.

\NALYTICAL BIOCHEMISTRY 12, 219-229 (1965)

On the Calorimetric Biuret Method of

Protein Determination1

R. PARVIN, S. V. PANDE, AND T. A. VENKITASUBRAMANIAN

From the Vallabhbhai Pate1 Chest Institute, University of Delhi, Delhi, India

Received December 7, 1964

The calorimetric biuret procedure has been widely employed for

estimation of protein from a variety of biological materials but studies
concerning the limitations and validity of this method have been few.
Pande et al. (1) noted that, for extracts of Aspergillus niger, the biuret
method of Gornall et al. (2) was not as satisfactory as the phenol method
of Lowry et al. (3). Khanna et al. (4) reported that the use of the biuret
procedure gave misleading high values for proteins in different biological
extracts. The present study confirms and amplifies the above observa-
tions. In addition, experiments pertaining to the elucidat,ion of the cause
of interference in the biuret method, its removal, and the applicability
of the phenol method are also described. While this manuscript was in
preparation a study of similar nature appeared (5).
MATERIALS AND METHODS

Chemicals. Sufficiently pure chemicals of either British Drug House,

England, or E. Merck, Germany, were used. Bovine serum albumin
(BSA) (fraction V) was a product of Sigma Chemical Co.
Preparation of Cell-Free Extracts for Study: A. niger and Mycobac-
terium 607 were grown and harvested as described by Pande et al. (1)
and Parvin et aZ. (6), respectively. These organisms were stored at -15
before use. The extracts were prepared by hand grinding with sand in
glass mortar and pestle. After 10 min grinding with water to obtain about
40% (wet w/v) disrupted cell suspension, the first extract was separated
by centrifugation at 10,000 X g for 10 min. The residue obtained was
reground similarly but for 15 min, and the second extract was obtained by
centrifugation. Further grinding of the residue for 20 min and centrifuga-
tion gave the third extract. Homogenates of animal tissues (10% wet,
w/v) were prepared in either 0.25 M sucrose (rat) or water (guinea-pig)

Taken from a thesis (of R. Parvin) submitted in partial fulfillment of the

requirements for the Ph.D. degree of Delhi University.
219
220 PARVIN, PANDE, AND VENKITASUBRAMAIGIAN

by using a Potter-Elvehjem homogenizer followed by centrifugation at

600 X 9 for 20 min. Whenever cell-free preparations contained a floating
fatty layer at the surface, it was removed by filtration through muslin.
All homogenate preparations and centrifugations etc. were carried out
between 2 and 5.
Trichloroacetic Acid Precipitation: TCA was added to homogenate to
5% final concentration and heated for 15 min at 100. The residue ob-
tained after cooling and brief centrifugation was washed once with 5%
TCA at room temperature and dissolved in alkali either as such, or after
delipidation as described below.
Delipidation: The TCA precipitate was suspended well in ca. 20 vol of
chloroform:methanol 2: 1 (v/v) for lipid extraction (7)) warmed (below
boiling) for 5 min, and allowed to stand overnight at 4. The residue was
separated by prolonged centrifugation (at 3,000 rpm for 1 to 2 hr) and
removal of solvent. It was re-extracted for lipid as above but only 1 hr
contact with solvent at room temperature was allowed. The residue from
this step was freed of adhering solvent by drying in vacua.
For dissolution, a paste of precipitate (TCA precipitate or delipidated
residue) was made with a few drops of 0.2 N NaOH by rubbing with a
glass rod and more alkali was added gradually with constant mixing. If
the solutions were not clear, the contents were heated for 5 min at 100.
In some cases, especially after delipidation, for complete dissolution, the
alkali concentration was increased up to 1 N and the heating time pro-
longed up to 20 min. By dilution, the NaOH concentration was lowered
to 0.1 N before performing the biuret calorimetry. For the phenol colors,
these solutions were further diluted 50-fold. The small amount of alkali
present in the protein solutions did not affect the calorimetry.
This hot alkaline treatment considerably affected the subsequent color
in both the biuret and the phenol methods, as tested with BSA. While
5 min prior heating with 0.2 N NaOH at 100 had no detectable effect,
10 min heating in 1 N NaOH lowered the biuret color by about 10% and
the phenol color by about 20%. A simultaneously run standard took care
of such effects to allow the necessary correction to be applied.
Biuret Method
Biuret Reagent. Four times as concentrated reagent as described by
Gornall et al. (3) was prepared. Powdered 1.50 gm CuSO,*4H,O and
6.0 gm sodium potassium tartrate (NaKC4H,0s .4H,O) were dissolved in
ca. 100 ml water. To this was added with constant mixing 100 ml of
carbonate-free 30% NaOH. The final volume was brought to 250 ml.
It was stored in an airtight polyethylene bottle and remained stable for
months.
 COLORIMETRIC PROTEIK DETERMINATION 221

Alkaline Tartrate Reagent. This differed from the hiuret reagent

described above in that no cupric sulfate was present.

