Enzymes 157 



indophenol blue under the conditions of the nadi M reaction 

 can be explained satisfactorily by the presence of fat perox- 

 ides in the myeloid granules. The simultaneous presence of 

 aldehydes would greatly accelerate the reaction.**^ The in- 

 hibition by cyanide, often quoted as an evidence of the en- 

 zymatic nature of the reaction, actually does not prove any- 

 thing. Cyanide is known to poison many inorganic catalysts; 

 moreover, it may act simply as a powerful reducer. In the 

 model experiments mentioned, cyanide completely inhibited 

 the staining of the paper slips and very greatly slowed down 

 the spontaneous oxidation of the nadi mixture in air. 



This interpretation of the reaction renders the arguments 

 meaningless as to whether the distribution of the reaction 

 does or does not correspond to the true localization of the 

 enzyme.*^ 



One version of the enzymatic theory, however, cannot be 

 refuted with certainty. Although the reaction will take place 

 even in the absence of any enzyme, a uniquely hardy per- 

 oxidase, capable of utilizing fat peroxides, might be present 

 and contribute its catalytic eflFect. 



Methods 



1) For cytochrome oxidase {labile or G nadi reaction).— 

 Use small fragments of fresh tissue or razor-blade slices. 

 Frozen sections of fresh unfixed tissue can also be used, al- 

 though freezing may cause partial inactivation of the en- 

 zyme. To about 25 ml. of a phosphate buflFer of pH 7.2-7.6 

 add 1-2 ml. of a 1 per cent solution of alpha-naphthol in 40 

 per cent alcohol and the same amount of a 1 per cent solu- 

 tion of dimethylparaphenylenediamine ( p-aminodimethyl- 

 anilin) hydrochloride. Incubate in a shallow dish for about 

 10 minutes or until the reaction is clearly visible. Rinse sec- 



45. Feigl, F.: Qualitative analysis by spot tests (New York: Elsevier Pub- 

 lishing Co., 1946), p. 345. 



46. Dietrich, A.: Zentralbl. f. allg. Path. u. path. Anat., 19:3, 1908; 

 Hollande, A. C: Compt. rend. Acad, sc, 178:1215, 1924; Prenant, M.: 

 Bull, d'histol. apphq. a la physiol., 2:329, 1925. 



