The Biuret Assay For Determining Total Protein

All proteins are composed of amino acids joined by peptide bonds in a linear sequence. There are ~20 naturally occurring amino acids found in proteins. The amino acids are joined to each other by peptide bonds formed by a condensation reaction that occurs between the terminal amine of one amino acid and the carboxyl end of the next. Peptides containing three or more amino acid residues will form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate. A similar colored chelate complex forms with the organic compound biuret (NH2-CO-NH-CO-NH2) and the cupric ion. The reaction in which a colored chelation complex is formed with peptide bonds in the presence of an alkaline cupric sulfate solution became known as the biuret reaction (Fig. B1.1.5). Thus, the biuret protein assay reagent gets its name from the above reaction even though it does not actually contain the organic compound biuret. Single amino acids or dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light-blue to violet complex that absorbs light at 540 nm. One cupric ion forms the colored coordinate complex with 4 to 6 nearby peptides bonds. The intensity of the color produced is proportional to the

nh2

nh2

O=C

C = O

\

/

NH

HN

/ \

/ \

O=C

\ / C = O

nh2

\u'2+ NH2

nh2

nh2

/ \ NH2

nh2

O=C

O=C

/ \ C = O

180°C

\

Cu2+ \ /

\ /

C = O -►

NH + NH3

->- NH

HN

/

/

V ~

nh2

O=C

O=C

C = O

nh2

nh2

nh2

Urea (in excess)

Biuret

Cu Complex

Figure B1.1.5 The schematic of the Biuret reaction.

Figure B1.1.5 The schematic of the Biuret reaction.

Table B1.1.3 Troubleshooting Guide for Biuret Protein Assay

Problem Possible cause Solution

No color in any tubes Sample contains copper Dialyze or dilute the sample chelating agent

Blank A540 is normal, but Color measured at the Measure the color at 540 nm standards show less color than wrong wavelength expected

All tubes (including the blank) are Sample contains a reducing Dialyze or dilute the sample dark purple agent number of peptide bonds participating in the reaction. Thus, the biuret reaction is the basis for a simple and rapid colorimetric method of quantitatively determining total protein concentration.

Because the working range for the Biuret assay is from 5 to 160 mg/ml, the Biuret reagent has found utility in the clinical laboratories for the quantitation of total protein in serum. The formulation employed in the Biuret total protein reagent (Sigma Diagnostics) was developed by Doumas et al. (1981) as a candidate reference method for the determination of serum total protein in the clinical lab. Using Sigma's Biuret reagent, the expected range for total protein in serum is from 63 to 83 mg/ml. Bilirubin, lipids, hemoglobin, and dextran are known to interfere in the Biuret assay for total serum protein. Outside of this application, other copper chelating agents such as EDTA, EGTA, citrate, Tris, imino-diacetic acid, and nitrilotriacetic acid will interfere. The working range of this assay is 5 to 160 mg/ml. Table B1.1.3 is a brief troubleshooting guide for this technique.

Materials

Standard protein (see Commentary)

Measurement of Protein Content

Biuret Reagent Standard Curve 540

0 10 20 30 40 50 60 70 80 90 100 Protein concentration (mg/ml)

0 10 20 30 40 50 60 70 80 90 100 Protein concentration (mg/ml)

Figure B1.1.6 Graph of the color response curves obtained with Sigma's Biuret Total Protein Reagent using bovine serum albumin (BSA) and bovine gamma globulin (BGG). The standard tube protocol was performed and the color was measured at 540 nm in a Hitachi U-2000 spectro-photometer.

Sample (unknown) protein

Biuret total protein reagent (Sigma Diagnostics; also see recipe)

1. Select a protein to use as the standard (see Strategic Planning) and prepare a dilution series with buffer to cover the range 10 to 160 mg/ml.

If possible, use the same diluent or buffer cocktail for the blanks and for diluting the standard that was used with the samples.

2. In duplicate, add 20 | l standard, sample, or diluent (blank) to appropriately labeled test tubes.

3. Add 1.0 ml biuret reagent to each tube. Mix well by vortexing 2 to 3 sec.

4. Incubate tubes at ambient room temperature (18° to 26°C) for 10 min.

5. Measure the color of each tube with a spectrophotometer at 540 nm (A540). Compare to the blank.

6. Plot a standard curve by graphing the blank-corrected A540 values for the standards versus protein concentration in milligrams per milliliter.

Example color response curves for BSA and BGG are shown in Figure B1.1.6.

7. Determine the sample concentration by interpolation from the standard curve (see Strategic Planning).

The Colorimetric Detection and Quantitation of Total Protein

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Responses

  • Klaus
    Why single amino acids and dipeptides do not give the biuret reaction,?
    7 years ago
  • biniam
    What are interference with the biuret assay for total protein?
    2 years ago

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