314 CHEMISTR Y OF THE DIGESTIVE PROCESSES. 



Method of mechanical precipitation. When an indifferent precipitate 

 is produced in a solution containing an enzyme, this is often carried 

 out of solution with the precipitate, probably in a condition of mechanical 

 adhesion. This observation was made by Briicke, who utilised the 

 property to free pepsin as far as possible from other substances. The 

 method has been extended to the preparation of purified forms of other 

 enzymes, and, as applied by Briicke * to pepsin, may be quoted as an 

 example of a general method. It is as follows : 



The mucous membrane of a pig's stomach is submitted to partial self- 

 digestion, in water acidulated with phosphoric acid ; the products of this 

 first digestion are rejected, being too rich in products of digestion, and not 

 containing much pepsin, which clings in^reat part to the mucous membrane. 

 The residue of the mucous membrane is again digested in water made acid 

 with phosphoric acid, and after some days is filtered from insoluble residue, 

 and just neutralised by the addition of lime water. The insoluble calcium 

 phosphate so precipitated carries down with it all the pepsin ; it is collected on 

 a filter paper, just dissolved by cautious addition of very dilute hydrochloric 

 acid, filtered off, and once more precipitated by the addition of just sufficient 

 lime water. This double precipitation is to free the pepsin of proteid, which 

 also has the property of being mechanically carried down, though more feebly 

 than pepsin. To this somewhat purified solution of pepsin a solution of 

 cholesterin in four parts of alcohol and one part of ether is added. On this 

 solution mixing with the water the cholesterin becomes insoluble, and is thrown 

 out of solution in a finely divided condition, carrying the pepsin mechanically 

 adhering to it just as it did to the calcium phosphate. The mixture is well 

 shaken up, and then filtered ; the precipitate is washed first with water, then 

 with water acidulated with acetic acid, and finally with water alone. It is 

 next, without drying, shaken up with ether, free of alcohol, but saturated 

 with water. The ether extracts the cholesterin, while the pepsin remains in 

 the watery layer beneath ; the extraction is repeated with fresh portions of 

 ether until all the cholesterin has been removed, and finally the watery 

 solution containing the pepsin is filtered. In this manner a solution is 

 obtained, which actively peptonises, but contains so little proteid as not to give 

 many of the proteid reactions. 



Method of auto-digestion. Kiihne and Chittenden 2 have combined 

 auto-digestion with precipitation by ammonium sulphate as a means of 

 preparing purified solutions of pepsin and trypsin. The following is an 

 outline of their methods : 



For the preparation of pepsin the mucous membrane of a pig's stomach is 

 taken, and allowed to undergo auto-digestion for several days, until peptonisa- 

 tion has far advanced, and but comparatively little albumose is left. The 

 solution, after filtration from undigested debris of nuclein, etc., is next satu- 

 rated with ammonium sulphate. The pepsin is thus completely thrown out 

 of solution along with the albumoses ; this precipitate is dissolved again, after 

 pressing in filter paper, in dilute hydrochloric acid, and allowed to go on 

 finishing the digestion of the albumoses for some days. 



The process is repeated as often as is necessary to remove the albumose, 

 and finally the pepsin, after being dissolved by addition of water, is freed from 



1 Sitzungsb. d. k. Akad. d. Wissensch., Wien, 1862, Bd. xliii. S. 601 ; " Vorlesungen ii. 

 Physiologic," Wien, 1885, S. 308. See also v. Heltzl, Jahresb. it. d. Fortschr. d. ges. Med., 

 Erlangen, 1864, Bd. i. S. 138. 



2 Ztschr. f. Biol., Miinchen, 1886, Bd. xxii. S. 428. Such a method of auto-digestion 

 can obviously only be employed in the case of proteoly tic enzymes. 



