K from yeast 781 - d e t e r m i n a t i o n in serum,
Histaminase - colorimetric assay 607
- in serum in pregnancy 58 - UV-assay, a u t o m a t e d 611
L-Histidine - UV-assay, m a n u a l 603
- determination - in liver t u m o u r s 24
- colorimetric assay 1669 - in m u s c u l a r d y s t r o p h y 39
- m a n o m e t r i c assay 1662 - in myocardial infarct 33
L-Histidine decarboxylase 1663 - n o r m a l values in serum 606, 610
Histogram 322 D-3-Hydroxybutyrate
H M G - C o A lyase - co ncentration in animal tissues 2292
- isolation 2029 -determination 1836
H M G - C o A reductase - n o r m a l values 1838
- assay of activity by a radiochemical assay 303, D - 3 - H y d r o x y b u t y r a t e dehydrogenase
304 - assay of activity 475
Homogenates - characteristics a n d commercial p r e p a r a t i o n s 475
- moist 401 - specificity 1839
-dry 407 DL-3-Hydroxybutyric acid
Homogenizers 401-404 - characteristics a n d commercial p r e p a r a t i o n s 543
H y a l u r o n a t e lyase L-3-Hydroxybutyryl-CoA
- assay of activity 944 - determination
-isolation 1163 - - other m e t h o d s 2025
- Michaelis constants 1161 --UV-assay 2022
H y a l u r o n i c acid - n o r m a l values 202*1
- a b s o r p t i o n spectrum 1158 3-Hydroxykynurenine
- competitive inhibition of d e g r a d a t i o n 1158 - n o r m a l values 1734
- determination 3-Hydroxy-3-methylglutaryl-CoA
- colorimetric assay 1162 - c o n c e n t r a t i o n in a n i m a l tissues 2291
- dependence of the UV-assay of the degree of - determination
polymerization 1161 - - other m e t h o d s 2029
--UV-assay 1157 - - UV-assay 2026
- n o r m a l values 1160 - n o r m a l values 2029
Hyaluronidase L-Hydroxyproline
- see h y a l u r o n a t e lyase - determination 1723
Hydrazine - n o r m a l values in urine 1725
- reaction with N A D 3-Hydroxypropionyl-CoA
- - light a b s o r p t i o n of 107, 1706 - determination 2031

Hyd-Inver Index LX
3-Hydroxypropionyl-CoA dehydrogenase - in two-substrate reactions 155

- isolation 2033 - of type of inhibition 152
Hydroxypyruvate -theory 151
- determination with Inhibitors for
- D-glycerate dehydrogenase 1457 -chymotrypsin 1074
- - LDH 1451 -kallikrein 1077
- n o r m a l values in rat tissues 1459 -plasmin 1066, 1077
3 a-Hydroxysteroid d e h y d r o g e n a s e -thrombin 1077
- assay of activity 476 -trypsin 1066
- characteristics a n d commercial p r e p a r a t i o n s 476 Inhibitor unit
-isolation 1874 -definition 1064
3 a, 20 ^ - H y d r o x y s t e r o i d dehydrogenase Inosine
- assay of activity 477 - characteristics a n d commercial p r e p a r a t i o n s 544
- characteristics a n d commercial p r e p a r a t i o n s 477 -determination 1932
3 j?, 17 j8-Hydroxysteroid d e h y d r o g e n a s e Inosine-5 ' - m o n o p h o s p h a t e
- assay of activity 477 -determination 2168
- characteristics a n d commercial p r e p a r a t i o n s 477 Inosine-5 '-triphosphate
-isolation 1874 - concentration ( + G T P ) in
Hypoxanthine - - rat heart 2086
- a bsorption curve 1941 - - rat liver 2086
- characteristics a n d commercial p r e p a r a t i o n s 543 - - rat skeletal muscle 2086
- determination - determination
- colorimetric assay 1945 - - with P G K , G A P D H 2158
--UV-assay 1941 - - together with G T P 2078
- n o r m a l values 1945,1949 - - other m e t h o d s 2161
Inositol d e h y d r o g e n a s e
-isolation 1336
I ( - )-Inositol-3-phosphate
- hydrolysis by myo-inositol-1 -phosphatase 1340
Ice b a t h 160 myo-Inositol
Identification of sample 205 - characteristics a n d commercial p r e p a r a t i o n s 544
Iditol -determination 1333
- reaction with sorbitol dehydrogenase 572,1326 myo-Inositol-1 - p h o s p h a t a s e
Immobilized enzymes - isolation 1340
- for determination of glucose 119 myo-Inositol-1 - p h o s p h a t e
- in enzymatic analysis 119, 120 -determination 1337
I n d e p e n d e n t variables 328 Insecticides
Indicator enzyme 109 - d e t e r m i n a t i o n with
Indicator reaction - cholinesterase 2249
-definition 109 - - other m e t h o d s 2257
- examples 109 et seq. International U n i t s 121, 311, 421
-preceding 112 Inulin
- succeeding 111 - cleavage by saccharase 1179
Individual steps -determination 1149
- in quantitative chemical analysis 205 Inulin clearance 1149
Individual values Invertase
- for uric acid 383 - assay of activity 450, 923
Inhibitor constants - characteristics a n d commercial p r e p a r a t i o n s 450
- determination - n o r m a l values 927
- in one-substrate reactions 152 - see also /?-fructofuranosidase
LXI Index Iso-Lac
Isobutyraldehyde 2-Ketoglutarate dehydrogenase

- oxidation by aldehyde d e h y d r o g e n a s e 1508 - see 2-oxoglutarate d e h y d r o g e n a s e
Isocitrate 2-Keto-n-valerate
- c o n c e n t r a t i o n in a n i m a l tissues 2281 - see 2-oxo-n-valerate
- determination 20-Ketosteroids
- fluorimetric 1573 - determination
--UV-assay 1570 - fluorimetric 1864
- n o r m a l values 1576 - UV-assay 1858
Isocitrate d e h y d r o g e n a s e - n o r m a l values 1862, 1867
- assay of activity, Kinases
- colorimetric assay 627 - relative rates with nucleoside di- a n d tri
- - UV-assay 624 phosphates 2081
- characteristics a n d commercial p r e p a r a t i o n s 479 Kinetics
- in b l o o d diseases 54 - chemical reactions 95-96
- in hepatitis - enzyme reactions 96 et seq.
- acute 15 - of enzymatic cycling 135
- chronic a n d cirrhosis 16 Kynureninase
- in m o n o n u c l e o s i s , infectious 19 -isolation 1735
- in m u s c u l a r d y s t r o p h y
- in muscle 39, 46
- in n e p h r o n s of h u m a n kidney 64 L
- isoenzymes 11
- in muscle 49 D-Lactate
- n o r m a l values in h u m a n serum 626, 631 - d e t e r m i n a t i o n with D - L D H 1492
Isoenzymes D-Lactate dehydrogenase
- characterization a n d m e a s u r e m e n t after - assay of activity 480
separation 272 - characteristics a n d commercial p r e p a r a t i o n s 480
- definition 261 L-Lactate
- electrophoretic s e p a r a t i o n 262 - concentration in animal tissues 2278
- importance 262 - determination
- in medicine 11 - fluorimetric 1468
Isotope dilution principle 296 - with a u t o m a t i c analysers 1479
Isotope l a b o r a t o r i e s - - with L D H a n d N A D 1464
- furnishing a n d o p e r a t i o n 283-285 - - with L D H from yeast 1483
- - with L D H , G P T a n d N A D 1475
- n o r m a l values 1468, 1471, 1474, 1479, 1487
K - stability in b l o o d 166
L-Lactate dehydrogenase
Kallikrein - assay of activity
-characteristics 1031 - - colorimetric assay with lactate, N A D a n d
- determination phenazinemethosulphate 579
- fluorimetric 1037 - UV-assay with pyruvate a n d N A D H 574
- UV-assay, m a n u a l 1033 - with a u t o m a t i c analysers 582
- UV-assay with autoanalysers 1034 - equilibrium c o n s t a n t 481
3-Ketoacid C o A - t r a n s f e r a s e - in b l o o d diseases 51
- see 3-oxoacid CoA-transferase - in diseases of kidney a n d urinary tract
2-Ketobutyrate - for diagnosis 62 et seq.
- see 2-oxobutyra te - in gynaecological diseases 57
2-Ketoglutarate - in heart diseases
- see 2-oxoglutarate - differential diagnosis 36
as D , L , ( + ), ( —), nt, fi. Where necessary reference to other s y n o n y m s is given.

