Mendel and Underhill — Papa'in-digestion. 11 



his pupils, and more recently by Fr. Alexander. 1 The latter employed 

 the method of fractional precipitation introduced by E. P. Pick 2 for 

 the albumoses. We have followed their scheme of analysis quite 

 closely, and refer to the papers of the writers mentioned for the 

 details of the method. The separation of the individual caseoses was 

 made in the neutralized and somewhat concentrated digestion filtrates, 

 after removal of the characteristic albumose-like substance which has 

 already been referred to as precipitating when heat is applied. 

 Instead of reproducing our protocols at length, we give an outline of 

 one of several experiments with casein and then add a brief resume 

 of the main results ascertained from all the trials. 



Experiment A. In this experiment li kilos of moist casein obtained from 

 skimmed milk and purified by re-precipitating three times were treated with 

 2£ liters of 0.25 per cent. Na 2 C0 3 ,4 grams of papain A and strong alcoholic 

 thymol solution. The mixture was kept at 38° C. for 11 days. Daring this inter- 

 val portions had repeatedly been withdrawn and examined for leucin, tyrosin and 

 tryptophan (as described on page 9) with negative results. Therefore 4 grams of 

 papain were again added. After digesting for 7 days longer, during which time 

 samples had again been withdrawn and examined for leucin, etc., with negative 

 outcome, the material was filtered and neutralized with acetic acid, whereupon 

 a very slight precipitate was obtained. The filtrates were then concentrated as 

 already indicated, until they contained about ten per cent, of dissolved substance. 

 On treatment of the carefully neutralized fluid with saturated ammonium sul- 

 phate solution, Fraction I. , which began to be precipitated when a content of 

 2.6 c.c. of saturated ammonium sulphate solution in a total volume of 10 c.c. 

 was reached, was completely separated when 6 c.c. of the sulphate solution were 

 present. In a large portion of digestion material this fraction was then precipi- 

 tated by mixing ten volumes of the digestive solution with nine volumes of 

 ammonium sulphate solution (following Alexander), 3 and after standing, this frac- 

 tion was filtered off completely. In this filtrate the lower limit of precipitation 

 was found to be 5.1 c.c, and the upper limit at 6.7 c.c. of ammonium sulphate 

 solution. Fraction II. was then separated from a larger quantity of the original 

 material by adding one volume of it to three volumes of saturated ammonium sul- 

 phate solution. For this filtrate obtained therefrom, lower and upper precipitation 

 limits of 7.8 c.c, and about 9.5 c.c of ammonium sulphate solution respectively 

 were ascertained. Fraction III. was therefore removed by saturating the remainder 

 of the original digestion material with ammonium sulphate crystals and filter- 

 ing after some hours. When the salt-saturated fluid thus obtained was further 

 treated with ^ s n sulphuric acid (saturated with ammonium sulphate) a precip- 

 itate, Fraction IV., separated. It was relatively large in quantity and was 

 removed by adding one-half volume of the salt-saturated acid to the entire fluid. 

 The filtrate still gave a strong biuret reaction, indicating the presence of pep- 



1 Alexander : Zeitschrift fur physiologische Chemie, 1898, xxv, p. 411. 



2 Pick : Zeitschrift fur physiologische Chemie, 1897, xxiv, p. 246. 



3 Alexander : Zeitschrift fur physiologische Chemie, 1898, xxv, p. 418. 



