Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase

• Published in: Analytical biochemistry Vol. 169; no. 2; pp. 262 - 267
• Main Authors: Storch, J., Ferber, E.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.03.1988; Elsevier
• Subjects: Acridines; Analytical, structural and metabolic biochemistry; Biological and medical sciences; chemiluminescence; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; lucigenin; Luminescent Measurements; macrophage; Microsomes - enzymology; NADH, NADPH Oxidoreductases - analysis; NADPH oxidase; NADPH Oxidases; Octoxynol; Oxidoreductases; Polyethylene Glycols - pharmacology; Solubility; superoxide anion; Superoxides - metabolism; xanthine oxidase; Xanthine Oxidase - analysis; macrophage; xanthine oxidase; superoxide anion; chemiluminescence; NADPH oxidase; lucigenin; Amplification; Assay; Enzyme; Non ionic surfactant; Xanthine oxidase; Chemiluminescence; Microsome; Macrophage
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(88)90283-7

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O 2, 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 O 2 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 O 2 −. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid and inhibited by Ca 2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a K m value of 1.35 μ m, which decreased to 0.95 μ m after the addition of 12% ( g g ) Triton X-100. The K m and V max 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.