Lac-Lip Index LXII
L-Lactate dehydrogenase, in heart diseases Lactose

— myocardial infarct 31 - determination 1180
— in liver diseases - oxidation by G a l - O D 1286
- - acute hepatitis 16 Lambert-Beer's Law 183
— chronic hepatitis a n d cirrhosis 16 L D H - 1 -isoenzyme
— in serum 26, 27 - after electrophoretic separation 593
— liver obstruction 20 - see also 2-hydroxybutyrate dehydrogenase
— liver t u m o u r s 23 - stability in serum 170
— obstructive j a u n d i c e 21 LDH-5-isoenzyme
— toxic liver d a m a g e 19 - after electrophoretic separation 593
— in mononucleosis, infectious 18 Lecithin
— in muscular dystrophy - determination 1813
— in muscle 39, 45 - values in foodstuffs 1817
— in serum 39 Leucine a m i n o p e p t i d a s e
— in myopathies, various 43 - assay of activity 954
— in pig as a sign of meat quality 75 - characteristics a n d commercial p r e p a r a t i o n s
— in serum 482, 952
— healthy subjects 9 - general 950
— n o r m a l values 9 - in diseases of kidney a n d urinary tract 66
— of rats after exercise 8 - in liver diseases
--stability 169 - acute hepatitis 15
— in urine 62 - chronic hepatitis a n d cirrhosis 16
— isoenzymes - in serum 26, 27
— assay of activity in serum 590, 593, 603, 607, - liver obstruction 21
611 - liver t u m o u r s 24
— in heart diseases 33 - obstructive j a u n d i c e 21
— in n e p h r o n s of kidneys 65 - in mononucleosis, infectious 18
— in skeletal muscle 48 - in serum in pregnancy 58
— in h u m a n tissues 11 - isoenzymes
— in urine 66 - in obstructive j a u n d i c e 22
— m e a s u r e m e n t s after electrophoretic separation - n o r m a l values 957
593 Leucine nitroanilidase
— myocardial infarct 31 - assay of activity 958
— separation on D E A E - S e p h a d e x . 590 - n o r m a l values 962
— of kidney Linear regression
— in n e p h r o n s 65 - c h e c k of 359
— in the structures 65 Lineweaver-Burk plot 146, 147
— total activity of h u m a n liver 14 Lipase
L-Lactate dehydrogenase from beef heart - assay of activity
— characteristics a n d commercial p r e p a r a t i o n s - photometric 819
482 - - titrimetric 814
L-Lactate dehydrogenase from rabbit muscle - in milk 74
— assay of activity 481 - in oil-containing seed 84
— characteristics a n d commercial p r e p a r a t i o n s 481 - n o r m a l values
L-Lactate dehydrogenase from yeast - in d u o d e n a l juice 822
— assay of activity 1490 - - i n serum 818,822
— isolation 1489 - see also post-heparin lipase
Lactate oxidase L i p o a m i d e dehydrogenase
-apoenzyme 2181 - assay of activity 448
LXIII Index Lip-Mano
Lipoxidases - in n e p h r o n s of kidney 64
- in foodstuff chemistry 75 - in serum
- in oil-containing seeds 84 - n o r m a l values 616, 622
Lipoxygenase --stability 170, 615
- assay of activity 483 - in structures of kidney 65
- characteristics a n d commercial p r e p a r a t i o n s 483 -isoenzymes 48,618
Liquid content of tissues - m e a s u r e m e n t s after electrophoretic separation
-calculations 314 618
Liver Maleate
- enzyme activity p a t t e r n s in h u m a n liver 12 - determination 1622
Liver aldolase M a l e a t e isomerase
-isolation 1312 -isolation 1624
L o g a r i t h m i c - n o r m a l distribution 350 Malonyl-CoA
L o n g i t u d i n a l assessment 383 - determination 2034
Luciferase - concentration in a n i m a l tissues 2293
-isolation 2125 Malonylsemialdehyde-CoA
L-Lysine - determination 2034
- determination Maltase
- colorimetric assay 1669 - assay of activity 459
- m a n o m e t r i c assay 1662 - characteristics a n d commercial p r e p a r a t i o n s
- with a u t o m a t i c analysers 1701 459
L-Lysine decarboxylase - see also a-glucosidase
- assay of activity 484 Maltose, d e t e r m i n a t i o n 1185
D-Mannitol
- characteristics a n d commercial p r e p a r a t i o n s 484 - content of in spores of A. oryzae 1273
D-Mannitol dehydrogenase
M
-isolation 1274
M a c e r a t i o n juice 411
- substrate specificity 1273
Mailing
D-Mannitol-1 -phosphate
- of biological samples 167
- concentration in E. coli 1278
L-Malate
-determination 1275
- c o n c e n t r a t i o n in a n i m a l tissues 2282
D - M a n n i t o l - 1 - p h o s p h a t e dehydrogenase
- determination
-isolation 1278
- - fluorimetric with M D H a n d A P A D 1600
-specificity 1278
- - with M D H a n d A P A D 1593
D-Mannosamine
- - with M D H a n d G O T 1589
- reaction with H K 1232, 1255
- - with M D H a n d N A D 1585
D-Mannose
- - with M D H in coupled assay 1596
-determination 1263
- n o r m a l values 1592, 1599, 1603
- oxidation by G O D 1212
L-Malate d e h y d r o g e n a s e
D-Mannose-1 -phosphate
- assay of activity,
-determination 1268
- after electrophoretic separation 618
D-Mannose-6-phosphate
--UV-assay 485, 613
-determination 1263
M a n o m e t e r capillaries 252
- equilibrium c o n s t a n t s 485
Manometer fluids 253
- in blood diseases 5 1 , 53, 54
Manometry
- in hepatitis, acute 16
- calculations 249
- in m u s c u l a r d y s t r o p h y
- in microtechniques 248
- in muscle 39, 46
- review 248
- in serum 39
as D , L , ( + ), ( - ) , a, /?. Where necessary reference to other s y n o n y m s is given.

