Demonstration of cytochrome oxidase activity with diaminobenzidine. A biochemical and electron microscopic study.

Abstract

The relationship between cytochrome oxidase activity assayed biochemically with 3,3'-diaminobenzidine (DAB) as substrate and the resulting electron microscopic staining reaction has been investigated. Mitochondria isolated from both fresh and perfusion-fixed (1% glutaraldehyde and phosphate buffer for 1-2 min) rat liver showed a significant increase in oxygen consumption, as measured by an oxygen electrode, after addition of DAB and were further stimulated by adding cytochrome c. 2,4-Dinitrophenol and antimycin had no effect on the oxidation of DAB; cyanide and azide completely inhibited DAB oxidation, resulting in the absence of any mitochondrial staining reaction. Already after incubation for only 2 min mitochondria exhibited electron-dense deposits on the surface of the inner mitochondrial membrane away from the matrix. The results indicate that DAB donates electrons at the cytochrome c level to the electron transfer system of the respiratory chain and demonstrate the direct relationship between the electron microscopic staining reaction and cytochrome oxidase activity.