REVERSION OF HYDROLYSIS 429 



solution of the products of the hydrolysis of protamin, to which 

 no ferment had been added, yielded no trace of protamin after 

 standing for several months. 



My experiments were conducted as follows: Four hundred cc. 

 of N/10 potassium hydroxide were neutralized (or rendered 

 faintly acid) to litmus by the addition of casein and the solu- 

 tion was subjected to the action of pepsin for a considerable 

 period, fresh pepsin (^ g. per litre) being added from time to time, 

 at 40° C. The digest at the end of this period, contained no 

 casein, but a considerable quantity of paranuclein had been 

 precipitated and had not undergone hydrolysis. The digest was 

 then heated to 100 degrees for about 10 or 15 minutes to destroy 

 the ferment, and filtered while hot. The solution was then 

 evaporated on a water-bath to about 70 cc. (the total concen- 

 tration of the system having been thus multiplied by about 6). 

 This concentrated solution of the products of the peptic digestion 

 of casein (and its first product of hydrolysis, paranuclein) is a 

 clear brown syrup which is strongly acid and gives no precipitate 

 or opalescence upon the addition of acetic acid, or upon the 

 addition of acetic acid in excess after previously rendering the 

 solution alkaline by the addition of KOH or NaOH. Both casein 

 and paranuclein are therefore absent from it. To 70 cc. of this 

 solution were added 30 cc. of a concentrated (approximately 

 10 per cent) solution of Gruebler's pepsin puriss. sice, which had 

 previously been filtered through rapid-filtering paper. The 

 mixture of the two solutions is a clear brown syrupy fluid which 

 yields no trace of a precipitate with acetic acid either before or 

 after neutralization with alkali. The mixture was set aside at 

 40° C. in the presence of excess of toluol to prevent bacterial 

 infection. Within two hours a thick white precipitate had formed 

 in the solution; after 48 hours the solution was filtered and the 

 precipitate thus collected on the filter was dissolved in a minimal 

 amount of sodium hydroxide and the filter so arranged that the 

 alkaline solution dropped directly into water acidified with acetic 

 acid. The precipitate thus obtained was reprecipitated twice 

 and was washed by decantation several times, employing several 

 litres of water at each decantation. Finally it was collected on 

 a hardened filter, washed with several litres of alcohol and ether, 

 and dried, first over CaCl2 and then over H2SO4 at 60° C. In 

 this way 1.02 grams of a friable greyish-white hygroscopic powder 