Mano-NAD Index LXIV
M a n o m e t r y , technical details 252 Methyl glyoxal

- two-vessel m e t h o d 249-251 -determination 1496
Mean 4-Methyl umbelliferone
- probability distribution 337 -asaglycone 721
- arithmetic 330 - as substrate for U D P - g l u c u r o n y l transferase
Measurements 721
- against the reaction equilibrium 107 Michaelis constants
- continuous 122 - determination
-kinetic 131 - according t o Florini & Vestling, two substrates
- new techniques 117 149
- of concentrations by activation or inhibition of - according to Frieden, two substrates 150
enzymes 134 - according to Hofstee 147
- of enzyme activities (schematic) 122 - according to Lineweaver v Burk 146
- of metabolites on a kinetic basis 131 - according to Wilkinson 147
- of metabolites (schematic) 105 Michaelis-Menten equation 100, 145
- of protein c o n c e n t r a t i o n 171-176 Microanalyser for gases 248
- two-point m e t h o d 122 Microanalysis, a p p a r a t u s for 230
- with aid of coupled reactions 109 Microanalytical balances 235
- with preceding indicator reaction Microanalytical systems 236
--definition 112 Microcentrifuges 235
- details a n d calculations 112 et seq. Microcuvettes 238
- with succeeding indicator reaction 109 Microdissection techniques 238
- with unspecific enzymes 108 Microgasometry 242
M e a s u r e m e n t s , presentation of p h o t o m e t r i c Microgenerator for gases 248
- analogue 193 Micromethods 237
-digital 193 Microphotometry 237
M e a s u r i n g techniques Microtechniques 228 et seq.
- automated 202 et seq. Microtest tubes 235
- calorimetry 118 M i t o c h o n d r i a from yeast
-conductivity 118 - preparation 407
- immobilized enzymes 119 Mixers for microanalysis 235
- manometry 248 et seq. Mixing of samples
- microtechniques 228 et seq. - in a u t o m a t i o n 206
- photometry 180 et seq. Model, additive 343
- radiometry 118 M o n o c h r o m a t i c light 185
- review of new m e t h o d s 117 et seq. myo-Inositol, see u n d e r I
- with electrodes 118, 254 et seq. Myokinase
3-Me rc a ptopyruvate - assay of activity 486
-determination 1460 - characteristics a n d commercial p r e p a r a t i o n s
Messenger R N A 486
- determination of the activity in peptidizing - in muscular d y s t r o p h y
system 1901 - in muscle 39
Metabolites - in serum 39
- as biochemical reagents 523 - relative rates of reaction with nucleoside di
- concentration in animal tissues 2266 et seq. phosphates 2081
- distribution in intracellular c o m p a r t m e n t s 398
- stability in blood 166
N
- stability to deproteinizing agents 165
6-O-Methyl-D-galactose NAD-analogues
- oxidation by G a l - D H 453 - use in metabolite assays 107
LXV Index NAD-Null
NAD-pyrophosphorylase - in foodstuff chemistry 75

- assay of activity 487 p-Nitrophenyl-a-D-glucoside
- characteristics a n d commercial p r e p a r a t i o n s - hydrolysis by a-glucosidase 1188
487 N o r m a l distribution
Nicotinamide-adenine dinucleotide - logarithmic 350
- a b s o r p t i o n curve 104, 184 - probability density 336
- characteristics a n d commercial p r e p a r a t i o n s - test of 350
545 N o r m a l range
- c o n c e n t r a t i o n in animal tissues 2298 - c o m p a r i s o n of d a t a in literature 391
- n o r m a l values in rat tissues 2072 - definition 385
N i c o t i n a m i d e - a d e n i n e dinucleotide, reduced - dependence
- a b s o r p t i o n curve 104, 184 - - on age 388
- characteristics a n d commercial p r e p a r a t i o n s 545 - on sex 388
- conc e ntra tion in animal tissues 2298 - determination 385
- c o n c e n t r a t i o n in rat tissues 2072 - diurnal variations 384
- determination - selection of p r o b a n d s 386
- by enzymatic cycling 2066 N o r m a l values
- fluorimetric 2058 - determination methods 385
- other m e t h o d s 2053 - deductive 384
- UV-assay 2052 - graphical 389
- extinction coefficient 184, 546 - - others 388
-stability 158 - probit analysis 388
Nicotinamide-adenine dinucleotide p h o s p h a t e " N o t h i n g " dehydrogenase 274
- characteristics a n d commercial p r e p a r a t i o n s 546 Nucleoside d i p h o s p h a t e kinase
- conc e ntra tion in animal tissues 2298 - assay of activity 488
- determination - characteristics a n d commercial p r e p a r a t i o n s 488
- by enzymatic cycling 2060 Nucleoside m o n o p h o s p h a t e kinase
- - fluorimetric 2058 - assay of activity 489
- - UV-assay 2050 - characteristics a n d commercial p r e p a r a t i o n s 489
N i c o t i n a m i d e - a d e n i n e dinucleotide p h o s p h a t e , Nucleoside phosphorylase
reduced - assay of activity 490
- c o n c e n t r a t i o n in animal tissues 2298 - characteristics a n d commercial p r e p a r a t i o n s 490
- determination 5'-Nucleotidase
- by enzymatic cycling 2066 - in liver diseases
- fluorimetric 2058 - chronic hepatitis a n d cirrhosis 17
- other m e t h o d s 2056 - obstructive j a u n d i c e 22
- - UV-assay 2054 - in m u s c u l a r d y s t r o p h y
Nicotinamide-adenine nucleotides - in muscle 39
- determination 5'-Nucleotidases
- by enzymatic cycling 2059 - assay of activity 871
- fluorimetric 2057 - general information 871
--UV-assay 2048 - n o r m a l values 874
- extraction Nucleotides
- - methods 2045 - concentration in
Nicotinamide-mononucleotide - a n i m a l tissues 2293
- determination 2073 - - rat heart 2086
Nitrate - - rat liver 2086
- determination 2260 - rat skeletal muscle 2086
- n o r m a l values 2264 Nuclides, physical properties 283
N i t r a t e reductase Null hypothesis (statistics) 348

Oestra-Pep Index LXVI
O 3-Oxoacid C o A transferase
- isolation 2044
Oestradiol de hydro genase 2-Oxobutyrate
- assay of activity in vivo with isotopes 306 - reduction by L D H 1451
Olive oil emulsion 2-Oxoglutarate
- stabilized dry p r e p a r a t i o n 816 - characteristics a n d commercial p r e p a r a t i o n s 548
Operating characteristic curve 373 - concentration in a n i m a l tissues 2281
O p e r a t i o n a l isomers 396 - determination
O p t i m u m conditions for m e t h o d 379 - - fluorimetric 1580
O p t i m a l system --UV-assay 1577
- for quality control 374 - n o r m a l values in b l o o d 1579
L-Ornithine - stability in b l o o d 166
- determination 2-Oxoglutarate d e h y d r o g e n a s e
- - colorimetric assay 1669 -isolation 1986
- m a n o m e t r i c assay 1662 2-Oxo-n-valerate
Ornithine carbamoyltransferase - reduction by L D H 1451
- assay of activity - reductive a m i n a t i o n by G I D H 1580
--manual 691 Oxytocinase
- - with a u t o m a t i c analysers 695 - assay of activity
- in liver d a m a g e , toxic 20 - with L-cystine-di-/?-naphthylamide 967
- in muscular d y s t r o p h y 39 - with S-benzyl-L-cystine -p-nitroanilide 971
- n o r m a l values in serum 695 -general 951
L-Ornithine decarboxylase 1663, 1670 - in serum
Orotic acid (orotate) - in pregnancy 58
- characteristics a n d commercial p r e p a r a t i o n s - n o r m a l values 970, 973
548
P
- determination
- with d i h y d r o o r o t a t e d e h y d r o g e n a s e 1963 Paired vessels
- with O - 5 - M P p y r o p h o s p h o r y l a s e 1959 - in m a n o m e t r y 250
- extinction coefficient 1961 Palmitoyl-coenzyme A
- n o r m a l values 1962 -determination 1986
Outliers (in statistics) 362 Papain
Overflow pipettes 233 - assay of activity 491
Oxalate - characteristics a n d commercial p r e p a r a t i o n s 491
- determination Pentose m o n o p h o s p h a t e s
- manometric 1543 - in chloroplasts 1389
- photometric 1544 - radiochemical d e t e r m i n a t i o n 1385
Oxalate decarboxylase Pentose p h o s p h a t e s
- isolation 1544 - concentration in a n i m a l tissues 2286
Oxaloacetate Pepsin
- concentration in a n i m a l tissues 2280 - assay of activity
- determination - with h a e m o g l o b i n as substrate 1046
- fluorimetric 1608 - - with N-acetyl-L-phenylalanyl-l-3,5-di-
--UV-assay 1604 iodotyrosine as substrate 1052
- - with [ C ] - a c e t y l - C o A
14
1611 - characteristics a n d commercial p r e p a r a t i o n s 493
- n o r m a l values - details for m e a s u r e m e n t s
- in m o u s e brain 1611 - in serum 1052
- in rat liver 1615 - in tissues 1052
Oxidases - isoenzymes 77
- assay after electrophoretic separation 274 - n o r m a l values in gastric juice 1051, 1055
LXVII Index Pep-Phos
Pepsinogen - chronic hepatitis a n d cirrhosis 16

