392 ENZYMES 



ISOLATION OF OXIDASES. 



The isolation of oxidase may be a difficult matter when it 

 exists in a tissue together with its substrate and other enzymes. 

 Bourquelot and Bertrand give the following method for Fungi 

 such as Russula. The tissue is chopped up, extracted with 

 water — which may be warmed — and filtered as quickly as may 

 be. The filtrate is then poured into an excess of strong 

 alcohol, whereby the enzyme is precipitated. The precipitate 

 is then filtered off and dissolved in water. 



When the oxidase is extracted together with other oxi- 

 dizing enzymes, separation may be effected, at any rate in part, 

 by adding to the aqueous solution of the oxidase, or to the 

 aqueous extract of the plant, two volumes of absolute alcohol 

 for each volume of extract. The oxidase will be precipitated, 

 whilst the other enzymes will remain in solution.* 



PEROXIDASE. 



Peroxidases which split oxygen from hydrogen peroxide, 

 organic peroxides, potassium permanganate, etc., are often 

 associated with other oxidases, and are very widely distributed ; 

 indeed, they have even been described as occurring in coal.f 



Preparation of Peroxidase. 



Appleman | recommends the following method of pre- 

 paring peroxidase. Potato tubers are grated into a pulp, 

 which is thoroughly mixed with calcium carbonate in order 

 to neutralize any acids. The mass is then ground with quartz 

 sand in a mortar for about two minutes and filtered through 

 butter muslin. The extract contains the peroxidase together 

 with oxidase ; the latter may be removed by raising the tem- 

 perature to 70° for ten minutes, whereby the oxidases are co- 

 agulated, or, according to Gruess,§ any oxidase present may be 

 destroyed by adding acetone which does not affect the peroxi- 

 dase. In many experiments where only a dilute solution of 

 peroxidase is required — i c.c. of extract to 300 c.c. of water 



*Aso: "Bull. Coll. Agric. Imp. Univ., Tokyo," 1902, 5, 207. 



fStoklasa, Ernst and Chocensky : " Ber. deut. bot. Gesells.," 1907, 25, 38. 



t Appleman: " Bot. Gaz.," 1911, 52, 306. 



§ Gruess : " Ber. deut. bot. Gesellr..,'" 1903, 21, 356. 



