Abstract

Lucigenin as a Substrate of Microsomal NAD(P)H-Oxidoreductases

I. A. Schepetkin

Institute of Oncology, Tomsk Research Center, Siberian Branch of the Russian Academy of Medical Sciences, Kooperativnyi Pereulok 5, Tomsk, 634001 Russia; fax: (3822) 224097; E-mail: kirp@flora.tsu.tomsk.su

Received June 25, 1998; Revision received September 20, 1998
NADPH oxidation and cytochrome c reduction with and without lucigenin as well as NAD(P)H/lucigenin-dependent chemiluminescence of rat liver microsomes were studied. An increased rate of NADPH oxidation and cytochrome c reduction in the presence of lucigenin was related to one-electron lucigenin reduction by microsomal NADPH reductases. The apparent Michaelis constant values for lucigenin (K_{m app}^Luc) were 3.6 and 5.0 µM in normoxygenic (p_O2 = 150 ± 5 mm Hg) and 8.7 and 8.3 µM in hypoxygenic (p_O2 = 45 ± 4 mm Hg) media in the reactions of lucigenin-dependent NADPH oxidation and cytochrome c reduction, respectively. The maximal level of NADPH/lucigenin-dependent chemiluminescence was registered at lucigenin concentration close to the mean K_{m app}^Luc in the lucigenin-reductase reaction. Increasing the lucigenin concentration from 5 to 100 µM was associated with a decrease in the chemiluminescence intensity; this could be due to the inactivation of cytochrome P450. In the presence of superoxide dismutase (SOD), the rate of lucigenin-dependent cytochrome c reduction and NADPH/lucigenin-dependent chemiluminescence were decreased by 10 and 30%, respectively. The addition of lucigenin to microsomes which contain the reduced hemoprotein--CO complex was followed by the disappearance of the differential absorption spectrum specific for the carboxy complex and by increase in chemiluminescence intensity versus the control (without carboxy complex). Thus, lucigenin-dependent chemiluminescence of microsomes may be due to some enzymes including lucigenin reductase (NADPH-cytochrome P450 reductase, NADH-cytochrome b₅ reductase), generation of O₂^-. in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of the cytochrome P450 heme on dioxetane decomposition followed by light quantum emission. Thus, lucigenin cannot be used to measure the basal O₂^-. formation in tissue homogenates with high levels of NAD(P)H-oxidoreductases.
KEY WORDS: chemiluminescence, lucigenin, cytochrome P450 reductase, cytochrome b₅ reductase, cytochrome P450