- activation 1046 - in serum 26, 27
-characteristics 1046 - liver t u m o u r s 23
Peptidases - obstructive j a u n d i c e 21
- assay of activity 950-999 - toxic liver d a m a g e 19
- after electrophoretic separation 275 - in milk 868
- in serum in p r e g n a n c y 58 - in mononucleosis, infectious 18
- review 949 - in m u s c u l a r d y s t r o p h y 40
Peptides - in serum
- characterization with enzyme 1625 - assay with a u t o m a t i c analysers 864
- stepwise d e g r a d a t i o n 1632 - c o n t i n u o u s assay 860
Percentile 325 - - in pregnancy 58
Peroxidases - n o r m a l values 860, 863
- assay of activity 495 --stability 170
- colorimetric assay 685 - in urine 67
- characteristics a n d commercial p r e p a r a t i o n s - see also alkaline p h o s p h a t a s e
- in b l o o d diseases 54 P h o s p h a t a s e s , general
- in milk 72 - acid a n d alkaline p h o s p h a t a s e in serum, t w o -
- in plant diseases 83 point m e t h o d 856
- in vegetables a n d fruit 74 - in meat a n d m e a t p r o d u c t s 75
- m e a s u r e m e n t s in tissues 689 - in milk 72
Peroxides, inorganic - n o r m a l values 860, 863, 870
- determination 2246 P h o s p h a t e , inorganic
Phenol oxidase, in vegetables a n d fruit 75 - co ncentration in a n i m a l tissues 2300
Phenolphthalein g l u c u r o n i d e - determination
- preparation 942 - chemical 2124
Phenyl-a-D-glucoside - - fluorimetric 2229
- cleavage by a-glucosidase 1188 - other m e t h o d s 2238
P h o s p h a t a s e , acid - - UV-assay 2234
- assay of activity 496 - n o r m a l values 2233
- - in serum, t w o - p o i n t m e t h o d 856 Phosphocreatine
- characteristics a n d commercial p r e p a r a t i o n s - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s
- in muscular d y s t r o p h y 40 529
- in serum - see also creatine p h o s p h a t e
- - in pregnancy 58 Phosphodiesterase
--stability 170 - assay of activity in isotope assay 304
- isoenzymes 11 - from beef heart
P h o s p h a t a s e , alkaline - assay of activity 497
- assay of activity 496 - characteristics a n d commercial p r e p a r a t i o n s
- - in milk 868 497
- in serum, c o n t i n u o u s m e t h o d 860 - from Crotalus terr. terr.
- in serum, t w o - p o i n t m e t h o d 856 - assay of activity 498
- in serum with a u t o m a t i c analysers 864 - characteristics a n d commercial p r e p a r a t i o n s
- in blood diseases 53 Phosphoenolpyruvate
- in diseases of kidney a n d u rin ary tract 68 - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 548
- in kidney - concentration in animal tissues 2276
- in h u m a n n e p h r o n s 64 -determination 1446
- in liver diseases 1 -Phosphofructoaldolase
- acute hepatitis 14 - assay of activity
as D , L, ( + ), ( - ) , a , fi. Where necessary reference to other s y n o n y m s is given.

Phos-Plas Index LXVIII
1-Phosphofructoaldolase, assay of activity - characteristics a n d commercial p r e p a r a t i o n s 503

--UV-assay 1109 - in b l o o d diseases 52
- n o r m a l values 1111 - in muscular d y s t r o p h y
Phosphofructokinase - in muscle 40
- in muscular d y s t r o p h y 40 2-Phosphoglycollate
- relative rates with nucleoside t r i p h o s p h a t e s 2081 - see glycollate-2-phosphate
- see also fructose-6-phosphate kinase P h o s p h o h e x o s e isomerase
Phosphoglucomutase - see phosphoglucose isomerase
- assay of activity 499 Phospholipase D
- - UV-assay 798 - assay of activity 504
- characteristics a n d commercial p r e p a r a t i o n s 499 - characteristics a n d commercial p r e p a r a t i o n s
- in muscular d y s t r o p h y 504
- in muscle 40 P h o s p h o m a n n o s e isomerase
- in serum 40 - assay of activity 505
- n o r m a l values 801 - characteristics a n d commercial p r e p a r a t i o n s 505
6-Phosphogluconate 5 '-Phospho-a-D-ribose-1 - d i p h o s p h a t e
- see gluconate-6-phosphate P h o s p h o r i c acid esters, with insecticide activity
6-Phosphogluconate d ehydro genase - determination 2249
- assay of activity 500 Phosphorylase a
--UV-assay 632 - assay of activity 505
- in blood diseases 53 Phosphorylases
- in muscular d y s t r o p h y - in muscular d y s t r o p h y 40
- in muscle 40 Phosphotransacetylase
- in serum 40 - assay of activity 507
- in plant diseases 83 - characteristics a n d commercial p r e p a r a t i o n s 507
- n o r m a l values in serum 634 Photometers
Phosphoglucose isomerase - principles of 184
- assay of activity 501, 1113 - review of available models 196-201
- characteristics a n d commercial p r e p a r a t i o n s 501 Photometric measurements
- in muscular d y s t r o p h y -errors 193
- in muscle 40 - in a u t o m a t i o n 209
- in serum 40 Photometry
- see also - in microtechniques 237
- glucosephosphate isomerase p H recording
- p h o s p h o h e x o s e isomerase - for enzymatic determination of metabolites 258
3-Phosphoglycerate Pipettes
- characteristics a n d commercial p r e p a r a t i o n s 540 - according to Sanz 233
- see also glycerate-3-phosphate - for micro-analysis 232
3-Phosphoglycerate kinase - standardization 161
- assay of activity 502 -tray 163
- characteristics a n d commercial p r e p a r a t i o n s 502 Plasma-specific enzymes 7
- in blood diseases 54 Plasmin inhibitors
- in muscular dystrophy - assay with azocasein as substrate 1071
- in muscle 40 - n o r m a l values 1074
- relative rates of reaction with nucleoside Plastic equipment 161
triphosphates 2081 Plastics
Phosphoglycerate m u t a s e - chemical stability 162
- assay of activity 503 - i n analysis 161
LXIX Index Plas-Pyri
Plastics Probability distribution

