Biuret Test

What is the Biuret Test?

The biuret test is a chemical test that can be used to check for the presence of peptide bonds in a given analyte. Therefore, the biuret test can also be used to gauge the amount of protein present in the analyte. In this test, the presence of peptides results in the formation of pale purple coloured (or mauve coloured) coordination compounds of the copper(II) ion (when the solution is sufficiently alkaline). An image detailing a positive biuret test and the characteristic pale purple colour that denotes it is provided below.

It can be noted that several variants of the biuret test have been developed. Notable examples of such variations include the modified Lowry test and the BCA test. It can also be noted that the intensity of the purple colour and, therefore, the absorption at 540 nanometers is directly proportional to the concentration of proteins in the given analyte (as a consequence of the Beer-Lambert law). A positive reaction for this test is also received when the analyte contains biuret molecules ([H₂N-CO]₂NH) since the bonds in this molecule are similar to peptide bonds.

Biuret Reagent

The biuret reagent is made up of hydrated copper sulfate, sodium hydroxide, and Rochelle salt (sodium-potassium tartrate). Here, the Rochelle salt acts as a chelating agent and stabilizes the copper(II) ions.

Biuret Test Principle

The copper(II) present in the biuret reagent binds itself to the nitrogen atoms that are present in the protein peptides. Now the copper(II) undergoes reduction and is converted to copper(I). Since this test is not greatly disturbed by the presence of amino acids in the sample, it can be used to gauge the concentration of proteins in whole tissue samples. However, the samples of proteins that are purified via ammonium sulphate ((NH₄)₂SO₄) precipitation are not ideal for this test since buffers like ammonia interfere with it.
The reaction between the copper(II) ions and the nitrogens belonging to the peptide bonds results in the displacement of peptide hydrogens (as long as the environment is sufficiently alkaline).

Now, four nitrogen atoms donate lone pairs to form coordinate covalent bonds with the cupric ion (illustrated above), resulting in the formation of a chelate complex. This chelate complex has the ability to absorb light with a wavelength of 540nm, which imparts a purple colour to it. Therefore, the formation of a purple-coloured complex indicates the presence of proteins in the analyte. Note that the concentration of peptide bonds in the analyte contributes to the intensity of the purple colour. Short-chain peptides often yield blue or pink colours in the biuret test.

Biuret Test Procedure

The procedure that can be followed to conduct a biuret test is provided below.

• An aqueous solution of the analyte must be prepared by dissolving it in water.
• A small amount of this aqueous solution must be treated with 1% sodium hydroxide or potassium hydroxide. To this mixture, a few drops of CuSO₄ (aq) must be added.
• If the solution turns purple upon the addition of copper(II) sulphate, the presence of protein in the analyte is confirmed.

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