One- and Two-Electron Oxidations of Luminol by Peroxidase Systems

The kinetics of luminol oxidation catalyzed by horseradish peroxidase (HRP), Arthromyces ramosus peroxidase (ARP) and lactoperoxidase (LPO) at pH 7.0 was investigated. One-electron oxidation of luminol by peroxidase systems was inferred from the detection of luminol radicals, luminol-mediated formation of ascorbate radicals, and the trapping of luminol-mediated GSH radicals. The catalytic intermediate of peroxidases in the steady state was Compound II and the rate constants of HRP, ARP, and LPO Compound II with luminol were 3.6 × 104, 1.1 × 107, and 2.5 × 104 M−1s−1, respectively. The intensity of luminol chemiluminescence (CL) generated by the peroxidases depended on the rate constants of the rate-determining step. The luminol CL catalyzed by peroxidases increased with an increase in the concentration of H2O2 and was inhibited in the presence of catalase. Neither oxygen consumption during the reaction under aerobic conditions nor a change of light intensity under anaerobic conditions was observed. The light emission and oxidation of luminol catalyzed by LPO was increased by trace amounts of iodide. LPO catalyzes two-electron oxidations of iodide to form iodinating intermediate (Nakamura, M.; et al. J. Biol. Chem. 260:13546–13552, 1985),[1] which subsequently oxidizes luminol. The results lead us to conclude that CL of luminol was initiated by peroxidase systems irrespective of one- or two-electron oxidations of luminol.