- properties a n d applications 162 - median 337
Point estimation p r o c e d u r e (statistics) 349 - dispersion 338
Poisson distribution 364 P r o d u c e r s ' risk 371
Polarography 245 n-Propanol
Polyol d e h y d r o g e n a s e - oxidation by A D H 1502
- see sorbitol d e h y d r o g e n a s e Propionaldehyde
Poly- a n d dipeptidases - reduction by aldehyde d e h y d r o g e n a s e 1508
- activity o p t i m a 979-980 Propionate
- classification 979-980 - p h o s p h o r y l a t i o n by acetate kinase 425, 1527
- general information 978 Propyleneglycol
- occurrence 979-980 - reaction with sorbitol dehydrogenase 1326
- o p t i m u m c o n d i t i o n s for m e a s u r e m e n t s 981 Prostaglandin d e h y d r o g e n a s e
- purification 979-980 -isolation 1883
- substrates 981 Prostaglandins
Polyphenoloxidase -determination 1887
- in plant diseases 83 Protease inhibitors
- in serum in pregnancy 58 - definition of inhibitor unit 1064
Polyribonucleotides - enzyme-inhibitor complex 1064
- determination of activities in peptidizing systems - general 1064
1901 - substrates for enzymatic assays 1065
- incorporation yield 1906 Proteinases
Polyunsaturated fatty acids - assay of activity with autoanalysers 1000
- determination 1807 Protein-binding assay
Polyuridylic acid - d e t e r m i n a t i o n of A - 3 : 5 - M P 2136
- characteristics a n d commercial p r e p a r a t i o n s 549 -principle 2137
Postheparin lipase Proteins
- assay of activity - characterization with enzymes 1625
- 1-mono-olein as substrate 828 - cleavage with
- triolein as s u b s t r a t e 825 - chymotrypsin 1629
- general 824 - other proteases 1631
- nomenclature 824 - pepsin 1630
- n o r m a l values 827, 830 - trypsin 1625
Precision - stepwise d e g r a d a t i o n 1632
-control 370 et seq. Pteroylglutamic acid
- from day to day 367, 378 - characteristics and commercial p r e p a r a t i o n s 552
- in the same series 367 Purine nucleotides
- of assay m e t h o d s 314 - analytical differentiation 2078
- of m e a s u r e m e n t s 314, 366 Purity criteria
- of m e a s u r e m e n t s of radioactivity 292 - for reagents 421 - 4 2 3
Prekallikrein Purity definitions
- determination with autoanalysers 1034 - for coenzymes a n d substrates 422
P r e p a r a t i o n of control samples 373, 376 - for enzymes 421
P r e p a r a t i o n of samples Pyridoxal-5-phosphate
- for microtechniques 229 - characteristics a n d commercial p r e p a r a t i o n s 550
Primary standard 373 -determination 2194
Probability 333 - n o r m a l values 2198
Probability density 336 - reduction to p y r i d o x a m i n e - 5 - p h o s p h a t e 2196
Probability distribution Pyridoxamine-5-phosphate
- mean 337 - determination 2194
as D, L, ( + ), ( - ) , a, p. Where necessary reference to other s y n o n y m s is given.

Pyri-Refe Index LXX
Pyridoxamine-5-phosphate Q
Pyrimidine nucleotides Quality
- analytical differentiation 2078 - biochemical reagents 418-420
P y r o p h o s p h a t a s e , inorganic Quality control
- assay of activity 508 - minimal system 374
- characteristics a n d commercial p r e p a r a t i o n s 508 - optimal system 374
P y r o p h o s p h a t e , inorganic - practical execution 374
- concentration in animal tissues 2300 - statistical 370
- determination 2239 Quantile
Pyruvate - of a probability distribution 338
- characteristics a n d commercial p r e p a r a t i o n s Q u a n t u m counter 2114
550 Q u a r t z fibre balance 236
- concentrations in a n i m a l tissues 2276 Quinolinate phosphoribosyltransferase
- determination - isolation 1346
- fluorimetric 1452 Q u i n o n e reductase
- - w i t h LDH 1446 - in plant diseases 83
- - with pH-stat 258
--UV-assay 1446
- formation of dimers in solution 1453 R
- reduction by glyoxylate reductase 1519 R a d i a t i o n , instruments for measuring 285-291
- stability in b l o o d 166 R a d i a t i o n , sources of
Pyruvate decarboxylase - for p h o t o m e t r y 184-187
- apoenzyme, isolation 2192 Radioactivity determination
- assay of activity 509 - precision 292
- characteristics a n d commercial p r e p a r a t i o n s Raffinose
509 - determination 1172
-isolation 2191 Range, normal 384-391
Pyruvate kinase (muscle) Ranked data 320
- assay of activity 510, 774, 778 Raw data 320
- characteristics a n d commercial p r e p a r a t i o n s Reaction curves, non-linear 309
509-510 Reaction kinetics 95
- in blood diseases 52 Reactions
- in muscular d y s t r o p h y -coupled 101
- in muscle 40, 45 - simple 99
- measurements in serum a n d erythrocytes Reagents
- n o r m a l values 777 - biochemical
- relative rates of reaction with nucleoside - description of 425-556
diphosphates 2081 - - h a n d l i n g of 158
Pyruvate kinase from yeast - manufacturers a n d suppliers 424
- assay of activity 778 - complete kits 557
- characteristics 778 - control of 158 et seq.
- Hill coefficients 778 - for procedures with radiobiochemicals 301
- measurements R e c o r d i n g of p h o t o m e t r i c measurements 193
- kinetic constants 781 Reductases
- - of cellular P K activity 780 - in milk 73
- molecular weight 778 Reference diver 243
LXXI Index Refe-Sor
Reference units 310 - c o n c e n t r a t i o n in a n i m a l tissues 2287

Regenerating systems 133 - determination
Regression - radiochemical 1385
- linear 328 --UV-assay 1359
Regression line Ribulose-5-phosphate kinase
- test of slope 359 -isolation 1389
R e p o r t i n g the n o r m a l range R u b b e r stoppers 159
- necessary information 389
Ribitol
- oxidation by S
- ribitol d e h y d r o g e n a s e 1354
- sorbitol de hydrogenase 1326 S-134-fraction
Ribitol de hydroge na se -preparation 1907
-isolation 1356 Sample collection 164, 396, 400
Ribonuclease Sample identification 205
- assay of activity 511 Sample storage 165, 168
- characteristics a n d commercial p r e p a r a t i o n s 511 Sarcosomes
- in blood diseases 54 - preparation 406
- inhibition by heparin 1151 Seasonal variation of results 389
- in muscular d y s t r o p h y 40 Secondary s t a n d a r d 377
- isoenzymes 11 Secreted enzymes 7
Ribonucleic acid Sedoheptitol
- characteristics a n d commercial p r e p a r a t i o n s 551 - oxidation by S D H 1384
- m R N A , d e t e r m i n a t i o n of activity in peptidizing D-Sedoheptulose-1,7-diphosphate
systems 1901 - determination 1193
- t R N A , d e t e r m i n a t i o n of acceptor activity for D-Sedoheptulose-7-phosphate
a m i n o acids 1894 - c o n c e n t r a t i o n in a n i m a l tissues 2287
D-Ribose-5-phosphate - determination 1189
- characteristics a n d commercial p r e p a r a t i o n s 551 DL-Serine
- determination -determination 1727
- radiochemical 1385 - n o r m a l values 1730
--UV-assay 1342 Sex dependence of n o r m a l range 388
R i b o s e - 5 - p h o s p h a t e isomerase Shewhart, statistical quality c o n t r o l 371
- isolation 1344 Side activities 108
Ribosomes Simple analysis of variants 353
- preparation Soluble ribonucleic acid
- - animal 405 - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 553
- from E. coli 1907 Solutions
D-Ribulose - of radiobiochemicals 292
-determination 1354 D-Sorbitol
L-Ribulose - determination
-determination 1350 - according to s t a n d a r d m e t h o d 1323
D-Ribulose-1,5-diphosphate - in wine 1326
- characteristics a n d commercial p r e p a r a t i o n s 552 - n o r m a l values 1326, 1330
- determination Sorbitol d e h y d r o g e n a s e
- radiochemical 1385 - assay of activity 512, 569
--UV-assay 1362 - characteristics a n d c o m m e r c i a l p r e p a r a t i o n s 512
Ribulose-1,5-diphosphate carboxylase - in heart diseases
-isolation 1389 - differential diagnosis 36
D-Ribulose-5-phosphate - i n liver 14
as D , L , ( + ) , ( - ) , a , /?. Where necessary reference to other s y n o n y m s is given.

