Section Advances in Biotechnology

PROTEIN AND PEPTIDE DETERMINATION BASED ON THE MODIFIED

BIURET PROCEDURE: IMPLICATIONS FOR VARIOUS
BIOTECHNOLOGIES

PhD Asst. Elena Mihalcea1,

Prof. Dr. Gabi Drochioiu1,
PhD Asst. Stefania-Claudia Jitaru1,
Dr. Violeta Mangalagiu1,2,
Assoc. Prof. Dr. Robert –Vasile Gradinaru1
1
Faculty of Chemistry, Al. I. Cuza University of Iasi, Romania
2
Faculty of Food Engineering, Stefan cel Mare University of Suceava, Romania

ABSTRACT
Spectrophotometric methods for total protein analysis are generally simple, rapid and
sensitive. Such sensitive protein assays may have applications in forensic science, in the
detection of protein contaminants in drugs and in a number of other applications of
research interest. Biuret reaction with proteins and peptides is widely used in clinical
and biological laboratories. In this work, instead of copper sulphate, sodium hydroxide
and Seignette salt, we used insoluble copper phosphate, potassium or sodium hydroxide
and ethyl alcohol for the preparation of the biuret reagent. Absorbance of the biuret
complex was recorded both at 219-230 nm (after dilution) and around 550 nm against a
reagent blank. Amino acid interference was investigated around 550 nm at the same
concentration as proteins. The sensitivity of the method at 226 nm was greater than
those of other spectrophotometric assays (old biuret method, Lowry, and BCA) with a
LOD of about 0.5 µg mL−1 BSA. The new variants of the biuret method for total protein
analysis eliminate the need for precise reagent addition and vortexing inherent in the
widely used Lowry method, providing flexibility of application. The method developed,
which uses an alkaline-alcoholic reagent and insoluble copper phosphate, is simple,
rapid, reproducible and sensitive; it is not influenced by detergents, solvents and buffers
containing ammonium and is flexible enough to change the analytical protocol when
necessary. A discussion was made on the applications of protein and peptide
determination with the new biuret assay.
Keywords: Protein determination, Peptide determination, Biuret reaction, Amino
acid interference, Spectrophotometry

INTRODUCTION
There are numerous methods for protein quantification. Protein determination
assays use progressively smaller amounts of material and modifications to classic
protocols were also introduced to provide a microvolume alternative to traditional
cuvette‐based methods [1-3]. However, classical protein assays are also widely used in
biotechnology and medical laboratories around the world [4,5]. Protein
concentrations in various

113
https://doi.org/10.5593/sgem2022/6.1/s25.14
22nd International Multidisciplinary Scientific GeoConference SGEM 2022

samples are estimated using a large body of techniques, including Dumas method,
Nessler method, Biuret method, Berthelot’s method, Lowry method, Kjeldahl method,
Folin-Ciocalteu method, Dye binding, Direct alkaline distillation, Near infrared
reflectance (NIR), Bradford method, Bicinchoninic acid (BCA) method etc., which have
been developed over past two decades [6,7]. The Kjeldahl and Dumas methods
determine nitrogen, which is proportional to the protein content of the samples [8,9].
The Bradford assay is based on a metachromatic shift of the dye on binding to a protein
(Bradford, 1976). The high sensitivity of this method is dependent on the quality of the
protein, the solvent and the reaction conditions. A linear range of 20–500 g mL-1 was
reported [7]. Given the importance of protein determination, it is significant to choose
the appropriate technique from the available methods [10]. It is of great importance to
know which particular range of protein concentration an assay is sensitive to. Besides,
several factors such as the nature of the protein, the nature of other components present
in the sample, and the preferred speed, accuracy, and sensitivity of assay are considered
[11]. Compounds containing two or more peptide bonds react with the biuret reagent to
form a purple colored complex [12]. The biuret reaction has been used for many
biological materials (Sapan & Lundblad, 2015) including cerebrospinal fluid (Cotton et
al., 1997), synovial fluid (Popko et al., 2006), and saliva (Wolf & Taylor, 1964).
Measurement of the total protein in serum using the biuret reaction was also
recommended [11]. Although simple, highly selective, and reliable, free amino acids,
peptides, Tris, dextrose, and dextran interfere with protein determination. EDTA can
also influence color development with the tartrate reagent. Therefore, we improved the
biuret method replacing tartrate and copper sulphate with insoluble copper phosphate. In
addition, the biuret absorbance was measured both in visible and UV region. To
eliminate completely the interference of amino acids and peptides, the protein was
precipitated with 10% TCA, centrifuged and the pellet re-dissolved using an alkaline-
alcoholic solution before the biuret reaction.

