Hydroxyl radical detection by ESR secondary spin-trapping with DMPO and by fluorescent method with TPA during UV irradiation of N-OHAAF. (a and b) ESR spectra of adducts of DMPO with ^•OH (a) and ^•CH₃, ^•CH(CH₃)OH, ^•COO^- and ^•N₃ (b) were observed when DMPO incubated with N-OHAAF was exposed to UV irradiation without (a) or with (b) DMSO, ethanol, formate and azide, respectively. (c and e) Both fluorescence intensity of 2-OH-TPA and ESR signal intensity of DMPO/^•OH were in a time-dependent and concentration-dependent manner. (d) Fluorescence of 2-OH-TPA was observed when TPA incubated with N-OHAAF was exposed to UV irradiation. The excitation and emission wavelength is 310 and 425 nm, respectively. (f) Fluorescence intensity of 2-OH-TPA was inhibited by DMSO and ethanol. Reaction mixtures contained 0.1 mM N-OHAAF (or changed as referred); DMPO (100 mM), NHPT (1 mM) and H₂O₂ (4 mM) for ESR detection and TPA (5 mM), NHPT (0.1 mM), Fe(II)-EDTA (0.1 mM) and H₂O₂ (1 mM) for fluorescence detection. Other regents including DMSO, ethanol, formate and azide were added as referred in the chart. All reactions were conducted with the compound in PB (100 mM, pH 7.4) under dark or UV irradiation (10.5 mW/cm² at 313 nm) for 1 min before ESR detection or UV irradiation (1.5 mW/cm² at 313 nm) for 10 min before fluorescence detection at room temperature. Each sample was determined by ESR or fluorescent methods at least in triplicate, and the SD was less than 5%.