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 + Luc2+ ⇔ O2 + Luc•+ is completely shifted to the right (Keq = 106); 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.