12 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



fe) Determination of the Completion of Hydrolysis. 



The completion of the hydrolysis has usually been ascertained by 

 performing the biuret test on the solution and on any residue remain- 

 ing when the time of hydrolysis is completed. Further hydrolysis is 

 required if the biuret test is positive. Many proteins are hydrolysed 

 with great slowness and, as has been pointed out by Osborne and 

 Jones [1910, 3], boiling with acids for a much longer time than stated 

 above is necessary ; in the case of caseinogen, Osborne and Guest 

 [1911] found that hydrolysis with concentrated hydrochloric acid was 

 only complete after three to five days. 



The biuret test is not altogether a satisfactory way of determining 

 the completion of hydrolysis. It is necessary to determine whether 

 an increase of ammonia and amino nitrogen occurs on a further period 

 of six or eight hours' boiling. This increase can be estimated either 

 by Van Slyke's amino nitrogen method or by Sorensen's method of 

 titration with formalin. 



(i) Van S fykes Amino Nitrogen Method. 



The hydrolysis is carried out in a tared flask under a reflux con- 

 denser ; after six or eight hours the heating is discontinued. A portion 

 of I or 2 c.c. is removed with a pipette and diluted to 10 c.c. The 

 amino nitrogen is estimated in this solution as described on pp. 89-96 ; 

 the reaction is allowed to proceed for five minutes in the stationary 

 apparatus and subsequently shaken for one minute. The flask in 

 which the hydrolysis is carried out is weighed, and the boiling con- 

 tinued for another six or eight hours. The flask is again weighed to 

 ascertain if the solution has become more concentrated ; water may 

 be added to replace the loss or a correction made for the decrease in 

 volume. The hydrolysis is continued until the amino nitrogen is 

 constant. 



(ii) Sorenseris Titration Method with Formalin. 



Sorensen [1907] proved that amino acids could be accurately 

 estimated in solution by titration with alkali after they had been treated 

 with neutralised formalin. The reaction of the amino acid with 

 formalin results in the formation of a methylene compound, the neutral 

 amino acid becoming a free carboxylic acid : 



CH 2 .NH 2 CH 2 .N=CH 2 



+ OHCH = | + H 2 O 



COOH COOH 



