ANALYTICAL BIOCHEMISTRY 9, 401-410 (1964)

A Micro-Biuret Method for Estimating Proteins

RUTH F. ITZHAKI I AND D. M. GILL2
From the Department of Radiotherapeutics, University of Cambridge, England

Received October 1, 1963

INTRODUCTION
Although there are many methods of estimating proteins, few are both
sensitive and nonspecific for type of protein (i.e., independent of
composition). In this laboratory we have used satisfactorily for several
years a method which is rapid, fairly sensitive, and reasonably non•
specific and which meets a further requirement of being unaffected by
the presence of high concentrations of deoxyribonucleic acid (DNA).
This method was adapted from a method of Zamenhof and Chargaff (1 )
and is based upon measurement of the ultraviolet absorption of the
complex formed between protein and copper in strongly alkaline copper
sulfate solutions.
MATERIALS
The following proteins were used for standardization of the method:
(1) Bovine plasma albumin (BPA), crystalline (Armour, England).
(2) Duck ovalbumin, lyophilized (kindly given by Dr. J. Williams).
(3) Calf thymus histone, lyophilized (kindly given by Dr. J. Hindley) .
(4) Casein, Hammarsten preparation (Merck, U.S.A.
(5) Trypsin, crystallized-lyophilized (Novo, Denmark
(6) Gelatin, granular (commercial preparation).
(7) Deoxyribonucleoprotein (DNP) prepared from rat thymus (2).
(8) Lysozyme, crystalline (BDH, England) .
All concentrations quoted below, of proteins and of other materials,
are values in the final reaction mixture of 3 ml, unless otherwise stated.
The approximate water content of the above proteins was found by
drying to constant weight at 105 0 C, and all protein concentrations given
are corrected accordingly. The amount of protein in the DNP preparation
was found by subtracting the DNA content from the dry weight;
1
Beit Memorial Research Fellow.
2
Holder of Medical Research Council Scholarship for training in research methods.
402 ITZHAKI AND GILL
401
the DNA was estimated from the ultraviolet absorption at 260 mg of a
dilution of the DNP preparation taking the extinction coefficient
cm) of DNA as 210.
Other materials used were:
(1)DNA from calf thymus (California Corporation for Biochemical
Research) .
(2)Urea, recrystallized from absolute alcohol.
(3)Copper sulfate (CuS04•5H20) AnalaR (all concentrations given
below refer to the hydrated salt) .
Dipeptides :
(4)Glycylglycine (Roche, England) .
(5)Glycyl-L-leucine (Roche, England) .
(6)L-Leucylglycine (Hoffmann-La Roche, Switzerland).
(7)Glycyl-L-proline (Mann, U.S.A.) .
(8)L-Prolylglycine (Mann, U.S.A.).
(9)L-Prolyl-L-proline (a gift from Dr. W. Rittel, Ciba Ltd.). All
other chemicals were of AnalaR grade.
EXPERIMENTAL
To find the best conditions for measurement, the dependence of
absorption of the copper-protein complex on copper and sodium
hydroxide concentrations and on wavelength was investigated.
Correction for the absorption due to the protein alone, and for any other
ultraviolet-absorbing material present, was made throughout by
measuring the optical density of the protein solution in alkali (against an
alkali blank) and subtracting this value from the optical density in the
presence of copper. All absorption measurements were made on a
Beckman DU spectrophotometer, unless stated otherwise.
Effect of Copper Concentration
The variation of optical density with concentration of copper sulfate
was found by adding 2-ml samples of BPA solution to each of a series of
I-ml samples of alkaline copper sulfate at different concentrations (final
protein concentration 0.117 mg/ml, 12% sodium hydroxide). The optical
density of each mixture was measured at a range of wavelengths against
the corresponding alkaline copper sulfate blank. Figure 1 shows the
results. It is clear that there is a rapid increase of optical density up to a
copper sulfate concentration of about 0.00770 3 followed by a very slow
increase. Therefore, it was obviously desirable to use a concentration of
 PROTEIN ESTIMATION

This corresponds to a ratio of approximately one copper atom per four peptide bonds
in agreement with the ratio found by Mehl (3) .
0.6 280 me

o 002 004 0.06 008 0.10 0.12

Final concentration of CuS04 •5H20(gm/OOml)

FIG. 1. Dependence of optical density of copper-protein complex on CuSOe5H20

concentration and on wavelength, for BPA (0.117 mg/ml) in 12% NaOH. Values are
corrected for absorption of protein and of copper sulfate as described in text.

