18. Singlet Oxygen Dependent Hydroxyl Radical Formation during Uroporphyrin-Photosensitization in the Presence of NADPH
Keizo Takeshita, Claudio A. Olea-Azar Michiko Mizuno and Toshihiko Ozawa
Keywords: singlet oxygen, hydroxyl radical, NADPH, photosensitization, uroporphyrin
The conversion of singlet oxygen (1O2) to hy droxyl radical ( OH) during photosensitization of uroporphyrin (UP) in the presence of NADPH was examined by a spin-trapping technique using 5,5dimethyl-1-pyrroline-N-oxide (DMPO). Significant 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 resulted from freely diffusing OH produced during irradiation of UP and NADPH, but not from decay of the O2- adduct of DIMPO. The ESR signal almost completely disappeared when quenehers of 1O2 were added, and it was enhanced when the amount of deutrated solvent was increased. The appearance of 1O2 was deter mined by the oxidation of 2,2,6,6-tetramethyl-4 piperidone (TEMPD) to corresponding nitroxyl radical, 2,2,6,6 tetramethyl 4 piperidone N oxyl (TEMPON) . The ESR signal of TEMPON increased with an increase of irradiation time in the absence of NADPH. This signal significantly decreased when irradiation was performed in the pres ence of quenchers of 1O2 such as NaN .3 and Lhistidine although OH scavengers, catalase, and SOD had less or no effect, indicating that oxidation of TEMPD to TEMPON was specific to 1O2 under this condition. The presence of NADPH delayed the appearance of TEMPON signal. The lag time increased with an increase in the concentration of NADPH. On the other hand, formation of DMPO-OH increased with the concentration of NADPH, when DMPO was used as a spin trapping agent instead of TEMPD. These observations implied that conversion of 1O2 to ' OH occurred quickly in the presence of NADPH. Hydrogen peroxide (H2O2) was produced 1O2dependently during irradiation of UP in the presence of NADPH. However neither catalase nor desferrioxamine decreased the DMPO-OH signal, and addition of H2O2 did not increase the signal. SOD slightly increased the signal. These results suggested that the production of OH from 1O2 involved neither superoxide anion radical nor H2O2.
Publication:
Takeshita, K., Olea-Azar, C. A., Mizuno, M., and Ozawa, T. : Antioxidants and Redox Signaling, 2 355362 2000.