OXTDASES 395 



greatly impaired, and also that the presence of coagulable 

 proteins interfere with the stability of the peroxidase ac- 

 tivity, besides causing a low yield of enzyme. 



Willstatter and Stoll * have elaborated a process of extrac- 

 tion which in outline is as follows : Five kilos of finely sliced 

 horse-radish are first washed for some days in .running water, 

 to remove soluble substances by dialysis through the cell 

 membranes ; the material is then warmed for a few hours with 

 a 0-4 per cent solution of oxalic acid, whereby the enzyme is 

 precipitated ; the residue is next crushed and washed on a 

 filter with 1 5 litres of very dilute oxalic acid ; it is then 

 ground up with baryta water to liberate the enzyme and the 

 expressed liquid is treated with carbon dioxide to precipitate 

 the barium. The addition of alcohol precipitates slimy sub- 

 stances which are removed and the filtrate is then evaporated 

 to 50 c.c. ; the addition of five times this bulk of alcohol will 

 precipitate the crude enzyme ; the latter is purified by solution 

 in water containing a trace of sulphuric acid and reprecipita- 

 tion with alcohol. The resulting product is a mixture of the 

 enzyme with a nitrogenous glucoside ; the latter forms an in- 

 soluble compound with mercuric chloride, and is precipitated 

 out of solution by this means and the filtrate is once more 

 precipitated with alcohol ; after several more solutions and 

 reprecipitations, the enzyme is obtained free from adhering 

 glucoside. 



The best yield of enzyme from 5 kilos of horse-radish was 

 0-45 gram, which was about 60 per cent of the total enzyme 

 present. 



Identification. 



1. Guaiacum tincture in the presence of oxidase turns 

 blue provided oxygen be present. 



2. In cases where the blueing of the guaiacum tincture 

 does not take place immediately, the addition of hydrogen 

 peroxide may bring it about. 



3. Tetramethyl-/>-phenylenediamine in the presence of 

 hydrogen peroxide gives a deep violet colour with an oxidase. 



* Willstatter and Stoll : " Annalen," 1918, 416, 21. 