copper sulfate above about 0.01%, but at the highest value investigated
(0.11%) the absorption of the copper blank was appreciable and so the
lower value of 0.07% was chosen for subsequent use.
Effect of Sodium Hydrocide Concentration
The absorption of the copper-protein complex was found to be
independent of sodium hydroxide concentration over the range 6 to
20% ; below about 6% a precipitate of copper hydroxide formed. A
concentration of 10% was subsequently used.
Effect of Wavelength
Figure 1 shows that the sensitivity increases with decreasing
wavelength down to at least 280 mu. At 270 mg, readings are even
higher but the absorption of the copper blank is so large at higher
copper concentrations that values become unreliable and are therefore
not shown. The choice of wavelength is limited, however, for protein
solutions containing DNA, by the very high absorption of the DNA
404 ITZHAKI AND GILL

relative to that of the copper-protein complex at wavelengths below

about 310 nut.
Figure 2 shows the absorption spectra in sodium hydroxide of
BPA (0.053 mg/ml), of BPA with added DNA (0.7 mg DNA/ml), and

260 270 280 290 300 310 320

Wavelength (mg )

FIG. 2. Ultraviolet absorption spectra of solutions used for estimation of protein, and
effect of added DNA. All solutions contain 10% NaOH and are read against distilled
water. Concentrations are final values in 3 ml. (O) Bovine plasma albumin (BPA), 0.117
mg/ml. (0) BPA in 0.07% CuSOe5H20. (O) BPA plus 0.7 mg/ml DNA. (u) BPA plus
DNA in 0.07% CuSOe5H20. (A) NaOH, 10%. (A) NaOH, 10%, plus 0.07%
CuSOe5H20.

of both in the presence of 0.07% copper sulfate. The spectra of the

reagent blanks are also shown, these and the other samples being read
against distilled water. Clearly 310 is the lowest wavelength which can
safely be used in the presence of high concentrations of DNA.
The absorption spectrum below 270 mg of the copper-protein complex
(not shown on graph) was read against the alkaline copper sulfate blank
solution on a Perkin-Elmer model 137 UV spectrophotometer. A broad
peak was found around 263 mg.
Effect of Temperature
Color development is very rapid at room temperature and is certainly
complete within 5 min. The color is not affected by heating at 100 0 C for
30 min.

FINAL METHOD

Reagents
(1) 0.21% CuS04 • 5H20 in 30% NaOH. (To prevent precipitation
of copper hydroxide, this should be prepared by adding dilute copper
sulfate (e.g., 1%) to the alkali).
 PROTEIN ESTIMATION

(2) 30% NaOH. When stored in Pyrex bottles, these reagents keep
satisfactorily for this reaction for at least 6 months.
406 ITZHAKI AND GILL

Procedure
The following mixtures are prepared:
(Al) 2 ml distilled water + 1 ml reagent (1).
(A2) 2 ml protein solution -4- 1 ml reagent (1).
(Bl) 2 ml distilled water + 1 ml reagent (2).
(B2) 2 ml protein solution + 1 ml reagent (2) .
All mixtures are shaken vigorously. The optical density at 310 me of
mixture (A2) is read against (Al) and of (B2) against (Bl) giving values
DA and DB, respectively. The difference, DA — DB, is referred to
below as ODD. Readings may be made 5 minutes after mixing since the
ultraviolet absorption of the copper-protein complex reaches its
maximum within this period. The absorption is then constant for at least
2.5 hr, for the proteins listed in Table 1. However, in the case of
lysozyme, the value of ODD increases during 2.5 hr by 11% of its initial
value (E l lcm% 20.6) at 15 min after mixing.

TABLE I
EXTINCTION COEFFICIENTSC AT 310 Mg OF COPPER-PROTEIN COMPLEXES

Protein El %

BPA '2
Ovalbumin 2.
1
DNP 1
8.
S
Histone 17
.1
Casein 1
Trypsin 7
.
4
Gelatin 1
5
.
6

a
Defined as ODD for protein concentration of I % (10 mg/ml) in final reaction mixture
of 3 ml.
 PROTEIN ESTIMATION 407

RESULTS

Proteins
The standard deviation of ODD was found from five aliquots of
protein solution to be ±2% of the mean value.
Figure 3 shows the calibration curve for BPA; this is linear in the
concentration range 0.026 to 0.53 mg/ml. The extinction coeffcient (of
the final reaction mixture of 3 ml) was measured from the gradient and is
given in Table 1 with values for other proteins standardized in a similar
way.
O.D. 1.48

1.22

0.26

0.3 0.53

0.9

0.8

0.7

06
$5
d 0.4
408 ITZHAKI AND GILL

0.3

0.2

0.1

Final protein concentration (mg/ml)

FIG. 3. Variation of OD310m„ with protein concentration, and effect of added

DNA. Concentrations are final values in 3 ml. Readings are made against blanks
described in text. All solutions contain 10% NaOH. OD values for 0.53 mg/ml are
specified since ordinate scale does not apply to these. (O) BPA (0.117 mg/ml). (O)
BPA in 0.07% CuSOe5HzO. (O) Difference between corresponding ordinates of these
two curves. (O) BPA plus DNA (0.7 mg/ml). (u) BPA plus DNA, in 0.07% cusoe
5H20. ( Difference between corresponding ordinates of these two curves.