Phenol Method
The procedure of Lowry et al. (2) was used. Here a marked deviation
from a linear relationship between optical density (OD) and protein
amount was seen; hence only those aliquots of unknown and standard
which gave an OD of 0.3 I+ 0.05 were used for the calculation of E:z I\/).
The protein colors in this method could vary with the lot of phenol
reagent.
In both the biuret (540 rnp) and the phenol (750 mp) methods, the OD
(Bausch & Lomb calorimeter) was recorded between 45 to 90 min of
color development.
Kjeldahl Method
The procedure of McKenzie and Wallace (8) was used.
Standard Protein
A freshly prepared aqueous BSA solution was used.
RESULTS
ilnalysis of Rat Tissues, Guinea-Pig Liver, and Bovine Serum Albumin.
The data obtained show (Table 1) that direct biuret calorimetry by the
Gornall (3) procedure with extracts and TCA-separated protein prepa-
rations gave much higher extinction values than the BSA. The possibility
of the contribution of opalescence to the biuret color measurements was
considered by treating homogenates with the alkaline tartrate reagent.
Tested in this way, all homogenates as well as TCA precipitates showed
marked absorption. In the AOD biuret method (Table 1)) which makes
allowance for such an effect, the color ext.inction values of the homoge-
nates and the TCA precipitates fell close to that of the BSA, compared
on the basis of protein nitrogen. After delipidation, the Gornall color
values became comparable to the AOD values for all tissues excepting
brain. For brain, the ultraviolet protein determination method is also
unsuitable (9).
When much of the work of this study was completed we came across
the report of Kayser and Vaughn (lo), in which recognition of inter-
ference due to turbidity in the biuret procedure was realized for serum
proteins. The application of the KCN method (Table 1) of these authors
to guinea-pig liver showed that the values by this method were similar
to the AOD biuret method, being comparable to the BSA.
The phenol color values, considered on the basis of protein nitrogen,
222 PARVIN, PANDE, AND VENKITASUBRAMANIAN

TABLE 1
RELATION BETWEEN BIUFLET COLORS, PHENOL COLORS, AND KJELDAHL NITROGEN x
6.25 VALUES IN PREPARATIONS OF RAT TISSUES, GUINEA-PM LIVER, AND BOVINE
SERUM ALBUMIN
E~~~Wy) refers to the extinction of final color solutions. The weight of protein is the
Kjeldahl nitrogen X 6.25. Protein nitrogen refers to TCA-separated nitrogen without
correction for remaining nucleic acid nitrogen, if any. In the biuret procedure, Gornall
values represent results obtained on treating protein (solution brought to 4.0 ml) with
biuret reagent (1.0 ml) and recording absorbancy (3). In the AOD method, the protein
solution (4.0 ml) was treated with alkaline tartrate reagent (1.0 ml) and absorbancy
recorded as in the Gornall method. The difference between Gornall color value and
alkaline tartrate value gave the AOD value. In the KCN method, after recording the
Gornall colors a pinch of KCN (ca. 100-120 mg) was added and contents mixed; 5 min
after discharge of color, absorbancy was recorded again. The difference in absorbancy
before and after KCN treatment gave the KCN value (10).
Original homogenate values based on
After TCA After
Source and method Total N Protein N precipitation delipidation

Bovine serum albumin:

Biuret
Gornall 2.94= 2.96 2.96 2.94
AOD 2.94
Phenol 222 222 220 226
%N 100 99.4 99.4 97.8
Percentage difference of E:~mWv) value from corresponding value
obtained with bovine ccrum albumin

