COBALT ACETYLACETONATE-DIAMINOBENZIDINE REACTION FOR MICROSPECTROPHOTOMETRY OF CYTOCHROME OXIDASE

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The cobalt acetylacetonate-diaminobenzidine (CAA-DAB) reaction for cytochrome oxidase has been examined. The medium consists of 2.5 mM CAA, 2.5 mM DAB・4HCl, 0.25 mg/ml cytochrome c and 0.1 mg/ml catalase dissolved in 0.1 M cacodylate buffer of pH 7.4. Hepatocytes were stained deep blue in granular form due to mitochondrial reaction in rat liver specimens incubated in this medium at 37℃ for 10 min. The reaction was inhibited by KCN, and by 10 min preheating at 80℃. It conformed to Michaelis-Menten's formula and was more sensitive than the color-unmodified DAB reaction processed in the complete medium minus CAA. The CAA-DAB reaction gave the reaction product whose absorption peak locates at 600 nm. The microspectrophotometry, carried out by scanning the specimens at 600 um, gave the following results; 1) the adaptability of Beer's law was proved, 2) a decreasing gradient in cytochrome oxidase activity was found along the radial axis of the hepatic lobule from the periphery to the center, 3) using a standard step wedge as a reference, cytochrome oxidase activity was expressed in terms of DAB equivalent units.
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