[57] Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria

Publisher Summary

The determination of the rate of O₂^- production is based upon the spectrophotometric measurement of oxidation or reduction reactions in which O₂^- is a reactant. The concentration of the spectrophotometric indicator that reacts with O₂^- is adjusted to compete effectively with the spontaneous dismutation of O₂^- so that nearly all O₂^- produced can be detected. The involvement of O₂^- is ascertained by the use of superoxide dismutase that inhibits the reaction rate specifically due to O₂^-. Cyanide, which is often used as inhibitor of the mitochondrial cytochrome oxidase (K_i about 3 x 10^-5 M) is also an inhibitor of the often used copper-containing superoxide dismutase (K_i about 3 × 10^-4 M). It is possible to use enough cyanide that partially inhibits cytochrome oxidase without inhibiting superoxide dismutase. Alternatively, bacterial or mitochondrial (manganese-containing) superoxide dismutase can be used. Mitochondria have Mn-superoxide dismutase in the matrix space, so to measure the total production of O₂^- by mitochondrial membranes, the dismutase should be inhibited or removed. Because effective inhibitors of Mn-superoxide dismutase are not known, matrical superoxide dismutase is usually removed by the repetitive washing of submitochondrial particles obtained by sonication or by other means. Mitochondrial O₂^- production is pH dependent and increases toward the alkaline region.