64 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



adhere to the sides of the flask and may therefore not be hydrolysed. 

 Even if there be apparent total solution a small amount may escape 

 hydrolysis by becoming enclosed in the " humin " which is formed to 

 a greater or lesser extent. The insoluble material should be filtered 

 off, washed, and tested with the biuret reaction. The absence of the 

 biuret reaction is not an absolute criterion that hydrolysis is complete, 

 for many polypeptides do not show it and are very resistant to 

 hydrolysis. The residue, if large, should therefore be hydrolysed 

 again. Complete hydrolysis should be tested for by Van Slyke's 

 amino-nitrogen method (p. 89). Osborne has found that hydrolysis 

 is sometimes only complete after boiling for two to five days. 



Henriques and Gjaldbak [1910] made an examination, by the 

 formal titration method of Sorensen, of the completeness of hydrolysis 

 of proteins by the action of enzymes and by boiling with acids. Hy- 

 drolysis by enzymes was seldom complete, but complete hydrolysis 

 was effected by boiling for twelve hours with 20 per cent, hydrochloric 

 acid, except in the case of certain proteins. They found that com- 

 plete hydrolysis occurred on heating the protein with 3N hydrochloric 

 acid for one and a half hours in an autoclave at 1 50. The completion 

 of hydrolysis could only be ascertained by testing the solution for in- 

 crease of amino groups whilst at the same time the amount of ammonia 

 must be minimal, i.e., arising only from amide groups and not by 

 deamination of the amino acids. Andersen and Roed-Muller [1915] 

 on account of the fact that more ammonia is slowly formed in a pro- 

 longed hydrolysis are inclined to attribute its origin to uramido groups 

 in the protein molecule. The rate of evolution of x ammonia was found 

 by Skraup and Hardt-Stremayr [1908] to be rapid at first and quite 

 slow subsequently, the greater part being evolved at the beginning of 

 the hydrolysis. Van Slyke [1912, 2] maintained that the maximum 

 amino nitrogen was evolved whether the hydrolysis was effected in an 

 autoclave at 150 or by boiling at 1 00 for forty-eight hours ; there 

 was less tendency for deamination at 100 than at 150. 



Pittom [1914] also found that ammonia is rapidly liberated in the 

 early stages of hydrolysis. Amino-nitrogen is liberated in the same 

 way during hydrolysis by acid, or by enzymes. Caseinogen differs 

 from egg-albumin in its hydrolysis. More amino acids are formed from 

 caseinogen than from egg-albumin in the earlier stages ; the reverse 

 holds good at later stages ; at a still later stage the rate of amino acid 

 formation is the same as in the early stage. There seems to be a defi- 

 nite point at which complex polypeptides are broken down into simpler 



