Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase.
The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.
Storch
J
J
Max-Planck Institut für Immunbiologie, Freiburg, Federal Republic of Germany.
Ferber
E
E
D000166:Acridines; D008163:Luminescent Measurements; D008861:Microsomes / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*; D019255:NADPH Oxidases; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology*; D012995:Solubility; D013481:Superoxides / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000032:analysis*
D000166:Acridines; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; D011092:Polyethylene Glycols; D017830:Octoxynol; D014969:Xanthine Oxidase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases