PROTEINS 99 



4. Biuret Test. To 2-3 c.c. of egg albumin solution in a test-tube add an 

 equal volume of concentrated potassium hydroxide solution, mix thoroughly, 

 and add slowly a very dilute (2-5 drops in a test-tube of water) copper sulphate 

 solution until a purplish-violet or pinkish-violet color is produced. The depth 

 of the color depends upon the nature of the protein; proteoses, and peptones 

 giving a decided pink, while the color produced with gelatin is not far removed 

 from a blue. 



This reaction is given by those substances which contain two amino 

 groups in their molecule, these groups either being joined directly 

 together or through a single atom of nitrogen or carbon. The amino 

 groups mentioned must either be two CONH 2 groups or one CONH 2 

 group and one CSNH 2 , C(NH)NH 2 or CH 2 NH 2 group. It follows 

 from this fact that substances which are non-protein in character but 

 which contain the necessary groups will respond to the biuret test. 

 As examples of such substances may be cited oxamide, 



CONH 2 



CONH 2 



and biuret, 



CONH 2 



NH. 



/ 

 CONH 2 



The test derives its name from the fact that this latter substance which 

 is formed on heating urea to i8oC. (see page 392) will respond to the 

 test. Protein material responds positively since there are two CONH 2 

 groups in the protein molecule. 



According to SchifF the end-reaction of the biuret test is dependent 

 upon the formation of a copper-potassium-biuret compound (cupri- 

 potassium biuret or biuret potassium cupric hydroxide). This sub- 

 stance was obtained by Schiff in the form of long red needles. It has 

 the following formula: 



OH 



K NH 2 -CO 



I 

 OH OH 



