PROTEASES 373 



ISOLATION OF THE ENZYME. 



The methods followed in isolating these enzymes differ in 

 details according to the material used ; the principle, however, 

 is the same in most cases. The enzyme is precipitated from 

 its solution by reagents, usually alcohol, filtered off, and washed 

 with alcohol. It may be partly purified by dissolving in water 

 and re-precipitating with alcohol. Following are some methods 

 which have been pursued in particular cases. 



To isolate the enzymes from the fluid contained within 

 the pitchers of Nepenthes, Vines * added to the liquid an equal 

 volume of absolute alcohol, then phosphoric acid followed by 

 lime water in order to increase the bulk of the precipitate. 

 Ammonium carbonate was added until the liquid gave a 

 neutral reaction, and the precipitate filtered off. For use, 

 the precipitate was shaken up with a '2 per cent solution of 

 hydrochloric acid and filtered ; the clear filtrate actively digests 

 fibrin. 



If it be desired to examine the contents of a tissue for 

 these ferments, the expressed juice may be used, or an aqueous 

 extract, the enzyme being separated as above if necessary. 

 But sometimes this is unsatisfactory for various reasons a 

 syrup-like consistency or high coloration, for example. In 

 such cases the tissues may be bruised in a mortar and placed 

 with water in the vessel in which the experiment is to be 

 carried out, together with the material fibrin, for example 

 to be acted upon.f Buscalioni and Fermi { used sterilized 

 gelatine, with -5-1 per cent carbolic acid as an antiseptic, in a 

 Petri dish. Fragments of the tissue to be tested are placed 

 upon the jelly ; the liquefaction of the gelatine in the neigh- 

 bourhood of the pieces indicates the presence of proteolytic 

 enzymes, but inasmuch as all proteases do not attack gelatine, 

 a negative result does not necessarily indicate the absence of 

 these enzymes. 



Dean prepared ereptase from the seeds of beans by ex- 

 tracting the cotyledons with water, filtering, and half saturating 

 the filtrate with ammonium sulphate. The precipitate thus 

 obtained is filtered off, dissolved in water and separated from 



* Vines: "Ann. Bot.," 1897, n, 573. 



t Vines: id., 1903, 17, 237, 597- 



Buscalioni and Fermi: "Ann. R. Inst. Bot. Roma," 1898, 7, 99. 



Dean : "Bot. Gaz.," 1905, 39, 321, 



