succinate + rotenone succinate + rotenone
+ antymicin a
Figure 16.1. Effects of inhibitors of the respiratory chain on the rate of H2O2 generation of rat heart mitochondria. (A) Addition of rotenone (complex I inhibitor) to pyruvate/malate-supplemented rat heart mitochondria increases H2O2 production. (B) Addition of antimycin A (complex III inhibitor) to succinate (+rotenone)-supplemented rat heart mitochondria increases H2O2 production.
instead of reducing oxygen to water at cytochrome oxidase. If mitochondrial H2O2 production and oxygen consumption are measured in parallel using the same incubation medium, temperature and concentrations of substrates and modulators in each mitochondria preparation, the free radical leak can be calculated. Since two electrons are needed to reduce one molecule of oxygen to H2O2, whereas four electrons are needed to reduce one molecule of oxygen to water, the free radical leak is easily calculated by dividing the rate of ROS production by two times the rate of oxygen consumption, the result being multiplied by 100.
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