METERIALS & METHODS

Chemicals All chemicals and solvents were purchased from Sigma Chemical Company
(Sigma Aldrich, Germany), Fluka (Switzerland), Roth (Carl Roth GmbH, Germany) and
deionized water (18.2 M·cm) was used for all the performed analysis (Milford, MA,
USA). Copper phosphate was prepared by treating copper sulphate with disodium
phosphate, washing the obtained precipitate several times and grinding it after drying.
Procedure. Previously, the reaction of biuret was first studied. Thus, 1 ml of zein
extract, a maize protein, was mixed with 0.2 ml of 6% KOH, to which approximately 20
mg of copper phosphate was added. The mixture was sonicated for 15 minutes and then
centrifuged at 18,000 rpm for 5 minutes. The absorption spectrum of the supernatant
was performed in the range 400-700 nm. In the present work, in eppendorf vials of
plastic we introduced each time 1 mL of 2 mg mL-1 aqueous solutions of albumin,
gelatin, and casein and 1 mL of 70% alcoholic solution of 2 mg mL-1 zein were mixed
with alkali-alcoholic solution containing 50% (v/v) ethyl alcohol (96%), 2 g of KOH
and water up to 100 mL. Approximately 20 mg of copper phosphate was added to each
vial and the mixture was sonicated for 15 minutes. Finally, the vials were centrifuged at
15,000 rpm, and the biuret absorbance read in a Biochrom® Libra S35 UV\Visible
spectrophotometer between 190 nm and 840 nm against a control containing the

114
 Section Advances in Biotechnology

reagents only. The calibration curves were drawn with absorbance values read at
different wavelengths such as 226 nm or 550 nm. If absorbance values in the ultraviolet
range are too high, both reference solutions and samples have been diluted appropriately
with distilled water, KOH solutions or alkaline alcoholic solutions respectively.

RESULTS
Biuret reaction: new developments
Modern spectrophotometers allow measurements in the 190-250 nm wavelength range,
where the protein biuret complex absorbs strongly. However, copper sulfate in the
classic biuret test interferes. Using insoluble copper phosphate, only proteins, the
copper-protein complex and the copper ions extracted by the proteins show interference.
In addition, we demonstrate here that several amino acids also interfere with the biuret
measurements even at 540-550 nm, and how to avoid their interference.
Protein determination
The sensitivity of the biuret method is applicable to protein samples with concentrations
between 0.01 to 5.00 mg mL-1 [10]. Therefore, we measured the biuret absorbance of
standard albumin solutions prepared from a solution of 5.00 mg mL-1 BSA (Fig. 1).

Figure 1. Calibration curve in protein determination. (a) Biuret spectra of bovine serum albumin
solution (BSA) solutions; (b) A 5 mg mL-1 BSA solution was used for calibration curve plotting.
Measuring the absorbance in the reaction of copper ions with BSA and serine, as well as
of their mixture after precipitation with 10% TCA, we found that interering amino acids
and other soluble compounds can be removed (not shown). Protein precipitation
followed by the biuret assay as it was described here can be used in protein
determination in sludge hydrolysates.
Peptide determination
The peptide content can also be determined by the biuret method, while the amino acids
are stained with ninhydrin (Yan et al., 2020). Here, we investigated the formation of the
biuret complex of a decapeptide, Arg-Cys-His-Gln-Tyr-His-His-Asn-Arg-Glu, 1 mg
mL-1 concentration, at pH 7.0 and pH 10.0 (Fig. 2). On increasing the pH values,
maxima shifted toward higher wavelength values. Therefore, this peptide binds copper
even at neutral pH. Glutathione (1 mL of 2 mg mL-1 GSH) was also treated with 0.5 mL
of alkaline alcoholic solution (2% KOH; 50% ethanol) and 50 mg of copper phosphate.

