A Biuret test is used to check the presence of protein in the sample.
Proteins are biomolecules made up of amino acids. Two amino acids are linked together by an amide-type covalent bond called the peptide bond. The quantitative estimation of protein is an essential step in various biochemical and molecular biology experiments. Ferdinand Rose in 1833 and Polish physiologist G. Piotrowski in 1857 independently discovered a protein estimation assay. The assay shows that in an alkaline solution, a copper (II) ion (Cu2+) forms a pale-purple-colored coordination complex in the presence of peptides. The protein estimation assay is also known as the Piotrowski test.
The protein estimation assay is a colorimetric assay. The intensity of the pale-purple color is directly proportional to the number of peptide bonds between two amino acids. Therefore, the concentration of protein can be measured using the Biuret test. The absorption maxima for the pale-purple-colored coordination complex is 540 nm. The instrument used to measure the absorption is a spectrophotometer. The Beer-Lambert law is used to calculate the concentration of protein.
The Biuret assay requires 1% NaOH or KOH for providing the alkaline environment, aqueous Copper(II) sulfate (CuSO4(H2O)x), and Potassium sodium tartrate. In alkaline conditions, the Cu2+ ion binds with nitrogen atoms present in the peptide bonds of proteins. A pale-purple color formed due to a tri- or tetra-dentate chelation complex between one Cu2+ ion and the four nitrogen atoms of the dipeptides molecules.