Singlet Oxygen-Dependent Hydroxyl Radical Formation during Uroporphyrin-Mediated Photosensitization in the Presence of NADPH

Abstract

The conversion of singlet oxygen (¹O₂) to hydroxyl radical (^.OH) during photosensitization of uroporphyrin (UP) in the presence of NADPH was examined by a spin-trapping technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Significant electron spin resonance (ESR) signals of DMPO-OH adduct were observed during irradiation of the UP-NADPH system with visible light. Scavengers of ^.OH reduced the signal intensity to 3–30% of control, indicating that more than 70% of DMPO-OH results from freely diffusing ^.OH. The ESR signal was almost completely lost when quenchers of ¹O₂ were added, and was enhanced when the amount of deutrated solvent was increased. The appearance of ¹O₂, as determined by the oxidation of 2,2,6,6-tetramethyl-4-piperidone (TEMPD), was delayed with an increase in the concentration of NADPH, whereas the production of ^.OH was upregulated. These observations indicate that conversion of ¹O₂ to ^.OH occurs quickly in the presence of NADPH. Hydrogen peroxide (H₂O₂) was produced ¹O₂-dependently during irradiation of UP in the presence of NADPH. However, neither catalase nor desferoxamine decreased the DMPO-OH signal, and addition of H₂O₂ did not increase the signal. SOD increased the signal only slightly. These results suggest that the production of ^.OH from ¹O₂ involves neither superoxide anion radical nor H₂O₂.