Biuret test

The biuret test is a chemical test used for detecting the presence of peptide bonds. In a positive test, a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. In simpler terms, a peptide bond breaks away to allow the nitrogens to bond to the starch in the biuret solution.

It is possible to use the Biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.

Procedure

The test can be done by adding an equal volume of 1 % of KOH (potassium hydroxide) and a few drops of 1 % of CuSO₄ (copper(II) sulfate) to the sample solution. If the solution turns purple, protein is present. 5-160 mg/ml can be determined.

Increasing the sensitivity of the biuret test

Cu⁺ is a strong reducing agent which can react for example with Mo(VI) in Folin-Ciocalteu's reagent to form molybdenum blue, a complex of 179410 Mo(V) and Mo(IV) ions. This is the basis of the Folin et al. [O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Mesurement With the Folin Phenol Reagent, J. Biol. Chem. 193 (1951) 265 - 275.] protein determination method, which can detect proteins in concentrations between 0.005 and 2 mg/ml. Molybdenum blue in turn can bind certain organic dyes (malachite green, Auramin O), resulting in further amplification [Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal. Biochem. 163 (1987) 476-481.] of the signal.

Cu⁺ forms a bright pink complex with bicinchoninic acid (BCA) [Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1985) 76-85.] , this allows proteins in the range of 0.0005 to 2 mg/ml to be determined. This assay is often referred to as "Pierce assay" after the manufacturer of a reagent kit.

References

# Gold. 1990. Organic Compounds in Biological Systems, 2nd ed. John Wiley & Sons, Inc.