Figure 1. (a) Rates of superoxide production (measured as H₂O₂ production) by isolated rat skeletal mitochondria respiring on succinate and (b) effects of internal (matrix) pH (pH_in) and ΔpH on H₂O₂ production rate.

(a) Standard incubation conditions were 120 mM KCl, 3 mM Hepes, 1 mM EGTA, 0.3% (w/v) BSA (pH 7.2 and 37 °C), 50 μg PHPA·ml⁻¹, 4 units·ml⁻¹ horseradish peroxidase, 30 units·ml⁻¹ SOD and 1.875 μM TPMP⁺. Where shown, 4 mM succinate, 4 μM rotenone, 2 μM piericidin, 2 μM FCCP and 100 nM nigericin were also present. *Significant (P<0.0001) difference from succinate; **significant (P<0.05) difference from succinate+nigericin. Bars show means±S.E.M. of measurements on four to ten separate mitochondrial preparations. (b) Incubation conditions were as for (a), except that the pH of the buffer was adjusted to 6.6–7.4 in steps of 0.2 pH units. Open symbols, nigericin absent; closed symbols, plus 100 nM nigericin. pH_in in the absence of nigericin was determined in parallel incubations using the TPMP⁺ electrode, as described in the Materials and methods section. In the presence of nigericin, pH_in=pH_out. Points represent means±S.E.M. of measurements on four separate mitochondrial preparations.