COLOR REACTIONS OF THE PROTEINS 107 



3. Color Reactions of the Proteins. a. Millon's Reaction. To about 

 5 c.c. of a dilute solution of egg albumin in a test-tube add a few drops of 

 Millon's reagent. A white precipitate forms which turns red when heated. 

 This test can be used with advantage on solid proteins. In this case the 

 reagent is added to suspensions of the solid substance. Such proteins as are 

 not precipitated by the mineral acids yield a red solution instead of a red 

 precipitate. Millon's reagent consists of one part mercury dissolved in two 

 parts by weight of concentrated nitric acid; the resulting solution is diluted 

 with two volumes of water. 



This reaction is due to the presence of the hydroxyphenyl group or 

 C 6 H 5 OH in the protein molecule. Accordingly, certain non-protein sub- 

 stances give this reaction; i.e., tyrosin, phenol (carbolic acid), thymol, etc. 

 The reaction 'given by the protein is due to the presence of the amino acid 

 tyrosin, and it is evident that the test is really an indication of the presence 

 of the tyrosin complex in the protein molecule. 



b. Xanthoproteic Reaction. To 2-3 c.c. of egg- albumin solution or of 

 some dry casein in the test-tube, add concentrated nitric acid and heat until 

 the protein dissolves, forming a yellow solution. Cool the solution and care- 

 fully add ammonium hydroxide in excess. The yellow 7 color changes to an 

 orange. This reaction is due to the presence in the protein molecule of the 

 phenyl group in phenyalanine, tyrosine, or tryptophane; with the phenyl 

 group nitric acid forms certain nitro-derivatives of benzene. 



c. Adamkiewicz or Hopkins-Cole Reaction. Mix a couple of c.c. of con- 

 centrated sulphuric acid with 4 or 5 c.c. of glacial acetic acid in a test-tube. 

 Add a few drops of egg-albumin solution and warm gently. A reddish- 

 violet color is produced. This reaction is due to the presence of trypto- 

 phane in the protein and the test depends on the presence of glyoxylic acid 

 (CHOH 2 COOH) in the acetic acid. The Hopkins-Cole reagent, a glyoxylic 

 acid solution, may be used instead of the glacial acetic acid. The reagent 

 is prepared by adding sodium amalgam to a saturated solution of oxalic 

 acid and allowing the mixture to stand until the evolution of gas ceases. 

 In making the test the protein solution and Hopkins-Cole reagent are mixed 

 thoroughly in a test-tube, and concentrated sulphuric acid poured gently 

 into the tube which has been inclined somewhat so that it forms a layer in 

 the bottom of the test-tube. The acid and protein solutions will be strati- 

 fied and a reddish-violet ring is developed where the two fluids come in 

 contact. 



This reaction is due to the presence of tryptophane in the protein mole- 

 cule. Gelatin does not respond to this test, for it does not yield this sub- 

 stance as a cleavage product. 



d. Liebermanri* s Reaction. Add a few drops of egg-white solution or a 

 little dry casein to about 5 c.c. of concentrated hydrochloric acid in a test- 

 tube and boil the mixture until a pinkish-violet color results. It was for 



