Lucigenin-dependent chemiluminescence as a new assay for NAD(P)H-oxidase activity in particulate fractions of human polymorphonuclear leukocytes.
The enzyme responsible for the respiratory burst in human neutrophils is an oxidase that catalyzes the reduction of oxygen to superoxide anion (O-2). Superoxide anion production may be measured by chemiluminescence (CL) in the presence of lucigenin (10,10'-dimethyl-9,9'- biacridinium dinitrate). We established an assay of the oxidase, by measuring the CL of particulate fractions of PMN in the presence of lucigenin . This CL required the addition of NAD(P)H and was very low in fractions of resting cells. In particulate fractions of PMNs stimulated with PMA selectively, the NADPH-dependent CL was found to be increased. CL was linear with protein concentrations up to 100 micrograms and was shown to be at least 10 times more sensitive for the detection of O-2 than the assay based on the spectrophotometric determination of superoxide mediated cytochrome c reduction. CL was abolished by inactivating the enzyme at 56 degrees C.
D000166:Acridines; D000328:Adult; D006801:Humans; D007700:Kinetics; D008163:Luminescent Measurements; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D013347:Subcellular Fractions / Q000201:enzymology; D013482:Superoxide Dismutase / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000166:Acridines; C033472:10,10'-dimethyl-9,9'-biacridinium; D013482:Superoxide Dismutase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate