Colorimetric microdetermination of cytochrome c oxidase.

The Nadi reaction, discovered by Ehrlich (1885), has been used for many years as a test for indophenol oxidase. The reaction consists in the formation of indophenol blue from dimethyl-p-phenylenediamine and cr-naphthol and is given by most animal and plant cells. It is now generally held that indophenol oxidase is identical with cytochrome c oxidase, and the Nadi reaction is often employed for the histochemical detection of cytochrome oxidase. During an attempt to determine cytochrome oxidase in cellular fractions which could be prepared only in small amounts, tests were made in order to learn whether the Nadi reaction would permit the quantitative determination of micro amounts of cytochrome oxidase. Mitochondrial suspensions from the kidney of the rat were used as a source of the enzyme. Unexpectedly, the formation of indophenol blue by small aliquots of mitochondrial suspensions containing 10 to 60 y of total N was found to be proportional to the concentration of enzyme and to the time of incubation during the first few minutes. These observations seemed to make it worth while to study the reaction in more detail and to establish optimal conditions for a quantitative test.