Figure 4. Effect of H₂O₂ on intermembrane space Cu,Zn-superoxide dismutase activation.

(A) Mitochondria were incubated with the indicated concentrations of H₂O₂ for 5 min at 37 °C, washed with sucrose/Tris buffer, pH 7.4, and incubated with digitonin in the presence of IAM for 1 h at room temperature. Cu,Zn-superoxide dismutase activity was assayed in the mitochondrial intermembrane space preparation by following the reduction of XTT at 470 nm, and was expressed as percentage of fully activated enzyme prepared by rupturing the outer membrane with digitonin alone. (B) Mitochondria were incubated with indicated concentrations of H₂O₂ for 5 min at 37 °C. Intermembrane space Cu,Zn-superoxide dismutase activity was then measured spectrophotometrically in intact mitochondria following XTT reduction at 470 nm. Reactions were carried out in the sucrose/Tris buffer consisting of 20 m-units/ml xanthine oxidase, 50 μM xanthine and 750 μM XTT. Where indicated, intact mitochondria were added at a concentration of 100 μg of protein/ml.