6] Assay for singlet-oxygen generation by peroxidases using 1270-nm chemiluminescence

Abstract

Publisher Summary Singlet oxygen ( l Δ g ) represents one possible product of systems composed of a peroxidase, hydrogen peroxide, and a halide. Both heme-containing peroxidases and nonheme vanadium bromoperoxidases can produce singlet oxygen. Peroxidases can also oxidize iodide anion. However, with hypoiodous acid, the reaction is endothermic and singlet oxygen is not produced. To distinguish singlet oxygen from a variety of other reactive products generated by peroxidases, including radicals and various oxidized halide species, a technique with extreme selectivity is required. The measurement of singlet oxygen phosphorescence at 1270 nm is one such technique. Even in complex biochemical systems, chemiluminescence near 1270 nm from other excited species is extremely uncommon. Singlet oxygen ( l Δ g ) is an electronically excited oxygen molecule with 23 kcal per mole more energy than ground-state oxygen. This energy can be released as a 1270-nm photon, but the transition, which requires an electron spin change, is not allowed by the rules of quantum mechanics. Thus, the radiative rate is low. Most deactivating collisions of singlet oxygen with water molecules generate heat via a nonradiative transition. The lifetime of singlet oxygen in water is only 3.1μsec. The modest singlet oxygen production rates found in peroxidase systems and the short singlet oxygen lifetime result in low steady-state singlet oxygen concentrations. The combination of a low steady-state concentration and of a low radiative rate causes the 1270-nm emission from peroxidase systems to be very weak. Thus, measuring the weak 1270-nm emission from singlet oxygen generated by peroxidases would appear to represent a significant technical challenge, but modern solid-state near-infrared detectors have made the measurement of singlet oxygen production by various biochemical systems straightforward.