The biuret test (Piotrowski's test) is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-colored coordination complexes in an alkaline solution. Several variants on the test have been developed, such as the BCA test and the Modified Lowry test.
The biuret reaction can be used to assess 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.
Despite its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The test is named so because it also gives a positive reaction to the peptide-like bonds in the biuret molecule
The Biuret reagent is made of sodium hydroxide (NaOH) and hydrated copper(II) sulfate, together with potassium sodium tartrate, the latter of which is added to chelate and thus stabilize the cupric ions. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of the peptide hydrogen atoms under the alkaline conditions. A tri- or tetra-dentate chelation with the peptide nitrogen produces the characteristic color. This is found with dipeptides (Datta,S.P., Leberman,R., and Rabin,B.R., Trans.Farad.Soc. (1959), 55, 2141).
The reagent is commonly used in the biuret protein assay, a colorimetric test used to determine protein concentration by UV/VIS spectroscopy at wavelength 550 nm.