Excited species generation in horseradish peroxidase-mediated oxidation of glutathione

Abstract

Photoemissive excited species are produced by the horseradish peroxide (HRP)-catalyzed oxidation of reduced glutathione (GSH), without exogenously added hydroperoxide under aerobic conditions. The emitted low-level chemiluminescence consisted of two phases. Ligh emission occured at wavelengths beyond 610 nm (≥90 intensity), indicative of singlet oxygen 1O 2. Deuterium oxide enhanced photoemission 4.4-fold. Ascorbate inhibited chemiluminescence completely. In the absence of GSH or when GSH was replaced by the disulfide, no red chemiluminescence was observed. The glutathionyl radical GS· is most likely to be involved in both phases of light emission. Further, the superoxide radical plays a role, as substantiated by the inhibitory effect of superoxide dismutase. Both phases of photemission were abolished by glutathione peroxidase: thus hydroperoxides are regarded as essential intermediates for the formation of excited species. Catalase abolished phase I and did not affect phase II. In contrast, glutathione S-transferase 1–2 (showing peroxidase activity towards organic hydroperoxides but not towards H 2O 2) inhibited phase II, whereas phase I was still present. Glutathione sulfonate and the disulfide GSSG were detected as oxidation products from GSH under conditions where phase II chemiluminescence was observed. HRP Compound III accumulated during the reduction. It is concluded that phase I is dependent on exogenously added or ednogenously generated H 2O 2, whereas phase II does not require H 2O 2 but an organic peroxy species. A mechanisms based on chain reactions involving oxygen addition to the thiyl radical is proposed. Sulfenyl peroxy Species are suggested as transient intemediates in reactions finally leading to the generation of excited states such as singlet molecular oxygen.