TABLE 2
MOLAR EXTINCTION COEFFICIENTS AT 310 M"
OF COPPER-DIPEPTIDE COMPLEXES

Dipeptide
EIM
1 cma

L-Prolylglycine•H20 176
Glycylglycineb 82
L-Leucylglycineb
107
Glycyl-L-leucine 113
L-Prolyl-L-proline
Glycyl-L-proline o
For 1 M dipeptide concentration in final reaction mixture of 3 ml.
b
ODD for these dipeptides increased with time after mixing reagents. The value given
iB for 15 min after mixing,
Peptides
Table 2 shows values of molar extinction coeficient for several
dipeptides. The value of ODD was zero for L-prolyl-L-proline and for
glycylL-proline even at concentrations three times that of the other
peptides.
Effect of Various Additives
The effect of DNA (0.1 mg/ml) on the estimation of ovalbumin was
studied. No interference occurred over a range of protein concentrations.
Figure 3 shows the effect of DNA (0.7 mg/ml) on values of ODD at
various concentrations of BPA. It is clear that DNA, even at this high
concentration, does not interfere with the estimation.
 PROTEIN ESTIMATION 409

The effect of various compounds which are commonly used in work

on proteins was investigated. All concentrations quoted below are those
in the initial aliquot of 2 ml. No change in ODD was observed when the
protein solution contained 1.5 N sodium chloride, 0.1 N sodium acetate,
0.75 N sodium formate, 0.67 N perchloric acid, or 0.01 M disodium
hydrogen phosphate. Ammonium sulfate decreased ODD by about 10%
at 20% saturation and by about 20% at 40% saturation. It would therefore
be essential to standardize the method in the presence of ammonium
sulfate if protein solutions containing this salt were to be estimated. Urea
at 2.1 and 4.2 M caused small increases in ODD which were corrected by
subtracting the corresponding values of ODD for the urea alone from the
observed values of ODD. None of the other compounds mentioned above
had a significant value of ODD in the absence of protein.
DISCUSSION
These results show that the method is both simple and sensitive. For
the proteins studied here, it is reasonably nonspecific for type of protein,
the difference between the extreme values of extinction coeffcient of the
final reaction mixture (15.6 and 22.1) for the seven proteins studied
being about 34% of the mean value (18.9).
The relatively low extinction coeffcient of the copper-gelatin complex
appears to be related to the high proline and hydroxyproline content of
this protein (about 30%). The "biuret" reaction between copper and
protein is believed to occur through coordination of one copper atom to
four peptide bond nitrogens, with accompanying loss of a proton from
each of the four substituted amide groups (4). In the case of dipeptides,
the copper is thought to be coordinated to the peptide nitrogen (again
after loss of a proton) and to the free amino and carboxyl groups (5). Of
the dipeptides listed in Table 2, only the first four have true peptide
bonds; the remaining two, the proline peptides, have no ionizable amide
hydrogen. The latter both have a zero extinction coeffcient whereas of
the first four, the prolyl peptide (containing proline linked through the
acyl group and having therefore an ionizable hydrogen on the amide
group) has a relatively high extinction coeffcient.
Thus since the proline (and presumably the hydroxyproline) residues
of gelatin—comprising about 1/3 of the total residues—do not form
ultraviolet-absorbing complexes with copper, the extinction coeffcient of
the gelatin-copper complex should be about % of the value for BPA,
which has only about 4% of these residues, and Table 1 shows that this is
in fact the case.
410 ITZHAKI AND GILL

