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  • Title: Chemiluminescence studies on the generation of oxygen radicals from the interaction of NADPH-cytochrome P-450 reductase with iron.
    Author: Puntarulo S, Cederbaum AI.
    Journal: Arch Biochem Biophys; 1987 Nov 01; 258(2):510-8. PubMed ID: 2823718.
    Abstract:
    The ability of NADPH-cytochrome P-450 reductase to interact with iron and generate oxygen radicals was evaluated by assaying for low level chemiluminescence. The basic reaction system which contained the reductase, an NADPH-generating system, ferric-EDTA as an electron acceptor, and t-butyl hydroperoxide as the oxidant acceptor, resulted in the production of chemiluminescence. Omission of any of these components resulted in a complete loss of chemiluminescence. The light emission was completely sensitive to inhibition by glutathione and butylated hydroxytoluene, partially sensitive (about 60% decrease) to catalase and hydroxyl radical scavengers, and relatively insensitive (about 20% decrease) to superoxide dismutase. The ability of other ferric chelates to replace ferric-EDTA in catalyzing the reductase-dependent chemiluminescence was evaluated. Ferric-citrate, -ADP, -ATP, and ferric-ammonium sulfate were ineffective in promoting chemiluminescence, whereas ferric-diethylenetriaminepentaacetic acid was even more effective than ferric-EDTA. Thus, the ferric chelates, which catalyze reductase-dependent chemiluminescence, are those which are efficient electron acceptors from the reductase and were previously shown to be those capable of catalyzing hydroxyl radical production by microsomes and the reductase. It is suggested that chemiluminescence results from (a) the direct interaction of the reduced iron chelate with the hydroperoxide (Fenton-type of reaction) to generate alkoxyl and peroxyl radicals, and (b) the generation of hydroxyl radicals, which subsequently react with the hydroperoxide to generate secondary radicals. The latter, but not the former, would be sensitive to inhibition by catalase and competitive hydroxyl radical scavengers, whereas both would be sensitive to antioxidants such as butylated hydroxytoluene. Chemiluminescence appears to be a versatile tool for studying the reductase-dependent generation of oxygen radicals and for the interaction of reductase with iron.
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