Sor-Tar Index LXXI1
Sorbitol d e h y d r o g e n a s e Steroid alcohols in urine

- in liver diseases, toxic 19 -determination 1868
- in m u s c u l a r d y s t r o p h y 40 - n o r m a l values 1872
- in o r g a n s 572 Steroid conjugates
- in serum -hydrolysis 1848
- n o r m a l values 572 Steroid sulphates
--stability 170 -hydrolysis 1854
- myocardial infarction 31 Stirrers for micro-analysis 235
D-Sorbitol-6-phosphate Stopper diver 243
-determination 1331 Student distribution 353
L-Sorbose-6-phosphate Subcellular particles
- determination 1320 - isolation 407
Specific activity Substances
- o f enzymes 311,422 - h a n d l i n g of biochemicals 158
Specific radioactivity 294 - storage, stability a n d control 158
Spectrophotometer Substrates
- review of m o d e l s 198-200 - as biochemical reagents 5 2 3 - 556
Spectrum-line p h o t o m e t e r Succinate
- review of m o d e l s 197 - concentration in animal tissues 2282
Spermidine -determination 16.16
- determination 1740 - n o r m a l values i 620
- n o r m a l values 1747 Succinate thiokinase
Spermidine oxidase - in d e t e r m i n a t i o n of succinate 1616
- isolation 1742 Succinyl-CoA
Spermine - determination 2041
- determination 1744 Sucrose
- n o r m a l values 1747 - determination 1176
Stability Sulphite inhibitions of enzymes 88
- of biochemical reagents 421 Systematic errors 363
- of enzymes in serum 168-170
- of metabolites in b l o o d a n d deproteinized
solutions 166 T
Stachyose
- cleavage by a-galactosidase 1174 t-Distribution 353
Standard t-Test
- primary 373 - for paired differences 353
- secondary 377 - t o check for similarity of two expected values
S t a n d a r d curves 310 354
S t a n d a r d deviation 314, 338, 366 D-Tagaturonate
- empirical 325 - determination 1299
S t a n d a r d i z a t i o n of biochemical reagents 418 L-Tartrate
Statistical assessment of results 381 et seq. - determination with L-tartrate dehydrase 1397
Statistical quality c o n t r o l 370 meso-Tartrate
- basic principles 370 - determination with L-tartrate dehydrogenase
- practical execution 374 1400
Statistical tests 332 et seq. L-Tartrate d e h y d r a s e
Statistical terms a n d m e t h o d s 319 et seq. -isolation 1403
Stepwise d e g r a d a t i o n L-Tartrate d e h y d r o g e n a s e
- of peptides and proteins with carboxypeptidase -isolation 1493
and L A P 1632 - Michaelis~covMd.nis 1400
LXXIII Index Tern-Trio
Temperature d e p e n d e n c e Tissue disintegration

- of enzyme reactions 127 - chemical 305
Test - thermal 404
- of equality between t w o variances (t-Test) 354 Tissue fixation 400
- of l o g a r i t h m i c - n o r m a l distribution 350 Tissue fractionation 407
- of n o r m a l distribution 350 Tissue, functional state 397
- of type of distribution 350 Tissue slices 399
Tetrahydrofolate d e h y d r o g e n a s e Titrimetric m e t h o d s
- see Dihydrofolate reductase - in microtechniques 245
Tetrahydrofolate formylase Total glycerol
- assay of activity - n o r m a l values in h u m a n serum 1830
- - other m e t h o d s 1123 Transaldolase
--UV-assay 1118 - assay of activity
- n o r m a l values 1123 - UV-assay 514,710
Tetrahydrofolate synthetase - characteristics a n d c o m m e r c i a l p r e p a r a t i o n 513
-isolation 1550 - in rabbit tissues 710
Tetrahydrofolic acid Transamidinase
- preparation 1124, 1550 - colorimetric assay 699
Tetrahydropteroylglutamic acid Transfer ribonucleic acids
- see tetrahydrofolic acid - characteristics a n d commercial p r e p a r a t i o n s 551
Tetrazolium salts - d e t e r m i n a t i o n of acceptor activity for a m i n o
- application in electrophoresis 274 acids 1894
- application in enzymatic reactions 139 Transketolase
- ditetrazolium salts 137 - assay of activity 703
- m o n o t e t r a z o l i u m salts 137 - in m u s c u l a r d y s t r o p h y 40
- structural formulae 138 - in rabbit tissues 710
-reduction 137 - in thiamine deficiency 708
- redox potentials 138 - isolation a n d crystallization 1380
Thiamine pyrophosphate - n o r m a l values in b l o o d 708
- characteristics a n d commercial p r e p a r a t i o n s 553 Transmission 181
- d e t e r m i n a t i o n with pyruvate decarboxylase 2186 Transsulphurase
- n o r m a l values 2190 - isolation 1463
- other m e t h o d s 2190 Transverse assessment 391
T h i a m i n e p y r o p h o s p h a t e deficiency Triacetate
- transketolase activity 708 -determination 1844
Thin-layer electrophoresis 264 Triglycerides
DL-Threonine - alkaline hydrolysis 1825
- determination 1727 - average molecular weight 1829
- n o r m a l values 1730 - determination
T h r o m b i n inhibitors 1077 - after alkaline hydrolysis 1825
T h r o m b o p l a s t i n time - after enzymatic hydrolysis 1831
- n o r m a l values 1029 - enzymatic hydrolysis 1821
- original m e t h o d according to Quick 1027 - n o r m a l values 1830
- use of citrated b l o o d 1029 Triosephosphate isomerase
- reaction m e c h a n i s m s 1026 - assay of activity 515
Thymidine - characteristics a n d commercial p r e p a r a t i o n s 515
- see deoxythymidine - in b l o o d diseases 52
Tie (in statistics) 321 - in m u s c u l a r d y s t r o p h y
Tissue brei 399 - in muscle 40
as D , L , ( + ), ( - ) , a, p. Where necessary reference to other s y n o n y m s is given.