115
https://doi.org/10.5593/sgem2022/6.1/s25.14
22nd International Multidisciplinary Scientific GeoConference SGEM 2022

A large absorption band with maximum at 595 nm (A = 0.421 A.u.). The 1: 20 dilution
of the biuret solution thus obtained resulted in a maximum at 228 nm (A = 2.088 A.u.),
while at 585 nm the absorbance was reduced to only 0.047 A.u. Thus, the absorbance
value of the GSH biuret at 228 nm was 44.42 times higher than reading in the visible.
0.14
527 nm

0.12 Ref pH 7
Ref pH 10
0.10 P10+CuPh 7
P10+CuPh 10
0.08
Absorbance

0.06 517 nm

0.04

0.02

0.00
450 550 650 750
Wavelength (nm)
Figure 2. The biuret reaction of decapeptide RCHQYHHNRE (Here: Ref 7 & 10 = absorbance
of the peptide at pH 7.0 & pH 10.0 and CuPh = insoluble copper phosphate).
UV measurements of protein concentrations
The biuret method is less sensitive than other methods. Therefore, we used -casein, a
protein from milk, which was treated with copper phosphate (CuPhos) in 0.1 N KOH
solution and observed a high absorption band around 230 nm.

Figure 3. UV spectrum of 1 mL of casein solution (Cas, 20 g mL-1), containing 350 L of

copper phosphate supernatant (CuPhos) and 50 L of 0.1 N KOH.

116
 Section Advances in Biotechnology

However, copper ions released from CuPhos also interfere due to their absorbance
around 210-215 nm. The protein concentration can be measured at about 226 nm, but
another band is important for UV biuret reading, at about 245 nm, where Cu2+ ions
interfere less. In our experiments, a linear relationship was found between casein
concentration (2-20 g mL-1) and absorbance at 226 nm (r = 0.996). So far, the
absorption band of the biuret at 245 nm was not fully investigated.
Protein hydrolysates
The proteins extracted from environmental samples have a high content of polypeptides
and amino acids. The peptide bonds connected with tryptophan (Trp) were more
inclined to be decomposed (Zhu et al. 2020). In addition, extraction of protein from
excess sludge was made by hydrolysis, which also contain amino acids and peptides
(Gao et al. 2020). Therefore, we first measured the biuret absorbance of some proteins
and amino acids (Fig. 4). We thus found that, at a concentration of 2 mg mL-1 of BSA,
the maximum absorbance of gelatin or zein, the characteristic wavelength differs
slightly (BSA - 547.0 nm; gelatin - 554.5 nm; zein - 542.5 nm). The maximum
absorbance values at the same concentration of 2 mg mL-1 were also different (BSA -
0.463 absorbance units (A.u.); gelatin - 0.325 A.u.; zein - 0.427 A.u.). Both sets of data
suggest that it is necessary to plot calibration curves with each of the proteins analysed
at the specific wavelength of each protein. However, BSA could be used as a standard,
with mention of its use in the determination of other proteins.
0.50
BSA
Gelatin
zein
0.40
Tryptophan
Absorbance (A.u.)

Serine

0.30

0.20

0.10

0.00
390 440 490 540 590 640 690 740
Wavelength (nm)

Figure 4. Absorbance spectra of some proteins and amino acids treated with an alkaline-
alcoholic reagent in the presence of insoluble copper phosphate. Legend: BSA, 2 mg mL-1 of
bovine serum albumin; Gelatin, 2 mg mL-1 aqueous solution; zein, 2 mg mL-1 in 70% alcoholic
solution; Tryptophan, 2 mg mL-1 aqueous solution; Serine, 2 mg mL-1 aqueous solution.
The same figure also shows that individual amino acids extract copper ions from
insoluble copper phosphate to form complexes with different absorption maxima
(tryptophan - 556.5 nm, serine - 605.5 nm; amino acid concentration - 2 mg mL-1). The
maximum absorbance values of these complexes were also completely different

