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ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 8, No.

2
Copyright 1978, Institute for Clinical Science

The Measurement of Total Serum Proteins


by the Biuret Method
M ICHAEL M. LUBRAN, M.D., Ph.D.
Harbor General Hospital Campus,
University o f California at Los Angeles,
School o f Medicine, Department o f Pathology,
Torrance, CA 90509

ABSTRACT
The biuret reaction for proteins provides a sim ple and precise m ethod for
m easuring serum proteins; Beers law is obeyed to at least 10 g per dl.
Several stable biuret reagents are available. H em oglobin is the only im por
tant cause of interference w hich cannot be m inim ized by use of a sample
blank. The m echanism of the b iu ret reaction is described and attention is
drawn to the heterogeneity of the serum proteins and to the use of a cer
tified album in standard.

Introduction

History of the B iuret Reaction

The addition of a cupric salt to an al


kaline solution of a protein produces a
r e d d is h -v io le t co lo r. T h is re a c tio n ,
known as the biuret reaction, is w idely
used for the determ ination of the concen
tration of proteins in serum , m ainly be
cause o f its convenience and good preci
sion. M odern tec h n iq u es use a single
b iuret reagent, w hich is stable and easy
to prepare. Interferences are few and eas
ily elim inated (except for hemoglobin).
However, there are some problem s as
sociated w ith the m ethod. T he b iu re t
rea c tio n occurs w ith the p o ly p e p tid e
chain, which accounts for only a part of
most protein molecules. Absorptivity will
not necessarily be the same per gram of
protein for the different serum proteins
(table I). There is the additional problem of
the selection of a standard for the method.
These problem s will be discussed.

W hat subsequently becam e known as


the biuret reaction was first described in
1833 by Rose,17 who was studying the
reactions of solutions of m etallic salts
w ith egg album en. H e noted that addi
tion of a solution of potassium hydroxide
or sodium carbonate to the precipitate
produced by adding a copper sulphate so
lution to egg album en resulted in a violet
color. He observed a sim ilar effect w hen
bovine blood serum was treated in the
same way. B iuret (N H 2C O N H C O N H 2)
w as first o b ta in e d in 1848 by
W ie d e m a n n 21 from th e p ro d u c ts of
pyrolysis of urea or urea nitrate. He d e
scribed the red color produced w hen a
solution of biuret was treated w ith a cu
pric solution and potassium hydroxide so
lution (the biuret reaction).
Ritthausen and Pott16 first applied the
biuret reaction to the study of proteins

106

107

M E A SU R E M E N T O F T O T A L SER U M P R O T E IN S BY B IU R E T M E T H O D

and H ofm eister8 was the first to describe


its use in the quantitation o f proteins and
peptones; but it was not until 1914 that
the b iu ret reaction was applied by Riegler15 to a biological fluid. He used it to
determ ine album in (i.e., total protein) in
urine by precipitating the protein w ith
/3-naphtholsulphonic acid, dissolving the
precipitate in sodium hydroxide solution
and adding copper sulfate solution. The
color of the filtrate was m easured. Egg
album en was used as a standard.
A u ten rieth and M ink2 im proved the
m eth o d an d u sed serum d isso lv ed in
urine as a standard. In 1917, A utenrieth1
extended the m ethod to serum and ascitic
fluid. H ow ever, the biuret reaction was
not w idely used as a quantitative proce
dure for serum proteins until 1942, w hen
Kingsley introduced a single biuret rea
gent, incorporating the copper salt and
the alkali. P recip itatio n o f cupric hy
droxide was prevented by the use of a high
concentration o f sodium hydroxide, but
the reagent was not very stable. M ehl11
u se d e th y le n e glycol to sta b iliz e th e
biuret reagent, but this reagent was dif
ficult to reproduce and underw ent au
toreduction. A stable b iuret reagent was
described by W eichselbaum 20 who added
sodium potassium tartrate (Rochelle salt)
as stabilizer and potassium iodide to pre
vent autoreduction of the alkaline copper
tartrate and separation of cuprous oxide.
G om all e t al6 carried out an extensive
and thorough study of the b iuret reagent.
They recom m ended a reagent containing
no iodide. Stability of this reagent d e
p ended on careful adherence to the p u b
lished details of the m ethod of prepara
tio n . A d d itio n o f one g o f p o tassiu m
iodide per liter of reagent was perm itted.
The latter version of the Gom all reagent
as w ell as the W eichselbaum reagent are
currently the most w idely used b iuret
reagents.
In addition to providing a stable rea
gent, various modifications of the b iuret
reagent w ere made to extend the range of
protein concentrations over which Beers

