The Biuret reaction can be used to assay the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. 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.
In spite of its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide bonds in the biuret molecule.
Procedure :
An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most often) followed by a few drops of aqueous copper(II) sulfate. If the solution turns purple, protein is present. 5–160 mg/mL can be determined.
Biuret reagent :
The biuret reagent is made of potassium hydroxide (KOH) and hydrated copper (II) sulfate, together with potassium sodium tartrate. The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides.
Not all biuret tests require the biuret reagent. The reagent is commonly used in a biuret protein assay, a colorimetric assay used to determine protein concentration—such as UV-VIS at wavelength 540 nm (to detect the Cu2+ ion).
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. In this way, proteins can be detected 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 of the of the signal.
Cu+ forms a deep purple complex with bicinchoninic acid (BCA), which 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.
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