Rat liver:
Biuret
Gornall $33 +52 1-46 -1
AOD -17 -6 -8 -2
Phenol -32 -2 -1 -1
%oN 100 89.1 89.1 83.4
Rat liver particulate fracttin:b
Biuret
Gornall += +35 +37 -6
AOD -12 -7 +7 -10
Phenol -12 -6 -5 -4
%oN 100 94 94 x7.s
Rat heart:
Biuret
Gornall +19 +40 +47 +2
AOD -16 -2 -10 +1
Phenol -23 -5 -6 -4
%N 100 85.8 85.8 78.4
 COLORIMETRIC PROTEIN DETERMINATION 223

TABLE 1 (Continued)

Original homogenate values based on

After TCA After
Source and method Total N Protein N precipitation delipidation

Percentage difference of J?:~wv value from correspondinK value

obtained with bovine serum albumin

Rat brain:
Biuret
Gornall +55 +78 c +41
AOD -26 -15 c -5
Phenol -19 -6 -s - 5
% pr 100 8i.l s7.1 $73
Guinea-pig liver:
Binret
Gornall i-85 +::
AOD -5 -5
ICCN +2 -9
Phenol +5 +3

a Average of four separate analysis.

b Homogenates were centrifuged at 10,000 X g for 30 min. The separated mat,erial was
resuspended by homogenizing with 0.25 M sucrose for use.
c Since the solutions were turbid, calorimetry coldd not be performed.

were about the same after TCA precipitation and delipidation as with
the original homogenates.
Analysis of Mycobacterium 607 and Aspergillus niger Preparations:
Analysis of the successive extracts of Mycobacterium 607 (Table 2) and
-4. niger (Table 3) showed that biuret calorimetry gave high extinction
values as compared to the BSA values. This was either unaffected
(Table 3) or slightly minimized (Table 2) by TCA precipitation. The
AOD method in all these cases gave better results. After delipidation, the
Gornall method gave satisfactory results with A. niger only; with M. 607
the AOD method still gave superior values. The values obtained by the
KCN method were comparable (Table 2) to the AOD method.
The pheno1 colors of original extracts in these cases were nearly com-
parable to those obtained after TCA precipitation. Delipidation did not
affect the phenol color extinction values. The Kjeldahl nitrogen of these
microbial extracts contained a considerable proportion of nitrogen that
did not contribute to calorimetric estimations, since nearly complete
rc>tcntion of the AOD biuret and the phenol colors was seen in the TCA
precipitates. In the ammonium sulfate separated material also (Table 2)
part of the nitrogen was removed by the TCA precipitation (perhaps due
to nucleic acid removal (see ref. 6)) without affecting the phenol or AOD
hiuret. values. Considered on the prot,ein nikogen basis, the Gornall
224 PARVIN, PANDE, AND VENKITASUBRAMANIAK

TABLE 2
RELATION BETWEEN BIURET COLORS, PHENOL COLORS, .~ND KJELDAHL NITROGEN X
6.25 VALUES IN PREPARATIONS Mycobacterium
OF 607
(Details as for Table 1)

Percentage difference ofY(y value from corresponding

value obtained with bovine serum albumin
Original h;m&& values
Preparation
and After TCA After
method Total N Protein N precipitation delipidation

First extract:
Biuret
Gomall -44 +33 +1s +10
AOD -51 -9 -7 -6
KCN -54 -11 -12 -7
Phenol -48 -1 +1 0
%oN 100 52.9 52.9 43.8
Second extract:
Biuret
Gornall +7 +60 +17 +10
AOD -37 -5 -10 -2
KCN -35 +7 -9 -2
Phenol -34 -1 +5 +3
%oN 100 66.S 66.8 53.2
Third eztract:
Biuret
Gornall +lQ +54 +15 +12
AOD -29 -8 -5 +4
KCN -27 -5 -4 +4
Phenol -27 -5 +4 +1
%N 100 77.1 77.1 62.1
Ammonium sulfate separated
materid:a
Biuret
Gornall 0 +lQ +Q +5
AOD -17 -3 -2 -1
KCN -16 -2 -3 -2
Phenol -15 0 -3 -1
%N 100 55.4 85.4 84.6

a A separately prepared aqueous extract was brought to full saturation with am-
monium sulfate and the separated material obtained after centrifugation (10 min at
10,000 X g) was dissolved in water. It was dialyzed 24 hr again& several changes of
large excess of water.

method here also gave high values as compared to the other methods.
It must be mentioned that the microbial extracts that were obtained by
centrifugation at 1000 X g for 10 min were too opalescent, for which
only the phenol method gave tolerably satisfactory results. With A. niger
 (OLORIMETRIC PROTEIN DETERMINATION 225