TABLE I
Biuret Values of Some Serum Proteins18

C o n c e n tr a ti o n i n
Serum (m g /d l)

Albumin
3,500 - 4,500
ai-Antitrypsin
210 500
y-Globulins
900 - 1,500
Haptoglobin
30 190
Hemopexin
80 100
a2-Macroglobulin
220 380
"Orosomucoid"
75 100
Prealbumin
28 35
Transferrin
200 320

B i u r e t V a lu e
(A lb u m in - 1 0 0 )

100
86
110
81
80
92
62
96
95

The relative biuret values depend upon the purity


of the proteins tested and the composition of the
biuret reagent. The values depend on the
proportion of the protein molecule made up of
polypeptides.

law was obeyed, and to perm it use o f the


reagent in the presence of salts, such as
sodium sulphate and sodium su lp h ite,
used in protein fractionation. R einhold14
described a reagent, essentially the same
as the W eichselbaum reagent, suitable
for use w ith these salts. Now that alb u
m in is m easured directly by dye-binding
and the b iuret reaction is reserved for the
m easurem ent of total protein in serum,
c o m p a tib ility o f th e re a g e n t w ith
protein-precipitating salts is no longer a
requirem ent. Doumas et al4 re-exam ined
the properties of various b iuret reagents
and finally adopted a m odification of the
Reinhold reagent.T heir reagent is stable,
is easy to prepare, has a w ide range of
linearity and is suitable for serum protein
determ inations.
In addition to the reagents described,
many other modifications, too num erous
to detail here, have been suggested but
have not proved popular.10 T he Doum as
reagent, the W eichselbaum reagent and
the G om all reagent (with iodide) are all
equally suitable for the determ ination of
total protein in serum. T heir suitability
for other biological fluids, such as urine
and cerebrospinal fluid, is not so certain;
nor are they necessarily suitable for pro
tein s o th er than serum p ro te in s, e.g.,
vegetable proteins and tissue proteins.13

108

LU BRAN

Chem istry of the B iuret Reaction


An extensive review of the early work
on the chem istry of the b iuret reaction is
given by Kurzer, and the crystal struc
tures of copper-peptide com plexes are
described by Freem an.5 T he nature of the
copper-protein complexes in solution has
been inferred from the crystal structures
and from a stu d y of th e ir ab so rp tio n
spectra. The characteristic reddish-violet
color of the b iu ret reaction for peptides
and proteins is due to the coordinate link
age of one copper atom to four nitrogen
atom s, w hich are d e p ro to n ated in the
strongly alkaline solution. The coordi
nated atoms are almost square-planar.
Three aminoacids at least are required
in the peptide for the characteristic color
to be produced. The copper atom binds to
the peptide N atoms w hich form the p ep
tide bond b etw een am inoacids, but it can
com bine w ith the N of the term inal N H 2
of the peptide chain, w ith the N of amide
groups and w ith the N of im idazole rings.
The copper atom may also link w ith the O
atom of a water m olecule in solution. The
intensity of the color produced in the
biuret reaction increases w ith increasing
alkalinity, because more deprotonated N
atoms are m ade available. At acid pH , the
copper atom links to O atoms derived
from the carboxyl groups. This com bina
tion does not result in a reddish-violet
color.
Absorptivity of the B iuret-Protein
Complex
The general shape of the absorption
spectrum of the biuret-protein complex is
the same for all serum proteins, showing
strong absorption in the ultraviolet and
yellow -green, but the exact w avelengths
of the absorption m axima and m inim a
and the specific absorptivities d epend
upon the composition of the b iuret rea
gent and the nature of the protein and its
concentration. As the ratio of copper to
protein increases from 1:70 to 1:10, the
absorption maximum moves from 540 to

