O₂⁻was introduced, at a constant rate, into buffered aqueous solutions, either by mechanical infusion of KO₂, dissolved in tetrahydrofuran, or by the in situ action of xanthine oxidase on xanthine plus oxygen. This O₂⁻ was allowed to react with ferricytochrome c or with tetranitromethane and the formation of the reaction products, ferrocytochrome c or nitroform, respectively, was monitored spectrophotometrically. That concentration of Superoxide dismutase, which competed equally with given levels of cytochrome c or tetranitromethane and which thus caused 50% inhibition of the rates of accumulation of ferrocytochrome c or of nitroform, was determined. The rate constant for the enzymatic dismutation of O₂⁻ by the copper and zinc containing enzyme from bovine erythrocytes was then calculated from the known rate constants for the reaction of O₂⁻ with ferricytochrome c and with tetranitromethane and was found to be 2 × 10⁹m⁻¹ sec⁻¹ at pH 7.8 and 8.5. This rate constant was obtained at steady-state concentrations of O₂⁻ in the 10⁻⁸m → 10⁻¹³m range and is in full agreement with the results of pulse radiolytic investigations which were performed at O₂⁻ concentrations in the 10⁻⁵m range. The second order rate constant for the enzymatic dismutation of O₂⁻ is thus independent of the concentration of O₂⁻ in the range 10⁻⁵ → 10⁻¹³m.

Several distinct types of Superoxide dismutase have been described. These include the mangano-enzymes from Escherichia coli and from chicken liver mitochondria and the iron-enzyme from E. coli. The rate constants for the dismutations catalyzed by these enzymes have also been investigated as a function of pH.