Biuret test
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.
In this assay, the copper(II) binds with nitrogens present in the peptides of proteins. In a secondary reaction, the copper(II) is reduced to copper(I). Buffers, such as Tris and ammonia interfere with this assay, therefore rendering this assay inappropriate for protein samples purified from ammonium sulfate precipitation. Due to its insensitivity and little interference by free amino acids, this assay is most useful for whole tissue samples and other sources with high protein concentration.
An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide) 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. Peptides with the chain length of at least 3 amino acids are necessary for a significant, measurable colour shift with these reagents.
The Biuret reagent is made of sodium hydroxide (NaOH) and hydrated copper(II) sulfate, together with [[potassium Chemical Reagents</ref> Potassium sodium tartrate 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 "biuret" color. This is found with dipeptides (Datta,S.P., Leberman,R., and Rabin,B.R., Trans.Farad.Soc. (1959), 55, 2141.)
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