The use of histochemical reagents for cytochrome oxidase in plant tissues

Abstract

The histochemical demonstration of cytochrome oxidase is based on the formation of a colored dye which appears at the site of enzyme activity upon oxidation of the histochemical reagent used. Positive reactions may result from the action of any oxidizing agent which is capable of performing this oxidation. There may be many such unspecific reactions in plant tissues, as exemplified by the many factors that can be involved in a positive G-Nadi reaction. Oxidation of this reagent has been considered to be due, in addition to the cytochromec-cytochrome oxidase complex, to: 1) the phenol-polyphenol oxidase system (Belval & Laeuffeur 1946, Suzuki 1957), 2) peroxidase in the presence of peroxides (Lipetz 1965a), 3) autoxidation accelerated by metal ions (Person & Fine 1961), 4) quinones (Suzuki 1957), 5) fatty peroxides (Gomori 1952), 6) diffusion of the spontaneously formed oxidation product, indophenol blue, into lipids (Lison 1960), and 7) aldehydes (Van Fleet 1952). Although rate and localization of a positive reaction may give an indication of the factor by which the reaction is brought about, it will be necessary to consider each of these factors in order to reach a valid conclusion concerning the localization and activity of cytochrome oxidase. In addition to the many unspecific, positive reactions that may occur with this reagent, negative results may be obtained even in the presence of cytochrome oxidase. This may be due to lack of penetration of the reagent into cells, to reduction of indophenol blue to a leucoderivative by dehydrogenases (Lison 1960), and possibly by other reducing substances, or too low an enzyme activity to be detected by the method.