MICRODETERMINATION OF CYTOCHROME OXIDASE IN RAT TISSUES BY THE OXIDATION OF N-PHENYL-p-PHENYLENEDIAMINE OR ASCORBIC ACID

Abstract

The cytochrome oxidase content of rat tissues was investigated, utilizing the accumulation of the free radical formed by the emzymatic, univalent oxidation of a stable, non-toxic substrate, N-phenyl-p-phenylenediamine (p-aminodiphenylamine). This procedure eliminated the non-enzymatic coupling of the radical with α-naphthol, found in the classic Nadi reaction. Using homogenates, the following decreasing order of enzyme activity was found: heart, kidney, diaphragm, liver, rectus abdominis. A more sensitive assay was also developed, involving the reduction of cytochrome c by ascorbic acid, and using 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride to analyze unoxidized ascorbic acid. The same order of tissue activity was demonstrated. The cytochrome oxidase content of a tissue may be a reflection of its physiological role.