VII] 



OXIDIZING ENZYMES 



109 



The above conception would represent a system which is capable of 

 absorbing molecular oxygen and of transforming it into active oxygen. 

 In this way substances can be oxidized which would not be affected 

 by molecular oxygen to any extent. 



Accordingly, on the basis of the above hypothesis, it is assumed that 

 an organic compound which can act as a peroxide is present in the 

 expressed plant juices or extracts which give the oxidase reaction. When 

 this organic peroxide is absent, hydrogen peroxide must be added to 

 complete the oxidase system. 



The above definition of oxidases and peroxidases is based upon their 

 behaviour towards guaiacum. There are, however, a number of other 

 oxidizable substances, such as a-naphthol, benzidine, p-phenylenediamine, 

 pyrogallol, etc. : 



NH, 



HoN 



OH NH 2 



a-Naphthol Benzidine p-Phenylenediamine 



which are only oxidized by oxidases with the assistance of hydrogen 

 peroxide, the oxidase system being insufficient ; they are also of course 

 oxidized by the hydrogen-peroxide-peroxidase system. Such substances 

 (in the presence of hydrogen peroxide) are used as tests for oxidizing 

 enzymes. 



Expt. 115. Additional tests for oxidizing enzymes. To a few c.c. of an extract of 

 Potato tuber (Expt. 112) in a small evaporating dish, add a few drops of the following 

 reagents, and subsequently a few drops of hydrogen peroxide : 



(a) A 1 % solution of a-naphthol in 50 / alcohol. A lilac colour is developed. 



(6) A 1 o/o solution of benzidine in 50 % alcohol. A blue colour is developed. 



(c) A 1 % solution of jo-phenylenediamine hydrochloride in water. A greenish 

 colour is developed. 



The peroxidases, like other enzymes, can be extracted either with 

 water or dilute alcohol and precipitated from solution by strong alcohol. 



Expt. 116. Preparation of peroxidase from Horse-radish (Cochlearia) roots. Mince 

 up the Horse-radish roots in a mincing machine. The product is allowed to stand for 

 24 hrs. to enable the glucoside, potassium myronate, to be hydrolyzed by the enzyme, 

 myrosin. Then extract with 80 % alcohol. The alcohol is decanted off, and the 

 residue pressed free from alcohol in a press. The residue is next extracted with 40 % 

 alcohol for 48 hrs., filtered and precipitated with 90% alcohol. The precipitate, 

 which contains the peroxidase, is filtered off. Dissolve up in water and make the 

 test for peroxidases (Expt. 113). 



