1. The enzyme–substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H2O2 indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H2O2 generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H2O2. Succinate, the most effective substrate, yields H2O2 at a rate of 0.5nmol/min per mg of protein in state 4. H2O2 generation is decreased in the state 4→state 3 transition. 4. In the combined mitochondrial–peroxisomal fraction of rat liver the changes in the mitochondrial generation of H2O2 modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H2O2 at a rate (8.6–16.4nmol/min per mg of protein) that corresponds to 42–61% of the rate of uric acid oxidation. Addition of azide increases these H2O2 rates by a factor of 1.4–1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H2O2 (up to 1.7nmol/min per mg of protein) at a ratio of 0.71–0.86mol of H2O2/mol of NADP+ during the oxidation of NADPH. H2O2 is also generated (6–25%) during the microsomal oxidation of NADH (0.06–0.025mol of H2O2/mol of NAD+). 8. Estimation of the rates of production of H2O2 under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H2O2/min per g of liver at 22°C serves as a crude approximation to evaluate the biochemical impact of H2O2 on cellular metabolism.

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