Temperature-dependent free radical reactions were investigated using nitroxyl radicals as redox probes. Reactions of two types of nitroxyl radicals, TEMPOL (4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl) and carbamoyl-PROXYL (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl), were tested in this paper. Heating a solution containing a nitroxyl radical and a reduced form of glutathione (GSH) caused temperature-dependent decay of electron paramagnetic resonance (EPR) signal of the nitroxyl radical. Heating a solution of the corresponding hydroxylamine form of the nitroxyl radical showed EPR signal recovery. The GSH-dependent reduction of nitroxyl radicals at 70°C was suppressed by antioxidants, spin trapping agents, and/or bubbling N(2) gas, although heating carbamoyl-PROXYL with GSH showed temporarily enhanced signal decay by bubbling N(2) gas. Since SOD could restrict the GSH-dependent EPR signal decay of TEMPOL, O(2) (•-) is related with this reaction. O(2) (•-) was probably generated from dissolved oxygen in the reaction mixture. Oxidation of the hydroxylamines at 70°C was also suppressed by bubbling N(2) gas. Heating a solution of spin trapping agent, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) showed a temperature-dependent increase of the EPR signal of the hydroxyl radical adduct of DMPO. Synthesis of hydroxyl radical adduct of DMPO at 70°C was suppressed by antioxidants and/or bubbling N(2) gas. The results suggested that heating an aqueous solution containing oxygen can generate O(2) (•-).
Keywords: electron paramagnetic resonance; hyperthermia; nitroxyl radical; reactive oxygen species; redox probe.