60 ENZYMES OR SOLUBLE FERMENTS. 



calcium phosphate is obtained to which all the pepsin is adherent. 

 The precipitate is now filtered off, dissolved in a minimal amount of 

 dilute hydrochloric acid and again precipitated by the addition of lime- 

 water; this second precipitation frees the pepsin largely from the 

 proteids which were at first carried down with it. This second pre- 

 cipitate is now as before dissolved in dilute hydrochloric acid. From 

 this the pepsin is separated as follows. Cholesterin is dissolved in a 

 mixture of four parts of alcohol and one of ether, and this solution is 

 introduced below the solution of pepsin by means of a long thistle- 

 tube. As soon as the cholesterin comes in contact with the water it 

 separates out and the separation is completed, as a finely granular 

 mass, by violently shaking the vessel in which the mixture is con- 

 tained. The pepsin adheres now to the cholesterin, which is filtered 

 off, washed first with water faintly acidulated with acetic acid and 

 finally with pure water. On treating the mass with pure ether in a 

 separating-funnel the cholesterin goes into solution in the ether which 

 forms an upper layer, below which is an aqueous solution of pepsin, 

 which must be shaken up several times with renewed portions of ether 

 until all the cholesterin has been extracted. The aqueous solution of 

 the enz3 r me thus obtained is exposed to the air until it is free from 

 ether, and is then filtered. It may be further purified by dialysis, 

 and is now found to give none of the reactions characteristic of pro- 

 teids, and to be precipitable only by the acetates of lead. It yielded 

 no trace of opalescence on the addition of tannic acid, though this is 

 capable of detecting one part of proteid in 100,000 of solvent. 1 



From the reactions of the pepsin solution obtained by Briicke's 

 method, it seems justifiable to consider that the enzyme is not 

 really a proteid. The same conclusion may be deduced from the 

 more recent investigation of Sundberg. 2 No analyses of purified 

 pepsin appear to have been made as yet, so that the views as to 

 its non-pro teid nature are based solely upon the reactions of its 

 solutions as described by Briicke and Sundberg, reactions which, 

 as already pointed out, are not really conclusive. 



Preparation of peptic digestive fluids. If a few drops of a 

 glycerine extract of gastric mucous membrane be added to dilute 

 (2 p. c.) hydrochloric acid, or if the tissue be simply extracted for 

 a short time with the dilute acid and the extract be filtered, a 

 solution is obtained which suffices for demonstration and ordi- 

 nary purposes. 3 When however a peptic extract is required for 

 research purposes it is essential to adopt some more elaborate 

 method which yields a product as free as possible from admixed 

 substances ; one of the best is that of Maly. 4 The mucous mem- 

 brane is digested, as in Briicke's method, with phosphoric acid 

 and the fluid precipitated with lime-water. The precipitate of 



1 Hofmeister, Zt. f. physiol. Chem. Bd. 11. (1878), S. 292. 



2 Zt. f. physiol. Chem. Bd. ix. (1885), S. 319. But see Low, Pfliiger's Arch. Bd. 

 xxxvi. (1885), S. 170. 



3 See also Kiihne and Chittenden, Zt. f. Biol Bd. xix. (1883), S. 184. 



4 Pfliiger's Arch. Bd. ix. (1874), S. 592. 