Trio-Uri Index LXXIV
Triosephosphate isomerase in m u s c u l a r d y s t r o p h y Unspecificity 108,421

- in serum 40 Urea
Triphosphopyridine nucleotide - determination with
- see nicotinamide-adenine dinucleotide p h o s p h a t e - a u t o m a t i c analysers 1798
Trypsin - G 1 D H as indicator enzyme 1794
- assay of activity with - p h e n o l a n d hypochlorite as indicator reaction
- casein as substrate 1018 1791
- haemoglobin as substrate 1013 Urease
- N-benzoyl-L-arginine ethyl ester as substrate - assay of activity
516 --UV-assay 1081
- N a-p-toluenesulphonyl-L-argininemethyl - characteristics a n d commercial p r e p a r a t i o n s 517
ester as substrate 1021 - n o r m a l values 1084
- characteristics a n d commercial p r e p a r a t i o n s 515 Uric acid
-general 1013 - characteristics a n d commercial p r e p a r a t i o n s 554
- n o r m a l values 1017, 1020, 1023 - determination
Trypsin inhibitor - colorimetric 1954
- characteristics a n d commercial p r e p a r a t i o n s 517 --UV-assay 1951
Trypsin inhibitors - n o r m a l values 1054, 1057
- measurements with Uricase
- azocasein as substrate 1071 - assay of activity 518
- N-benzoylarginine-p-nitroanilide as substrate - characteristics a n d commercial p r e p a r a t i o n 518
1066 Uridine-5 ' - d i p h o s p h a t e
- n o r m a l values 1070, 1074 - determination 2172
Turanose Uridine-5 '-diphosphogalactose
- cleavage with a-glucosidase 1188 -determination 2221
Two vessel m e t h o d 249 - n o r m a l values 2224
L-Tyrosine Uridinediphosphogalactose pyrophosphorylase
- determination - assay of activity in radiochemical assay 304
- colorimetric assay 1669 Uridine-5 '-diphosphoglucose
- m a n o m e t r i c assay 1662 - characteristics a n d commercial p r e p a r a t i o n s 555
L-Tyrosine decarboxylase 1663 - c o n c e n t r a t i o n in a n i m a l tissues 2268
U U r i d i n e d i p h o s p h o g l u c o s e dehydrogenase
UDPG - characteristics a n d commercial p r e p a r a t i o n s 519
- see uridine-5-diphosphoglucose Uridinediphosphoglucose pyrophosphorylase
UDP-galactosamine - assay of activity 520
- reaction with uridyltransferase 802 - characteristics a n d commercial p r e p a r a t i o n s 519
UDP-galactose Uridine-5 ' - d i p h o s p h o g l u c u r o n i c acid
- see uridine-5'-diphosphogalactose - characteristics a n d commercial p r e p a r a t i o n s 555
UDP-glucuronyltransferase Uridine-5 ' - m o n o p h o s p h a t e
- assay of activity - determination
- fluorimetric 721 - - with N M P - k i n a s e , N D P - k i n a s e , U D P G - p y r o -
- n o r m a l values in liver 724 phosphorylase a n d U D P G - d e h y d r o g e n a s e
- other assay m e t h o d s 725 2172
Ultramicromethods 228 - - with N M P - k i n a s e , P K a n d L D H 2153
Ultrasonic generator 404 - n o r m a l values 2178
Units Uridine-5 '-triphosphate
- of enzyme activity 121, 311, 313, 421 - concentration in a n i m a l tissues 2297
LXXV Index Uri-Zymo
Uridine-5 '-triphosphate X a n t h i n e oxidase

- determination 2172 - assay of activity
- n o r m a l values 2178 - colorimetric assay 644
Uridyltransferase - - UV-assay 522
- assay of activity - characteristics a n d commercial p r e p a r a t i o n s 521
--UV-assay 521, 802 - in milk 73
- characteristics a n d commercial p r e p a r a t i o n s 521 - isoenzymes 11
- n o r m a l values 805 - n o r m a l values in b l o o d , serum a n d organs 648
Uropepsinogen Xylitol
-characteristics 1046,1056 - d e t e r m i n a t i o n with sorbitol dehydrogenase 1381
- see also pepsin Xylitol d e h y d r o g e n a s e
- NAD-dependent
V --isolation 1369
- NADP-dependent
Valeraldehyde - isolation 1369
- reduction by A D H 1508 D-Xylose
Values, true 363 - characteristics a n d commercial p r e p a r a t i o n s 556
Variability - d e t e r m i n a t i o n with D-xylose isomerase 1371
- biological 389 D-Xylose isomerase
Variables -isolation 1375
- dependent 328 D-Xylulose
- independent 328 - d e t e r m i n a t i o n with
Variance - NAD-xylitol dehydrogenase 1368
- analysis 327, 338 - D-xylulose isomerase 1371
Variation coefficient 312, 314, 327 L-Xylulose
Visualization -determination 1365
- N A D ( P ) - d e p e n d e n t reactions D-Xylulose d e h y d r o g e n a s e
--ethanol 142 -isolation 1369
- glucose 142 L-Xylulose d e h y d r o g e n a s e
--glutamate 1708 - isolation 1369
- glutamate dehydrogenase 142 D-Xylulose-5-phosphate
- lactate d e h y d r o g e n a s e 141 - co ncentration in a n i m a l tissues 2287
- malate dehydrogenase 142 - determination
- succinate d e h y d r o g e n a s e 141 - radiochemical 1385
Volume activity --UV-assay 1377
-calculation 313 Xylulose-5-phosphate epimerase
-definition 311 - isolation 1345
Water content of tissues 314, 315 Y

Wavelengths, o p t i m a l 181
Yeast L D H
-solation 1489
X
Xanthine
- characteristics a n d commercial p r e p a r a t i o n s Z
- determination
- colorimetric assay 1945 Zwischenferment
--UV-assay 1941 - see glucose-6-phosphate dehydrogenase
- n o r m a l values 1945, 1949 Zymogram 263
as D , L , ( + ) , ( —), a, /?. Where necessary reference to other s y n o n y m s is given.

Atomic Weights·
International atomic weights ba ed** on the value 12 for the relative atomic mass of the carbon isotope
12C (according to·**).
Sym- Atomic Atomic Sym - Atomic Atomic

Element Element number weight
bol number weight bol
Aluminium AI 13 26.9815 Molybdenum Mo 42 95.94

Antimony Sb 51 121.75 eodymium d 60 144.24
Argon Ar 18 39.948 eon e 10 20.183
Arsenic As 33 74.9216 ickel i 28 58.71
Barium Sa 56 137.34 Niobium Nb 41 92 .906
Beryllium Be 4 9.0 122 Nitrogen N 7 14.0067
Bism ut h Di 83 208 .980 Osmium Os 76 190.2
Boron D 5 10.811 Oxygen 0 8 15.9994
Bromine Br 35 79.909 Pa lla d iu m Pd 46 106.4
Cadmium Cd 48 112.40 Phosphorus P 15 30.9738
Cae ium Cs 55 132.905 Platinum Pt 78 195.09
Calcium Ca 20 40.08 Potassium K 19 39 .102
Carbon C 6 12.01115 Pra eodymium Pr 59 140.907
Cerium Ce 58 140.12 Rhenium Re 75 186.2
Chlorine CI 17 35.453 R hod iu m Rh 45 102.905
Chromium Cr 24 51.996 Rubidium Rb 37 85.47
Cobalt Co 27 58.9332 R ut he niu m Ru 44 101.07
Copper Cu 29 63.54 Samari u m Sm 62 150.35
Dysp ro sium Dy 66 162.50 Sca ndi um Sc 21 44 .956
Erbium Er 68 167.26 Sele niu m Se 34 78.96
Europium Eu 63 151.96 Silico n Si 14 28.086
Fluorine F 9 18.9984 Si lver Ag 47 107.870
Gadolinium Gd 64 157.25 Sodium a II 22.9898
Gallium Ga 31 69.72 Strontium Sr 38 87.62
Germanium Ge 32 72.59 Su lphur S 16 32.06
Gold Au 79 196.967 Tantal um Ta 73 180.948
Hafnium Hf 72 178.49 Tellurium Te 52 127.60
Helium He 2 4.0026 Terbium Tb 65 158.924
Ho lm ium Ho 67 164.930 T hallium TI 81 204.37
Hyd rogen H I 1.00797 T hori um Th 90 232.038
Ind ium In 49 114.82 T huli um Tm 69 168.934
Iod ine I 53 126.9044 T in Sn 50 118.69
Irid iu m Ir 77 192.2 T ita n ium Ti 22 47 .90
Iron Fe 26 55.847 Tungsten W 74 183.85
Kr ypton Kr 36 83 .80 Uran ium U 92 238.03
Lantha num La 57 138.9 1 Vanadium V 23 50.9 42
Lead Pb 82 207 . 19 Xe non Xe 54 131.30
Lithi um Li 3 6.939 Y tterb ium Yb 70 173.04
Lut etium Lu 73 174.97 Ytt rium Y 39 88.905
Magn esium Mg 12 24.312 Zi nc Zn 30 65.37
Man ga nese Mn 25 54.9381 Zirco niu m Zr 40 91.22
Me rc ury Hg 80 200 .59
• Lanthan ides a nd ac tinide a rc incomplet e.