TABLE 3
RELATION BETWEEN BIURET COLORS, PHENOL COLORS, AND KJELDAHL NITROGEN X
6.25 VALUES IN PREPARATION OF A. niger
(Details as for Table 1)

Percentage difference of E::: value from corresponding valuej

obtained with bovine serum albumin
Original homogenate values based on
After TCA After
Preparation and method Total N Protein N - precipitation delipidstion

First extract:
Biuret
Gornall 1-79 +276 $243 +3
AOD -51 f3 +3 +1
Phenol -51 f4 -1 -4
%N 100 47.5 47.5 47.3
Second extract:
Biuret
Gornall $99 1-318 +308 f7
AOD -54 -4 -3 f5
Phenol -55 -3 -3 +2
%N 100 47.4 47.4 47
Third extract:
Biuret
Gornall $130 f376 +336 +5
AOD -46 +12 $12 +5
Phenol -48 +9 -1 +1
%N 100 48.2 48.2 48.2

it has been found (11) that the ultraviolet protein determination method
is also unsatisfactory as compared to the phenol method.
Analysis of Spinach Preparation: The Gornall biuret method gave high
extinction values in the homogenate as well as with the TCA precipitates
(Table 4). Delipidation and acetone treatment, which also removed the
pigments, lowered the Gornall color values though the AOD procedure
even in these cases gave better results. With acetone-washed TCA pre-
cipitates of Cuctm also, the direct biurct calorimetry gave high values
(12). In acetone treatment, complete protein precipitation (as judged
by both the phenol and the AOD biuret colors which were comparable)
was obtained (1) by adding a minimum of 10 vol of acetone directly to
homogenate or (2) by washing the TCA precipitates with excess acetone,
and or (3) by adding TCA in acetom to provide 5 vol of acetone with
575 TCA. With wheat germ, comparable protein valued were obtained by
the biuret and the phenol methods after separating proteins with 30 vol of
acetone (13). Both the phenol and the AOD biuret color ext,inctions were
higher in spinach homogenates than those obtained after TCA precipita-
226 PARVIN, PANDE, AND VENKITASUBRAMANIAN

TABLE 4
RELATION BETWEEN BIURET COLORS, PHENOL COLORS, AND KJELDAHL NITROGEN x
6.25 VALUES IN SPINACH LEAF PREPARATIONS
(Details of acetone treatment are described in text; other details as for Table 1)

Percentage difference of E;~:~~) value from corresponding value

obtained with bovine serum albumin
Original h;;;~,nnte* values
After TCA After After acetone
Method Total N Protein N precipitation delipidation treatment

Biuret
Gornall +124 +186 + 150 +18 +15
AOU +7 +37 -12 -4 - 3
Phenol -11 $42 +2 fl -1
%N 100 78.1 78.1 72.4 75.6

QLeaf homogenate (30% wet w/v) was obtained using a Waring Blendor and clarifying
the resulting suspension by cenkifugation at 10,000 X g for 10 min.

tion. A need for the TCA precipitation of proteins prior to calorimetry

was felt with Euglena also (14).
DISCUSSION
The inapplicability of the direct biuret calorimetry has been realized
in many instances (12-26). Thus the Cleland and Slater (17) procedure
of washing TCA precipitates with ethanol before color development has
been adopted by several workers (21-24). Deoxycholate (15, 18, 25, 26)
and Triton (19) treatments have also been made to improve the
calorimetry. However, use of the Gornall biuret method is being con-
tinued (9, 27-34), perhaps because a systematic study urging caution in
the use of direct biuret calorimetry has not appeared. Since this procedure
gives wrong results, as emphasized in the present study, it is a potential
source of error in comparison and evaluation of results of studies in dif-
ferent laboratories. As the AOD or the KCN procedure improved the
results, the cause of interference is the persistence of opalescence which
contributes to the OD measurement (cf. ref. 5). Although carbohydrates
have been considered responsible (1, 4)) TCA treatment which completely
extracts glycogen (35) did not minimize interference. Removal or lower-
ing of interference by delipidation shows that lipids are mainly responsi-
ble for this trouble. In separate experiments with the mitochondria-free
fraction of rat liver preparations, it was also found that interference in
the biuret procedure increased when the liver lipid content increased
(cf. ref. 20).
Layne (36) has recommended ether extraction of the biuret colors to
 COLORIMETRIC PROTEIN DETERMINATIOX 227

