Assay of Cytochrome Oxidase Activity of Sunflower Leaf Tissue in Relation to pH Value and Cation Concentration of the Buffer.

Cytochrome oxidase probably occurs in the tissues of most plants and mediates a major portion of terminal respiration. Webster (11, 12) recently demonstrated the presence of a cytochrome oxidase in the tissues of 54 plant species, representing 23 families of dicotyledonous plants. Investigations in the plant physiology laboratory at Rutgers University have indicated the presence of a cytochrome oxidase in sunflower, tomato, soybean, and wheat leaf, and in dodder (Cuscuta subinclusa) tissues. There is evidence that a low or negligible cytochrome oxidase activity reported by some investigators for a number of plant species is at least partly due to the use of buffers at a molarity and a pH which are not optimum for the activity of this enzyme. It has been well established by Howell and Sumner (6) for urease, by Massey (7) for fumarase, and by Gilbert and Swallow (5) for Q-enzyme that the pH optimum for maximum activity is dependent upon ion concentration of the reaction mixture, and that definition of the pH optimum or ion concentration alone does not satisfactorily characterize an enzyme reaction. Smith and Stotz (9) reported that the rate of oxidation of ferrocytochrome c by rat liver, heart, kidney, and intestine homogenates increased slightly from pH 6.5 to 7.0. Bertrand and Gavard (1) found the optimum pH of beef heart cytochrome oxidase to be 8.0. Quinlan-Watson and Dewey (8) reported a pH optimum of 7.1 to 7.3 for various animal tissues. Ducet and Rosenberg (4) reported a pH optimum of 7.4 and a phosphate buffer concentration of about 0.05M for potato leaf particulate material. Cooperstein, Eichel, and Wainio (2) found the optimum for beef heart cytochrome oxidase to be about 7.0. In a later study, Wainio et al (10) reported that the rate of oxidation of ferrocytochrome c is markedly influenced by the molar concentration of the Na2HPO4-KH2PO4 buffer used. Maximum activity of the beef heart cytochrome ccytochrome oxidase system was found to increase and the optimum molarity of the buffer to decrease as the pH was changed from 7.4 to 6.2. Wainio and his coworkers in more recent, unpublished work, have demonstrated that desoxycholate preparations of beef heart cytochrome oxidase assayed spectrophotometrically show maximum activity in a phosphate buffer of pH 6.0 with a cation concentration of 0.112 M. They found that the activity of the enzyme is not re-