Author: Shenzhou
Reviewer: Weiming

  The biuret reaction of proteins is based on a simple principle and is relatively easy to perform. This article explains the reaction, and the procedure is very safe, so anyone with the necessary materials can try it at home.

  First, when urea is heated to 180°C, two urea molecules condense, releasing one molecule of ammonia and forming biuret. The reaction equation is shown below:

  Biuret contains the -CO-NH- group. Under alkaline conditions, it reacts with Cu2+ ions to form a purplish-red chelate. Its structure is shown below:

  At this point, the basis for using this experiment to detect proteins should be clear. Proteins consist of many amino acids linked by peptide bonds, so dipeptides and larger polypeptides contain structures similar to those in biuret. Proteins, as well as dipeptides and larger polypeptides, can therefore react with Cu2+ under alkaline conditions to produce a purplish-red substance, and the intensity of the color is positively correlated with protein content. This also means, however, that substances with similar structures can affect the result. Compounds containing groups such as -CS-NH2, -CH2-NH2, or -CRH-NH2, as well as substances containing excessive ammonium salts, will interfere with the reaction.

  The principle above shows that amino acids cannot undergo this reaction, so it can also be used to determine how extensively a protein has been hydrolyzed.

Detailed Procedure

  ① Place a small amount of urea in a test tube and put a piece of moist litmus paper at the mouth of the tube. Heat it until the paper turns blue, then stop heating after the material in the tube has evaporated to dryness. Once the tube has cooled, use a graduated pipette to add 1 ml of 10% NaOH solution and shake well. Add 2–4 drops of 1% CuSO4, shake again, and observe the change in the solution’s color.

  ② Place diluted egg white in a test tube. Use a graduated pipette to add 1 ml of 10% NaOH solution and shake well. Add 2–4 drops of 1% CuSO4, shake again, and observe the change in the solution’s color. (Required materials: spatula, graduated pipette, pipette bulb, test-tube holder, test-tube rack, urea, NaOH, and CuSO4.)

  P.S. If you carry out the experiment at home, you can use diluted egg white to test for protein. When preparing the biuret reagent, take care not to add too much CuSO4, which would produce blue Cu(OH)2 and interfere with the observed result. If materials permit, you can also try the reaction with amino acids and biuret.