Figure 1.

H₂O₂ release during the forward and reverse electron transfer. Representative traces of respiration (top) and generation of H₂O₂ (bottom) by intact mitochondria from mouse brain. (a) The reaction was started by addition of mitochondria 0.16 mg of protein/ml to the medium composed of 125 mM KCl, 0.2 mM EGTA, 20 mM HEPES-Tris (pH 7.4), 4 mM KH₂PO₄, 2 mM MgCl₂, 2 mg/ml BSA, 10 μM Amplex UltraRed and 4 U/ml horseradish peroxidase at 37℃. Substrates were either 5 mM pyruvate and 2 mM malate (right) or 5 mM succinate and 1 mM glutamate (left). Black traces correspond to oxygen concentration (top) and Amplex UltraRed fluorescence (bottom). The rate of the change of these signals per minute is shown in red. (b) Release of H₂O₂ during coupled oxidation of 5 mM succinate and 1 mM glutamate as a function of oxygen concentration. 100% rate corresponds to 1400 pmol H₂O₂ × min⁻¹ × mg protein⁻¹. Oxygen concentration as measuring directly by the Oroboros respirometer was rapidly varied by continuously purging the headspace with nitrogen as described in Materials and Methods section.