282 Report of the Department of Chemistry of the 



EXPERIMENTAL 



This investigation was undertaken as an attempt to determine 

 the truth of the statements made above. In repeating the work of 

 Hammarsten and others a soluble substance which had not been 

 coagulated by rennin and could not be precipitated by dilute acetic 

 acid was always found in the filtrate. Casein solutions for such 

 investigations have been prepared, as a general rule, by shaking 

 pure casein with an excess of lime water or by grinding with moist 

 calcium carbonate. The casein solutions thus obtained were made 

 neutral to litmus and coagulated by the addition of rennin. The 

 curds were filtered off* and the filtrates examined for nitrogen. Sol- 

 uble nitrogen was always found, but the amounts were not constant 

 and seemed to have no relation to the amounts of casein or rennin 

 used. In the control experiments, to which no rennin had been 

 added, similar amounts of nitrogen which could not be precipitated 

 by dilute acetic acid were also found. 



Casemate solutions prepared in the manner described contain 

 basic casemates, either neutral or alkaline to phenolphthalein. As 

 Robertson 6 has shown that such caseinates in solution undergo an 

 autohydrolysis, the following experiment was carried out in order 

 to determine if this might account for the soluble nitrogen found. 



Five grams of casein were dissolved in 250 cc. of ~ calcium 

 hydroxide in the presence of toluol. After complete solution of 

 the casein, portions of the solution were withdrawn at intervals and 

 the casein precipitated by means of dilute acetic acid. The casein 

 was filtered off and the nitrogen in the filtrates determined by the 

 microchemical method devised by Folin. 7 The results are as follows: 



3 



hours 



15 



hours 



24 

 hours 



Milligrams of nitrogen in original solution 



Milligrams of nitrogen not precipitated by rennin . 



158 

 4.0 



158 

 10.0 



158 

 28.8 



Results of the same nature were obtained with solutions made 

 by grinding casein with moist calcium carbonate. The extent of 

 this autohydrolysis, temperature being constant, depends upon time. 

 As dry casein goes into solution very slowly and freshly precip- 

 itated casein is quite rapidly redissolved the following procedure was 

 adopted in order to circumvent autohydrolysis. 



Ten grams of pure dry casein were dissolved in 500 cc. of & cal- 

 cium hydroxide. The casein was then precipitated by adding about 



6 Robertson: Jour. Biol. Chem. 2:344; see also Osborne: Jour. Physiol, 27:398. 



7 Folin and Farmer: Jour. Biol. Chem., 11: 493. All nitrogen determinations 

 reported in this paper were made by this method. 