550 nm to 560 to 570 nm and, as the con


centration of alkali in the b iuret reagent
increases, th e absorptivity for a given
amount of protein increases. Further, the
absorptivity of the b iu ret-protein com
plex w ill d epend upon the polypeptide
content of the protein (table I). Serum al
bum in an d gam m a-globulin have fairly
close biuret values per gram of protein,
but the values differ m arkedly for some of
the other serum proteins.
The absorptive maximum is at 540 nm
for serum proteins using the b iuret rea
gents listed in table II. T here is also ab
sorption in the ultraviolet. Using 5 ml of
the Doumas reagent added to 100 fil of
protein solution, the absorptivity of the
protein-biuret complex of a 1 g per 1 solu
tion of hum an album in is 0.302 (corrected
for sam ple b lank); th e co rresp o n d in g
value for bovine serum album in is 0.292.
The rate of developm ent of the color
after m ixing b iu re t reagent and protein
solution is initially rapid; after 5 m inutes
the absorbance has alm ost reached its
maximum value. After 30 m inutes, the
rate of increase in absorbance is barely
m easurable. T here are slight differences
in the rate o f color developm ent betw een
bovine and hum an serum album in. Be
tw een 5 and 10 m inutes after the reaction
has started, bovine and hum an serum al
bum in of equal concentrations give the
same absorbance.
The absorbances quoted are those ob
tained at 24 to 26. Increasing tem pera
ture slightly accelerates the rate of color
developm ent, but not the m agnitude of
the absorbance at 30 m inutes. In practice,
10 m in u te s is a c o n v e n ie n t tim e for
m easuring the absorbance and, provided
samples, blanks and standards are all pre
pared at the same tem perature and read
at approximately the same time, the reac
tion can be carried out at room temperature.
Standards for Use w ith the B iuret
Reaction
On general principles, the best stand
ard for an analytical m ethod is a prim ary

109

M E A SU R E M E N T O F T O T A L SER U M P R O T E IN S BY B IU R E T M E T H O D

standard, i.e., a pure sample of the sub


stance to be m easured. However, serum
contains a mixture of proteins of some
w hat different b iuret values. Many pro
cedures em ploy pooled norm al hum an
serum as a standard, but this m aterial has
to be assayed, i.e., it is not a prim ary
standard. T he apparent concentration of
the protein in the pooled serum standard
depends upon the assay m ethod used. It
appears, therefore, that pure serum al
bum in, which can be w eighed to give a
solution of known strength independent
of any assay procedure, w ould provide
the best standard. Although the use of al
bum in as a standard leads to a small u n
derestim ate of the protein concentration,
in terms of mass concentration, so do all
the other m ethods based on measuring,
in some way, the polypeptide chain.
Bovine serum albumin is more conven
ient to use than hum an serum album in;
the latter, in addition to costing much
m ore, is u su a lly c o n ta m in a te d w ith
bilirubin. Album in solutions polymerize
on standing through oxidation of the sulphydryl groups in the protein. The Na
tional Bureau of Standards has produced
a standard album in12 low in dimer. This
certified album in is recom m ended as the
most suitable standard for the biuret pro
cedure, but may not be suitable for other
methods.
Interferences w ith the B iuret Beaction
Few substances in blood serum inter
fere w ith the biuret reaction. M arked tur
bidity owing to lipem ia may cause er
roneously high results, w hile the use of a
serum blank may overcorrect leading to
low results. Errors owing to lipem ia can
be red u c e d (1) by adding urea to the
biuret reagent20 to a final concentration of
6 m ol p er 1, (2) by ad d in g potassium
cyanide solution to decolorize the biuret
reagent3 or (3) by extraction of the lipid
from the serum using ether.
H em olysis causes significant errors,
each milligram of hem oglobin increasing
the apparent protein concentration by

TABLE I I
Composition of Some Biuret Reagents (g/L)
R eagent

CuSO4>5H20

NaOH

1.67
1.69

117
43

Kingsley9
Mehl11
Weichselbaum2 0
Gornall6
Reinhold*1*
Doumas4
*NaKC^H^Og*4H20

15
1.5
3
3

8
30
8
24

" T a r tr a te " *

Kl

O th e r

80 ml
ethylene
glycol
45

6
9
9

5
(1) +
5
5

tlnclusion of K1 is optional.

about 2 mg. This source of error cannot


be rem oved by a sam ple blank. Bilirubin
up to 20 mg per dl does not interfere.
Glucose does not interfere up to concen
trations of 200 mg per dl, b ut at higher
values may cause a slight error ow ing to
reduction of the reagent.4
Dextran in blood serum reacts with the
b iu r e t re a g e n t to give a p r e c ip ita te ,
which may be rem oved by centrifugation.
T he b iuret color may be m easured in the
supernatant to give the true protein con
centration. Some drugs which give a red
color in alkaline solution (e.g., phenolphthalein in some laxatives) may increase
the apparent protein concentration. T heir
effect is usually small and may be re
m oved by m eans of a sample blank.
Stability of the Biuret Reagent
The reagent is stable at room tem pera
ture for at least a year, if kept in a tightly
sealed, dark, heavy plastic bottle. D e
terioration is shown by the presence of a
black, granular precipitate. Although at
m ospheric carbon dioxide does not affect
th e re a g e n t, it is a d v is a b le to u se
bicarbonate-free sodium hydroxide and
freshly deionized w ater in its preparation.
The biuret reagent has a high salt content
and should, therefore, not be kept in the
refrigerator.
R eproducibility of the Biuret M ethod
Reproducibility depends on the accu
racy of the pipets used in the m ethod and
on the spectrophotom eter, provided that

110

LU BRA N

the m easurem ents are m ade betw een 5


and 10 m inutes after adding 0.1 ml of
serum to 5 ml of biuret reagent. A coeffi
cient of variation of less than 1 percent is
readily obtained over the protein concen
tration range of 2 to 10 g per dl.