The sensitivity of this method is 6 times that of the ordinary biuret

method (6) though only about 1/16 that of the Folin-Lowry modification
(7). However it is preferable to the latter in being far less specific for
type of protein and in giving a linear variation of optical density with
protein concentration. It is also more sensitive and much simpler than the
micro-Kjeldahl nitrogen estimation (8). Another advantage over all the
above methods is that it is relatively unaffected by high concentrations of
urea and only slightly by ammonium sulfate.
It should be stressed that, in the absence of DNA, the sensitivity can be
increased by measuring the absorption at a wavelength below 310 mg,
provided that the copper concentration is reduced to avoid a high reagent
blank (since its absorption increases rapidly with decreasing wavelength,
as shown in Fig. 2). At 280 my, using a copper sulfate concentration of
about 0.02%, the sensitivity is greater by a factor of two. 1At 270 the
sensitivity would be even greater but at concentrations suffciently low to
give a small reagent blank, the variation of ODD with copper
concentration is undesirably high. In the presence of low concentrations
of DNA (less than about 0.2 mg/ml), readings can be made at 300 mp
without reducing the copper concentration, thereby increasing the
sensitivity by about 40%.
It should be noted for comparison that the Warburg and Christian
method of estimating proteins in the presence of DNA (6) by direct
absorption measurements at 280 and 260 cannot be used if the DNA
concentration is greater than 20% of that of the protein, In the present
method, with DNA concentrations ten times greater than that of the
protein, no interference occurs. In any case, if the direct protein
absorption is measured at 280 mg, its value is about % to 1/2 that of the
copper-protein complex at this wavelength. The absorption at 280 mg has
the further disadvantage of depending on the tyrosine and tryptophan
content of the protein; this can vary greatly from one protein to another.
Since this method was developed, Ellman has
published a similar method (9) in which he
measures the absorption of the copper-protein
complex at 263 mp, using a copper sulfate
concentration of 0.04% and sodium hydroxide
concentration of 2 N. Although the copper
concentration is lower than ours, the alkaline
copper sulfate blank absorption at 263 mß is
extremely high (optical density 1.3 against
1 This wavelength has the disadvantage, however, of being near the peak of the
alkaline-protein absorption spectrum, but the peak is relatively small (see later).
 PROTEIN ESTIMATION 411

water). The absorption of the copper protein

complex is also greater at 310 but the increase
is not as great, proportionately, as that of the
copper alone (i.e., the ratio of absorption of
copper blank to copper protein complex is very
much greater at 263 than at 310 null). We think,
therefore, that the advantage of increased
sensitivity at 263 mg is greatly outweighed by
the increased liability to error through the very
high blank absorption. In any case, since this
wavelength is so close to that of the wavelength
of peak absorption of DNA (260 me), it could be
used only in the presence of very small amounts
of DNA.
Finally, since the method is satisfactory even
in the presence of very high concentrations of
DNA, it should prove useful for estimating
enzymes and other proteins in tissue extracts.
SUMMARY
A rapid and sensitive method for estimating
proteins using an alkaline copper sulfate reagent
is described. The method is reasonably
nonspecific for type of protein and can be used
for solutions containing DNA even at
concentrations of the latter as high as 0.7 mg/ml
in the final reaction mixture. In the presence of
DNA, 0.15 to 3.0 mg of protein can be estimated
and in the absence of DNA, as little as 0.075 mg.
The effects are described of various salts, of
perchloric acid, and of urea on the estimation.
Measurements on several dipeptides showed that
proline peptides do not form ultraviolet-
absorbing complexes with copper whereas prolyl
peptides do form such complexes. This explains
the finding that gelatin (which has a high
proportion of proline and of hydroxyproline
residues) is less reactive with copper than are
the other proteins studied here.
ACKNOWLEDGMENT
412 ITZHAKI AND GILL

We wish to thank Professor J. S. Mitchell for his

interest.
REFERENCES
1. ZAMENHOFF, S., in "Methods in Enzymology" (S. P. Colowick
and N. O. Kaplan, eds.), Vol. Ill, p. 702. Academic Press,
New York, 1957.
2. ITZHAKI, R. F., Nature 194, 1241 (1962).
3. MEHL, J. W., PAcovsKA, E., AND WINZLER, R. J., J. Biol.
Chem. 177, 13 (1949).
4. RßING, M. M., AND YANG, P. S., J. Biol. Chem. 99, 755
(1932-1933).
5. RABIN, B. R., Biochem. soc. sump. (Cambridge, Engl.) 21 (1958).
413 AND GILL

1'rZHAK1

6. LAYNE, E., in "Methods in Enzymology" (S. P. Colowick and N. O. Kaplan, eds.),

Vol. Ill, p. 450. Academic Press, New York, 1957.
7. Lowny, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol.
Chem. 193, 265 (1951).
8. MA, T. S., AND ZUAZAGA, G., Ind. Eng. Chem., Anal. Ed. 14, 280 (1942).
9. ELLMAN, G. L., Anal. Biochem. 3, 40 (1962).