JOHN H. NORTHROP 241 



filtered off. The solution was then evaporated in vacuum to 60 cc. 1 cc. of 

 this solution contained the equivalent of 0.7 gm. of gelatin and had a formol 

 titration of 16 cc. of 0.1 n NaOH. 



Preparation from Casein. — 200 gm. of commercial casein were dissolved in 3 

 liters of water and precipitated by the addition of sulfuric acid. The precipitate 

 was washed in water, suspended in 1 liter of water and heated to boiling. Ba(0H)2 

 was then added until the supernatant liquid had a pH of 9.0. The solution was 

 cooled and 5 cc. dialyzed trypsin added and the solution kept at 23° for 10 days. 

 It was then filtered, the filtrate titrated to pH 6.3 with sulfuric acid, evaporated 

 in vacuum to 100 cc, and precipitated by the addition of 800 cc. of 95 per cent 

 alcohol. A gummy precipitate forms which consists of higher products which 

 are still acted upon by trypsin. The filtrate is evaporated in vacuo to a thick 

 syrup to remove the alcohol and taken up in 50 cc. water. The solution so 

 obtained is a clear yellowish syrup. It is not further acted upon by trypsin and 

 contains no active trypsin. The formol titration per cubic centimeter is equiva- 

 lent to 15 cc. of 0.1 N NaOH. This solution was then accurately adjusted to 

 pH 6.3 with HCl and to a specific conductivity of 2 X 10~^ reciprocal ohms by 

 the addition of a small amount of KCl. The experiments described in this paper 

 were all made with the solution prepared in this way from casein. It is referred 

 to as inhibiting solution or inhibitor. 



Properties of the Inhibiting Solution. 



It had already been stated that no effect could be noted if amino- 

 acids were added to the trypsin, unless very much higher concentra- 

 tions were used than could be furnished by digestion of the protein. 

 Glycine, alanine, tryptophane, leucine, tyrosine, arginine, prohne, 

 and histidine were tested alone and in combination. It was also 

 found that gelatin or casein which had been completely hydrolyzed 

 by either acid or alkali was without effect. The results of some of 

 these experiments are shown in Table IV. The experiment shows 

 that the inhibiting substance is not formed in the acid or alkali hydroly- 

 sis of gelatin or casein, and that it is dialyzable. The evidence is 

 not sufficient to prove that the inhibiting substance is a specific result 

 of trypsin hydrolysis since the acid and alkali hydrolyses were only 

 tested when the reaction had continued far beyond the stage reached 

 by trypsin hydrolysis. It may be mentioned, however, that it has 

 been found by an entirely different method that the hydrolysis of 

 gelatin by acids or alkalies or trypsin in the early stages follows a 

 different course and must give rise to different products in each 