117
https://doi.org/10.5593/sgem2022/6.1/s25.14
22nd International Multidisciplinary Scientific GeoConference SGEM 2022

(tryptophan - 0.027 A.u.; serine - 0.282 A.u.). Hence, a serious interference of amino
acids in protein hydrolysates in the determination of pure proteins in the solution may
appear.
Interference of amino acids
Amino acids treated with the biuret reagents showed different absorption intensities in
the range of biuret absorption (Fig. 5). Thus, histidine binds copper ions most strongly,
followed by proline. Although the absorbance of copper-amino acid complexes has
maxima of absorption at higher wavelengths than 600 nm, some of them interfere much
with the readings at 550 nm.
Alanine
0.60 Serine
Threonine
Tryptophan
0.50
Histidine
Glutamic Ac.
0.40 Lysine
Absorbance (A.u.)

Glycine
Leucine
0.30 Arginine
Aspartic Ac.
Methionine
0.20
Phenylalanine
Proline
0.10

0.00
400 450 500 550 600 650 700 750 800 850
Wavelength (nm)

Figure 5. Absorbance spectra of amino acids treated with insoluble copper phosphate and
alkaline alcoholic solutions.

DISCUSSION
Protein determination based on the biuret reaction is commonly used as a reference
method [11]. Bovine and human serum albumin are normally used to standardize the
biuret method. However, the biuret method is sensitive in the range 0.5 to 2.5 mg
protein per assay, while the Lowry method is 1 to 2 orders of magnitude more sensitive
(5 to 150 μg) (Rodger & Sanders, 2017). Nevertheless, the main disadvantage of the
Lowry method is the number of interfering substances. Here, we showed new
advantages of the biuret method. Indeed, copper-peptide bond interactions contribute to
analysis by biuret, Lowry and bicinchoninic acid (BCA) methods. However, certain
amino acids are involved in Lowry, BCA, dye-binding and UV 280 nm methods. Some
authors consider that both the Lowry and biuret techniques, with such adjustments,
could nearly completely replace the reference Micro-Kjeldahl method that had
complicated procedures [13]. However, these authors used copper sulfate and sodium
potassium tartrate within their experiments. Indeed, there is no single protein assay
method that yields absolutely accurate results [14]. Each method has different

118
 Section Advances in Biotechnology

advantages and limitations. We have proposed here a new variant of the biuret method,
which utilises insoluble copper phosphate and ethyl alcohol instead copper sulphate and
potassium sodium tartrate tetrahydrate (Seignette's salt) and studied the biuret reaction
both with proteins, peptides and amino acids. Thus, we found that proteins mobilize
copper ions in alkaline alcoholic solutions to obtain the biuret complex whose colour
intensity is proportional to the concentration of compounds containing peptide bonds.
Amino acids do not form the biuret complex, but some of them form copper salts and
complexes that absorb visible radiation with an absorption maximum near the biuret
absorption band.
The applicability of the colorimetric biuret procedure for protein determination at 540
nm to a variety of biological preparation may be affected by the presence of lipids
(Parvin et al., 1965). This interference was eliminated by using ethyl alcohol. The starch
also interferes in the classical method, and not in our proposed variant.

CONCLUSION
The total protein in biological samples can be measured by the biuret method. In this
work we investigated the interference of some amino acids and peptides in the
determination of proteins found in biological media and in artificial solutions by a
modified biuret method. Thus, to 1 ml of protein solution, 0.5-1.0 ml of alkaline
alcoholic solution and insoluble copper phosphate powder were added. After sonication
and centrifugation, the UV-vis spectrum of the biuret colour of the supernatant is read.
Absorbance of the biuret complex is recorded both in the range from 190 nm to 1100
nm and at about 545 nm, as well as at 219-230 nm relative to a reagent blank. To
eliminate completely the interference of amino acids and peptides, the proteins can be
precipitated with 10% TCA, centrifuged and the pellet re-dissolved using an alkaline-
alcoholic solution before the biuret reaction. Protein precipitation followed by the biuret
assay here described could be used in protein determination in sludge hydrolysates or in
herbal samples containing a mixture of proteins, amino acids and peptides. In addition,
biuret absorption can be measured in both the visible and UV region, in the latter case
the sensitivity being greatly increased.

