Lucigenin is a mediator of cytochrome c reduction but not of superoxide production

The relevance of lucigenin (bis-N-methylacridinium nitrate) amplified chemiluminescence (CL) as a specific assay for superoxide ion has recently been disputed (S. I. Liochev and I. Fridovich, Arch. Biochem. Biophys. 337, 115-120, 1997). These authors suggested that the redox cycling of lucigenin can lead to the formation of additional amount of superoxide ion. However, thermodynamic consideration shows that the equilibrium for the reaction O-2(.-) + Luc(2+) double left right arrow O-2 + Luc(.+) is completely shifted to the right (K-eq = 10(6)); therefore, the redox cycling of lucigenin is of no importance. This conclusion is supported by the study of the effects of lucigenin on cytochrome c reduction by xanthine oxidase. It was found that lucigenin did enhance the rate of cytochrome c reduction with xanthine as a substrate, but it did not increase the rate of xanthine oxidation. When NADH was used as a substrate, lucigenin inhibited the SOD-dependent component of cytochrome c reduction and enhanced both the SOD-independent cytochrome c reduction and NADH oxidation, being a sole acceptor of an electron from the enzyme. All these findings indicate the extremely low probability of lucigenin redox cycling. In our opinion, lucigenin-amplified CL remains the most sensitive and highly specific test for superoxide formation in biological systems. (C) 1999 Academic Press.