4.

5.

Sensitivity of the B iuret M ethod


The m ethod has adequate sensitivity for
protein concentrations found in normal
and pathological sera. Below about 1 g per
dl, the precision worsens, as the absorb
ance of the reagent forms a large part of
the total absorbance. Addition of a larger
volume of a dilute protein solution (e.g.,
cerebrospinal fluid) changes the concen
trations of the constituents of the biuret
reagent. The reagent should, therefore, be
reform ulated and optim um composition
determ ined, according to the range of pro
tein concentrations to be m easured. A
reagent designed for blood serum protein
concentrations may not necessarily be
suitable for other fluids.

6.

7.

8.
9.

10.
11.
12.
13.

Conclusions
The biuret m ethod has stood the test of
tim e as a convenient and precise m ethod
for m easuring total serum proteins. Con
c e n tra tio n o f p ro te in s s h o u ld b e ex
pressed in term s of album in, either hum an
or bovine. The sensitivity of the m ethod is
inadequate for fluids of low protein con
centration and it may not be suitable (un
less modified) for tissue proteins. T he cor
rectly prepared reagent has a long shelf
life at room tem perature.
Beferences
W.: Colorimetric estimation of
serum albumin and globulin in urine, ascitic
fluid and blood serum . M unch, med.
Wochenschr. 64:241-245, 1917.
2. A u t e n r i e t h , W. and M in k , F.: Colorimetric
determinations: the quantitative determination
of albumin in urine. Munch, med. Wochenschr.
62:1417-1420, 1915.
3. B o d e , C., G o e b e l l , H., and S t a h l e r , E.: The
'
elimination of errors caused by turbidity in the
1.

14.
15.
16.
17.
18.

19.

A u te n rie th ,

20.

21.

determination of protein by the biuret method.


Z. Klin. Chem. u. Klin. Biochem. 6:418-422,
1968.
D o u m a s , B. T. and the Study Group on Proteins
of th e A m erican A ssociation of C linical
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assaysa collaborative study. Clin. Chem.
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F r e e m a n , H. C.: Crystal structures of metalpeptide complexes. Adv. Prot. Chem. 22:258424, 1967.
G o r n a l l , A. G ., B a r d a w i l l , C. J., and D a v i d ,
M. M.: Determination of serum proteins by
means of the biuret reaction. J. Biol. Chem.
177:751-766, 1949.
H o f f m a n n , J. P. and R i c h t e r i c h , R.: The
elimination of turbidity in the determination of
plasma proteins with the biuret reagent. Z. Klin.
Chem. u. Klin. Biochem. 6:595-598, 1970.
H o f m e i s t e r , F .: A method for the complete
removal of protein from animal fluids. Zeitschr.
f. Physiolog. Chem. 2:288-293, 1879.
K i n g s l e y , F . G. R.: The direct biuret method
for the determ ination of serum proteins as
applied to photoelectric and visual colorimetry.
J. Lab. Clin. Med. 27:840-845, 1942.
K u r z e r , F .: Biuret and related compounds.
Chem. Rev. 56:95-197, 1956.
M e h l , J. W .: The biuret reaction of proteins in
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157:173-180, 1945.
N ational B ureau of Standards SRM 926
(lyophilized bovine serum albumin) and SRM
927 (7.045 g per 1 albumin solution).
P arvin , R., P a n d e , S. V., and Venkitasubra MANIAN, T. A.: On the colorimetric b iu ret
m ethod of p ro tein d eterm in atio n . Anal.
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R e i n h o l d , J. G.: Total protein, albumin and
globulin. Stand. Methods Clin. Chem. 1 :88-97,
1953.
RlEGLER, E.: A colorimetric method for the de
term in atio n o f album in. Z. Anal. Chem .
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RlTTHAUSEN, H. and POTT, R.i Investigations
of the compounds of proteins with copper
oxide. J. Prakt. Chem. 7:361-377, 1873.
R o s e , F .: On the compounds of egg albumen
with metal oxides. Ann. Phys. Chem. 28:132142, 1833.
S c h u l z e , H . E. and H e r e m a n s , J. F .: Molecu
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