ACKNOWLEDGEMENTS
Financial support from the Romanian Government through UEFISCDI Bucharest (PN-
III-P2-2.1-PED2019-2484, Contract PED494, BioPASCAL G.D. also acknowledges
Contract no.11PFE/30.12.2021 from Romanian Ministry of Research, Innovation and
Digitization.

REFERENCES
[1] Razzino C.A., Serafín V., Gamella M., Pedrero M., Montero-Calle A., Barderas R.,
Colero M., Lobo A.O., Yáñez-Sedeño P., Campuzano S., Pingarrón J.M. An
electrochemical immunosensor using gold nanoparticles-PAMAM-nanostructured
screen-printed carbon electrodes for tau protein determination in plasma and brain
tissues from Alzheimer patients. Biosensors and Bioelectronics, vol. 163, Art. 112238,
2020;

119
https://doi.org/10.5593/sgem2022/6.1/s25.14
22nd International Multidisciplinary Scientific GeoConference SGEM 2022

[2] Moreira R.A., Chwastyk M., Baker J.L., Guzman H.V., Poma A.B. Quantitative
determination of mechanical stability in the novel coronavirus spike protein.
Nanoscale, vol. 12/issue 31, pp. 16409-16413, 2020;
[3] Seffernick J.T., Lindert, S. Hybrid methods for combined experimental and
computational determination of protein structure. Journal of Chemical Physics, vol.
153/issue 24, Art. 240901, 2020;
[4] Thiex N. Evaluation of analytical methods for the determination of moisture, crude
protein, crude fat, and crude fiber in distillers dried grains with solubles. Journal of
AOAC International, vol. 92/issue 1, pp. 61-73, 2009;
[5] Kamboj U., Guha P., Mishra S. Comparison of PLSR, MLR, SVM regression
methods for determination of crude protein and carbohydrate content in stored wheat
using near Infrared spectroscopy. Materials Today: Proceedings, vol. 48, pp. 576-582,
2022;
[6] Sáez-Plaza P, Michałowski T, Navas MJ, Asuero AG, Wybraniec S. An overview of
the Kjeldahl method of nitrogen determination. Part I. Early history, chemistry of the
procedure, and titrimetric finish. Critical Reviews in Analytical Chemistry, vol. 43/issue
4, pp. 178-223, 2013;
[7] Okutucu B, Dınçer A, Habib Ö, Zıhnıoglu F. Comparison of five methods for
determination of total plasma protein concentration. Journal of Biochemical and
Biophysical Methods, vol. 70/issue 5, pp. 709-711, 2007;
[8] Chang S.K., Zhang, Y. Protein analysis. In Food analysis. Springer, Cham., pp. 315-
331, 2017;
[9] Zhang Q., Qi D., Dong X., Li X., Cheng L., Liu H., Chen S., Rajora O.P., Li X.Q.,
Liu, G. Amino acid composition, protein content and accurate nitrogen-to-protein
conversion factor for sheepgrass (Leymus chinensis). Botany, vol. 98/issue 3, pp. 137-
146, 2020;
[10] Janairo G., Linley M.S., Yap L., Llanos-Lazaro N., Robles J. Determination of the
sensitivity range of biuret test for undergraduate biochemistry experiments, e -Journal of
Science & Technology, vol. 5/issue 6, pp. 77-83, 2011;
[11] Zheng K., Wu L., He Z., Yang B., Yang Y. Measurement of the total protein in
serum by biuret method with uncertainty evaluation. Measurement, vol. 112, pp. 16-21,
2017;
[12] Boyer R. Modern Experimental Biochemistry, 3rd ed.; Addison Wesley Longman,
Inc., California, 2000;
[13] Arunima S., Verulkar S. Comparative analysis of different protein estimation
methods. The Pharma Innovation Journal, vol. SP-11/issue 4, pp. 2091-2095, 2022;
[14] Olson, B. J., Markwell, J. Assays for determination of protein
concentration. Current Protocols in Pharmacology, vol. 38/issue 1, pp. A-3A, 2007.

120
Reproduced with permission of copyright owner. Further reproduction
prohibited without permission.