A critical reevaluation of some assay methods for superoxide dismutase activity

We have compared the direct method of pulse radiolysis to the indirect methods of cytochrome c and nitroblue tetrazolium for assaying the superoxide activity of a compound. We have shown that with pulse radiolysis, where high concentrations of O₂^- are generated, the "turnover" rate constant, k_cat, can be determined directly, while with the indirect methods, where relatively low steady state concentrations of O₂^- are formedl, the value of k_cat determined by these methods, can be orders of magnitiude lower than that determined directly. The main reason for the lower values obtained with the indirect methods is due to the fast reoxidation of the reduced compound by molecular oxygen. Additional problems which arise with the use of indirect methods for determining superoxide dismutase catalytic activity are discussed.