PROTEINS IOI 



Rosenheim claims the reaction is due to the presence of oxidizing material in 

 the sulphuric acid and that when pure sulphuric acid is used no reaction is obtained. 

 He advises the use of a slight amount of an oxidizing agent, e.g., ferric chloride or 

 potassium nitrate (0.005 gram per 100 c.c. of sulphuric acid) in order to facilitate 

 the reaction. Rosenheim further states that proteins respond to the formaldehyde 

 reaction because of the presence of the tryptophane group, a statement which Acree 

 does not accept as proven. 



8. Bardach's Reaction. 1 This is one of the most recent tests which have been 

 described for the detection of protein material. The test depends upon the property 

 possessed by protein substances of preventing the formation of typical iodoform 

 crystals through the interaction of an alkaline acetone solution with iodopotassium 

 iodide. Instead of the typical hexagonal plates or stellar formations of iodoform 

 there are produced, under the conditions of the test, fine yellow needles which are 

 apparently some iodine compound other than iodoform. The technic of the test 

 is as follows: Place about 5 c.c. of the protein solution 2 under examination in a test- 

 tube, add 2-3 drops of a 0.5 per cent solution of acetone and sufficient Lugol's solu- 

 tion 3 to supply a moderate excess of iodine and produce a red-brown coloration. 

 (The amount of Lugol's solution necessary will depend upon the content of protein, 

 sugar, and other iodine-reacting substances in the solution under examination and 

 may vary from one drop to several cubic centimeters.) Add an excess (ordinarily 

 about 3 c.c.) of concentrated ammonium hydroxide and thoroughly mix the solu- 

 tion. Place the tube in the test-tube rack, examine the contents at intervals of 

 five minutes, and when it is evident that crystals have formed, place a drop of the 

 mixture upon a microscopic slide, put a cover-glass in position, and examine the 

 mixture under the microscope. The formation of canary yellow crystals indicates 

 the presence of protein material in the solution examined. The crystals are ordi- 

 narily needle-like in appearance and show a tendency to assume rosette or bundle- 

 like formations, but under certain conditions they may show knobbed (nail-like) 

 and branching variations. 



If a moderate excess of iodine is used in making the test, a black precipitate 

 of iodonitro compounds is at once formed upon the addition of the ammonium 

 hydroxide, and yellow needles are subsequently deposited upon it. In case just the 

 proper amount of iodine is used, the solution soon assumes a yellow color and the 

 black precipitate formed upon the addition of the ammonium hydroxide is gradually 

 transformed more or less completely into the yellow crystals. In either case the 

 needles ordinarily form within an hour, and frequently in a much shorter time. 

 If too great an excess of iodine is employed the heavy black precipitate may obscure 

 or even prevent the reaction. The presence of insufficient iodine or excess protein 

 may likewise prevent the reaction. In tests in which a concentrated protein solu- 

 tion and an excess of iodine are used, the addition of ammonium hydroxide im- 

 mediately produces a grayish-green precipitate. In such instances, if the propor- 

 tions are favorable and the mixture be stirred with a glass rod for a few minutes, 

 the precipitate is gradually transformed into the crystals before mentioned. 



It is probable that all soluble proteins will respond to Bardach's reaction, but 

 the relative delicacy of the reaction as well as the value of the test as compared with 



1 Bardach: Zeitschrift fiir Physiologische Chemie, 54, 355, 1908; also Seaman and Gies: 

 Proceedings of the Society for Experimental Biology and Medicine, 5, 125, 1908. 



2 The solution should not contain more than 5 per cent of protein material. 



3 Dissolve 4 grams of iodine and 6 grams of potassium iodide in 100 c.c. of distilled water. 



