98 PHYSIOLOGICAL CHEMISTRY 



examination is strongly alkaline it should be neutralized inasmuch as 

 the alkali will precipitate yellow or black oxides of mercury. 



2. Xanthoproteic Reaction. To 2-3 c.c. of egg albumin solution in a test-tube 

 add concentrated nitric acid. A white precipitate forms, which upon heating 

 turns yellow and finally dissolves, imparting to the solution a yellow color. 

 Cool the solution and carefully add ammonium hydroxide, potassium hydroxide, 

 or sodium hydroxide in excess. Note that the yellow color deepens into an 

 orange. 



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

 phenyl group C 6 H 5 , with which the nitric acid forms certain nitro 

 modifications. The particular complexes of the protein molecule 

 which are of especial importance in this connection are those of tyrosine, 

 phenylalanine, and tryptophane. The test is not a satisfactory one for 

 use in urinary examination because of the color of the end-reaction. 



3. Glyoxylic Acid Reaction (Hopkins-Cole). 1 Place 1-2 c.c. of egg albumin 

 solution and 3 c.c. of glyoxylic acid, CHO.COOH + H 2 O or CH(OH) 2 COOH, 

 solution (Hopkins-Cole reagent 2 ) in a test-tube and mix thoroughly. In a second 

 tube place 5 c.c. of concentrated sulphuric acid. Incline the tube containing sul- 

 phuric acid and by means of a pipette allow the albumin-glyoxylic acid solution 

 to flow carefully down the side. When stratified in this manner a reddish-violet 

 color forms at the zone of contact of the two fluids. 



This color is due to the presence of the tryptophane group. Gelatin 

 does not respond to this test. For formula of tryptophane see page 

 77. Benedict 3 has suggested a new reagent for use in carrying out 

 the Hopkins-Cole reaction. 4 Nitrates (NaNO 3 and KN0 3 ) entirely 

 prevent the reaction whereas formaldehyde or nitric acid interfere 

 somewhat. 5 



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, THJT 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 



1 Hopkins and Cole: Journal of Physiology, 27, 418, 1902. 



2 Hopkins-Cole reagent is prepared as follows: To one liter of a saturated solution of 

 oxalic acid add 60 grams of sodium amalgam and allow the mixture to stand until the 

 evolution of gas ceases. Filter and dilute with 2-3 volumes of water. 



3 Benedict: Journal of Biological Chemistry, 6, 51, 1909. 



4 Benedict's modified Hopkins-Cole reagent is prepared as follows: Ten grams of pow- 

 dered magnesium are placed in a large Erlenmeyer flask and shaken up with enough dis- 

 tilled water to liberally cover the magnesium. Two hundred and fifty c.c. of a cold, satur- 

 ated solution of oxalic acid is now added slowly. The reaction proceeds very rapidly and 

 with the liberation of much heat, so that the flask should be cooled under running water 

 during the addition of the acid. The contents of the flask are shaken after the addition of 

 the last portion of the acid and then poured upon a filter, to remove the insoluble magnesium 

 oxalate. A little wash water is poured through the filter, the filtrate acidified with acetic 

 acid to prevent the partial precipitation of the magnesium on long standing, and made up to 

 a liter with distilled water. This solution contains only the magnesium salt of glyoxylic 

 acid. 



6 Mathewson: Dissertation (Columbia Univ.), Eschenbach Publishing Co., Easton, Pa., 

 1912. 



