title: Excited species generation in horseradish peroxidase-mediated oxidation of glutathione.
abstract: Photoemissive excited species are produced by the horseradish peroxidase (HRP)-catalyzed oxidation of reduced glutathione (GSH), without exogenously added hydroperoxide under aerobic conditions. The emitted low-level chemiluminescence consisted of two phases. Light emission occurred at wavelengths beyond 610 nm (greater than or equal to 90% intensity), indicative of singlet oxygen 1O2. 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 photoemission 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 H2O2) 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 reaction. It is concluded that phase I is dependent on exogenously added or endogenously generated H2O2, whereas phase II does not require H2O2 but an organic peroxy species. A mechanism based on chain reactions involving oxygen addition to the thiyl radical is proposed. Sulfenyl peroxy species are suggested as transient intermediates in reactions finally leading to the generation of excited states such as singlet molecular oxygen.
mesh_terms: D005609:Free Radicals; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism*; D019803:Glutathione Disulfide; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
chemical_list: D005609:Free Radicals; D013481:Superoxides; C040136:glutathione sulfonate; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases; D005978:Glutathione; D019803:Glutathione Disulfide