1. The enzyme-substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H(2)O(2) 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 H(2)O(2) generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H(2)O(2). Succinate, the most effective substrate, yields H(2)O(2) at a rate of 0.5nmol/min per mg of protein in state 4. H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) (up to 1.7nmol/min per mg of protein) at a ratio of 0.71-0.86mol of H(2)O(2)/mol of NADP(+) during the oxidation of NADPH. H(2)O(2) is also generated (6-25%) during the microsomal oxidation of NADH (0.06-0.025mol of H(2)O(2)/mol of NAD(+)). 8. Estimation of the rates of production of H(2)O(2) under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H(2)O(2)/min per g of liver at 22 degrees C serves as a crude approximation to evaluate the biochemical impact of H(2)O(2) on cellular metabolism.

A B S T R A C T The extinction of fluorescence of scopoletin during its oxidation by horseradish peroxidase (HPO) provides a highly sensitive and specific assay for small quantities of peroxide in solution. With this assay, the release of free H2sO into the extracellular medium by phagocytizing human granulocytes has been documented and quantitated, and some of the regulating factors have been determined. Under basal conditions granulocytes released less than 0.01 nmol/ml of H20. (2.5 X 10' polymorphonuclear leukocytes/ml). Upon the addition of phagocyte particles (latex, opsonized yeast, or staphylococci), an abrupt increase in extracellular peroxide concentration was observed (>50-fold above basal levels) after latencies as short as 10 s. Release reflected increased intracellular H202 production during phagocytosis in that it paralleled the respiratory burst and was absent when phagocytosis was prevented or when cells from patients with chronic granulomatous disease were utilized. Evidence that scopoletin oxidation occurred predominantly in the extracellular medium was obtained by demonstrating a marked inhibition when HPO was omitted from the reaction mixture or when exogenous catalase was added. Similarly, it was found that exogenous serum also inhibited scopoletin oxidation, apparently because of the presence of competing hydrogen donors.H202 formation and release were observed at rates which closely paralleled those of phagocytosis. With O2 consumption as an approximate index of H202 formation, the fractions released during maximal rates of Portions of this work were presented at the Eastern and National

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