Biuret test

In this assay, copper(II) binds with nitrogen atoms found in protein peptides. Copper(II) is reduced to copper in a secondary reaction (I). Buffers such as Tris and ammonia interfere with this assay, making it unsuitable for protein samples purified from ammonium sulphate precipitation. Because of its sensitivity and lack of interference from free amino acids, this assay is best suited for whole tissue samples and other sources containing a high protein concentration.

Principle of biuret test:

Copper (II) sulphate (CuSO4) reacts with the peptide bond nitrogen of peptides and proteins in alkaline medium to form a violet complex. The reaction is named after the substance biuret, which is obtained by condensation of two molecules of urea when heated to 1800 degrees Celsius.

This reaction takes place only if the peptide molecule has at least two peptide bonds (3 amino acids). This test does not apply to free amino acids or dipeptides. When diamides of oxalic, malonic, and succinic acid are present, the false-positive test result can also be observed.

Biuret test equation:

Procedure of biuret test:

• In separate test tubes, add 2 ml of each solution and 2 ml of 5% NaOH to each.

• Then, add 2 drops of 1% CuSO4 solution and mix well.

• The presence of peptide bonds in the molecule is indicated by the formation of a violet color.

What does a positive biuret test indicate?

If there is no color change, there are no proteins (i.e. solution remains blue ),and it confirms peptide bonds are present.

What does a negative biuret test indicate?

If the solution changes from blue to violet, protein or peptides are present (i.e. deep purple),and it confirms peptide bonds are absent.

Biuret reagent:

The biuret test employs a reagent made up of potassium hydroxide and copper sulphate. The biuret reagent has a blue colour under normal conditions. If peptide bonds are present, it changes colour to violet. The peptide bond is the chemical bond that connects amino acids. Copper sulphate and sodium hydroxide are two other biuret reagent substitutes. A and B of Fehling’s solutions can also be used.

The hydrated copper sulphate is in charge of the formation of Cu (II) ions, which form the chelate complex. Cu (II) ions are responsible for the reagent’s distinctive blue colour. The potassium hydroxide solution, on the other hand, is in charge of providing the alkaline medium. Finally, the potassium sodium tartrate causes the chelate complex to be stabilised.

Uses of biuret test:

• It can be used to determine the amount of protein in a urine sample.

• The biuret reaction with protein can be used to quantify total protein using spectrophotometric analysis.

When using Fehling’s A and B solutions, how do you perform the biuret test for protein?

• Make sure to make new Fehling’s A and B solutions. Keep in mind that A is a copper (II) solution, whereas B contains both sodium hydroxide and sodium potassium tartrate solutions.

• When testing a food sample, 1 cm3 of solutions A and B should be added to the specimen.

• To create a negative control, repeat the preceding steps with deionized water. Use albumin or egg white as a positive control.

• Shake the mixture gently and set it aside for five minutes.

• Keep an eye out for any color changes.

Using copper sulphate and sodium hydroxide solutions, conducting  a biuret test.

• Food samples are treated with a 1 cm3 solution of sodium hydroxide and a 1 cm3 solution of copper (II) sulphate (1 percent ).

• To create a negative control, repeat the steps outlined above, but this time use de-ionized water. The other is for positive control, which uses albumin or egg white.

• Allow the mixture to stand for about five minutes after shaking it.

• Keep an eye out for any colour changes.

High sensitivity variant of the biuret test:

The bicinchoninic acid (BCA) assay and the Lowry assay are two major modifications of the biuret test that are commonly used in modern colorimetric analysis of peptides. The Cu+ formed during the biuret reaction reacts further with other reagents in these tests, resulting in a deeper colour.

Cu+ forms a deep purple complex with bicinchoninic acid (BCA) in the BCA test, which absorbs around 562 nm and produces the signature mauve colour. The water-soluble BCA/copper complex absorbs much more strongly than the peptide/copper complex, increasing the sensitivity of the biuret test by a factor of 100,the BCA assay detects proteins at concentrations ranging from 0.0005 to 2 mg/mL.

Furthermore, the BCA protein assay has the important advantage of being compatible with substances such as up to 5% surfactants in protein samples.

MoVI in the Folin–Ciocalteu reagent oxidizes Cu+ back to Cu2+ in the Lowry protein assay, forming molybdenum blue (MoIV). Under these conditions, tyrosine residues in the protein also form molybdenum blue. Proteins can be detected at concentrations ranging from 0.005 to 2 mg/mL in this manner. Molybdenum blue, in turn, can bind certain organic dyes like malachite green and Auramine O, resulting in further signal amplification.

Conclusion:

The biuret method is a colorimetric technique that is used to analyse proteins and peptides. In alkaline solution, copper salts form a purple complex with substances containing two or more peptide bonds. The amount of absorbance produced is proportional to the number of peptide bonds that are reacting, and thus to the number of protein molecules present in the reaction system. As a result, the biuret reaction with proteins is appropriate for determining total protein by spectrophotometry (at 540–560 nm).