OXIDASES 503 



PEROXIDASE. 



Peroxidases which set free active or atomic oxygen from 

 hydrogen peroxide, or organic peroxides, are very widely 

 distributed, being, according to Wheldale-Onslow almost 

 universally present in the higher plants, indeed, they have 

 even been described as occurring in coal.* 



Preparation of Peroxidase. 



A crude preparation of horse-radish peroxidase f may be 

 obtained by mincing the material and setting it aside for 

 twenty-four hours in order to allow the myrosin to destroy 

 the sinigrin. The material is then extracted for some days 

 with three changes of 80 per cent alcohol and filtered. The 

 residue is washed with strong alcohol, pressed dry, and ex- 

 tracted with 40 per cent alcohol in which the peroxidase is 

 soluble ; on adding a mixture of three parts of alcohol and 

 one part of ether to the filtrate, the peroxide is precipitated. 



Willstatter and his fellow-workers % have elaborated a 

 method for obtaining a highly purified enzyme ; for this 

 purpose the horse-radish root is left in running water to remove 

 dialysable impurities ; it is then treated with dilute oxalic 

 acid whereby the enzyme is precipitated upon the protein ; 

 the peroxidase is then removed from the protein by elution 

 with alkali and is then successively adsorbed on to kaolin, 

 which removes carbohydrates, including a very tenacious 

 glucoside, and alumina, and is subsequently precipitated by 

 tannin ; a further treatment with alumina, followed by pre- 

 cipitation with alcohol, completes the process. 



In order to check the activity of the enzyme preparations 

 during the course of purification, they are allowed to act upon 

 an aqueous solution of pyrogallol in the presence of hydrogen 

 peroxide ; at the end of a given interval the reaction is stopped 

 by the addition of acid and the purpurogallin formed by 

 oxidation of the pyrogallol is extracted by means of ether and 

 estimated colorimetrically. The " purpurogallin number " is 

 * Stoklasa, Ernst, and Chocensky : " Ber. deut. bot. Gesells.," 1907, 



t Bach : " Ber. deut. chem. Gesells.," 1904, 37, 3787. 

 t Willstatter and StoU : " Annalen," 1918, 416, 21 ; 1920, 422, 47 ; 

 Willstatter and Pollinger : id., 1923, 430, 269. 