remove interfering turbidity. We find this practice unsuitable. In many

cases this minimized t.he extent of error only slightly; further, the repro-
ducibility was poor. Also, we did not find improvement simply by the
inclusion of deoxycholate (5-10 mg) 10 min before color development. In
addition, in certain cases, depending upon the source, the AOD or the
KCN method gave better results than did the procedure of Cleland and
Slater (18) because even after delipidation the opalescence persisted.
The present study reveals that with different sources, a variable degree
of protein nitrogen is lost on delipidation of TCA precipitates. This
emphasizes the conclusion of Munro and Downie (37) that the protein
content of delipidated residue may be significantly lower than the actual
protein present in the original extracts.
Both in the phenol and in the AOD biuret methods, the protein colors
of rat tissues and Mycobacterium 607 extracts were found to be com-
pletely retained in the TCA precipitates. This justifies the assumption
that TCA-separated nitrogen is a good measure of total protein and
emphasizes that, in t,he Kjeldahl method, use of the total nitrogen as an
index of protein can lead to serious errors. For A. niger and more so for
spinach preparations, however, the color values were higher with the
initial extracts than with the TCA precipitates. Since these color reactions
are not entirely specific to prot.eins, the possibility exist,s that, apart from
the presence of peptides and prot,eins, nonprecipitable by TCA, other
substances (1, 2, 38) could also contribute to color. The discrepancy due
to this effect will be minimized by the TCA precipitation of proteins. This
will also avoid interference due to (1) certain cations, such as K+ (391
Mn++ (6)) NH,+ (2,36), and (2) other substances, such as tris buffer (40))
reducing substances? (cf. ref. 41), etc. (42).
When homogenates develop turbidity on standing, the KCN method
appears unsatisfactory (10). This is avoided by the TCA precipitation
and subsequent mild alkaline treatment which gives homogeneous protein
solutions. At times this also reduced the contribution of the opalescence
to the OD measurement and, therefore, made the AOD method more
reliable (cf. ref. 5). This opalescence contribution could further be mini-
mized by the inclusion of ethanol--40cjo (v/v)-in the color solutions
without affecting the calorimetry.
With biological extract,s, unlike with pure proteins, it is desirable to
record the biuret colors without unnecessary delay since turbidities appear
on standing (cf. ref. 1). In the AOD biuret method, this development of
turbidity is less marked in the case of alkaline tartrate controls and hence

For this reason in the growth medium of A. Gger the hiuret method could not
he used though, surprisingly, the phenol method served well.
228 PARVIN, PANDE, AND VENKITASUBRAMANIAh-

this becomes all the more necessary. This is also unavoidable when alco-
hol, acetone, or deoxycholate is present. In such cases, development of
yellowish color on standing, probably due to Cu++ reduction, invalidates
the calorimetry.
Finally, whereas the chromogenicity of individual proteins differs in
the biuret procedure (3) and more so in the phenol method (2, 43), for
mixtures of proteins in biological extracts the chromogenicity was not
found to be markedly different, as this was similar to that of the BSA.
In such cases, therefore, the use of BSA as reference standard for colori-
metric protein determination methods appears adequate.

SUMMARY
A study of the applicability of a calorimetric biuret procedure for
protein determination to a variety of biological preparation has revealed
that the practice of using this procedure is undesirable because markedly
apparent high values are obtained in this way. In some cases, this inter-
ference was mainly due to lipids. Use of suitable controls to account for
the interfering opalescence in biuret color measurements considerably
improved the method. Conditions affecting the reliability of the procedure
are also described. For crude extracts the calorimetric phenol method
was found to be more reliable. However, a prior separation of protein by
TCA is often advantageous not alone for Kjeldahl analysis but also for
calorimetric estimations. The actual chromogenicity of the protein mix-
tures of different biological extracts studied was found to be comparable
to that of the commonly employed standard protein, bovine serum
albumin.

ACKNOWLEDGMENTS
This work was supported in part by funds from the Indian Council of Medical
Research, New Delhi, and Grant No. E-3427, National Institute of Allergy and
Infectious Diseases, U. S. P. H. S. We are grateful to Dr. R. Viswanathan, Director
of this 1nstitut.e for his interest in this work. The technical assistance of Mr. R. K.
Bhatnagar is gratefully acknowledged.

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