•• In tern ati onal U n ion of Pure and Applied C he m istry . Report o f th e 10th Gener al Assembly. Otta wa
1960, p . 24.
••• International U n io n of Pure a nd Applied C he mis try. Com pt. rend . d e la 24c Con ference 1967.
Butterworth. London 1967, p. 130.
of Enzymes (Continuation)
3.2.2 Hydrolysing JV-glycosyl compounds
\\~k ''V:* s v-'- .. V 3AI2 Peptftyfa^ licihtal^^ '

^ • S ^ ^ ? ^ V / ' l * | i i Dipeptide hydrolases
^ df n 0 r S 3 4 1 4 DiP^tidvlpeptide hydrolases
r transfers) ^^g^ffefPeptidvldipeptide h vdm
» for other substitnled ^ n t f b o 0 ^ ^ ^ A

2,7*9 Pho
^containing gratis
jM deling on etfar 3.5,99 In otter compotrnds

3.1 J Carboxylic ester hydrolases
3.6J In phosphoryl-cotitammg anhydrides
3,6.2 In sulphonyl-contaitiitig anhydrides
3.1.5 Triphosphoric monoester hydrolases 33 Acting on carbon-carbon bends W B I I B
3.74 In ketonic substances
3X7 Diphosphoric monoester hydrolases 3.8 A cting on haUde bonds
3.2 Acting on glycosyt compounds 3.8. f In C-halide compounds
3.2 J Hydrolysing O-glycosyl compounds 3.8.2 In P-halide compounds |

Hans-UIrich Bergmeyer (Eds.) - Methods of Enzymatic Analysis-Academic Press (1974) PDF

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METHODS

in collaboration with Karlfried Gawehn

Second English Edition

Verlag Chemie Weinheim

a) G e r m a n editions with the title " M e t h o d e n der enzymatischen A n a l y s e "

1. Auflage 1962, one volume

1st Edition 1963, one v o l u m e

2nd Edition (translated from the Third G e r m a n Edition), 1974, four v o l u m e s

W i t h 263 figures a n d 546 tables.

I S B N : 3-527-25370-X (Set Verlag Chemie)

Library of Congress C a t a l o g C a r d N u m b e r : 73-75657

©Verlag Chemie G m b H , Weinheim/Bergstr., 1974

C o m p o s i t i o n : H e l m u t Becker, 6232 Bad Soden, G e r m a n y

list o f a u t h o r s a n d a b b r e v i a t i o n s a n d t h e c o m p l e t e list o f c o n t e n t s . It is h o p e d that t h e s e

Tutzing/Oberbayern (Germany), M a r c h 1974 H a n s Ulrich Bergmeyer.

From the Preface to the 1st Edition

Tutzing/Oberbayern (Germany), M a r c h 1963 Hans Ulrich Bergmeyer

list o f a u t h o r s a n d a b b r e v i a t i o n s a n d t h e c o m p l e t e list o f c o n t e n t s . It is h o p e d that t h e s e

Tutzing/Oberbayern (Germany), M a r c h 1974 H a n s Ulrich Bergmeyer.

From the Preface to the 1st Edition

Tutzing/Oberbayern (Germany), M a r c h 1963 Hans Ulrich Bergmeyer

Bergmeyer, H a n s Ulrich Boulanger, Paul

Bevill, R a r d o n D . D e p a r t m e n t of Biological Chemistry

Buttner, H a n n e s Dahl, Katharina von

Cerioiti, G i o v a n n i Dahlqvist, Arne

Czok, Rudolf Egami, Fujio

Sandoz Forschungsinstitut G m b H Mitsubishi-Kasei Institute

Eggstein, Manfred Friebe, Ursula

Garland, Peter Bryan Goldberg, Nelson D .

Gerlach, Ulrich Grassl, Marianne

Clinica Delle M a l a t t i e Infetive

Goedde, Heinz W. Gutmann, Ingeborg

Hagen, Alexander Heinz, Fritz

Jones, Mary Ellen King, John

Medizinische Poliklinik D-8000 M u n i c h 15, G e r m a n y p. 2045

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Knappe, Joachim Kusche, Jurgen

Leuthardt, Franz Lowry, Oliver H.

Levine, Jacob B. Luhby, A. Leonard

Lohr, Georg Wilhelm Lynen, Feodor

Lorenz, Wilfried Matthaei, Heinrich

Maurer, Claus Nagel, Charles W.

Mollering, Hans Ohlenbusch, Hans-Dieter

Otto, Peter Preiss, Jack

Packmann, Paul M. University of California

Rosano, Carmen Louis Schmid, Ella

Schievelbein, H e l m u t Institut fur Biochemie

N u B b a u m s t r a s s e 20 D-6300 Giessen, G e r m a n y p. 1596, 1994

D-8000 M u n i c h 2, G e r m a n y p. 1731, 1736

Schreiber, Gerhard Staib, Wolfgang

Szasz, G a b o r Voigt, Klaus-Dieter

Tubbs, Philip K. Waller, H a n s D i e r c k

Wieme, Roger Jozef

W i l k i n s o n , J. H e n r y Zachau, Hans Georg

Zak, Bennie Ziegenhorn, Joachim

Abbreviations of Units of Mass and Constants

mm. Millimetre [10 ~ m.] 3

hr. Hour 1. Litre [1.]

sec. Second /il. Microlitre [10 " 1 . ] 6

rpm Revolutions per minute [ m i n . ] - 1

Abbreviations for Chemical and Biochemical Compounds

AA A m i n o acid arylamidase (micro­ AK A c e t a t e kinase

ATCase Aspartate transcarbamylase E-4-P D-Erythrose-4-phosphate

AA A m i n o acid arylamidase (micro AK A c e t a t e kinase

14. Carboxypeptidase weight about 300000). Commercial prepara

Carboxypeptidase: Carbobenzoxyglycylphenylalanine is used as substrate to assay the activity; the pro

A s the rate o f t h e e n z y m a t i c h y d r o l y s i s varies f r o m s u b s t r a t e t o s u b s t r a t e , t h e o p t i m u m c o n