Probing the Hydroxyl Radical-Mediated Reactivity of Peroxynitrite by a Spin-Labeling Fluorophore

The decomposition of peroxynitrite at physiological pH yielded a hydroxyl radical, which reacted rapidly with dimethyl sulfoxide (DMSO) to produce a methyl radical (·CH3), which was then trapped by a spin-label fluorophore nitroxide-linked naphthalene (NTEMPO), a carbon-centered radical probe with a low fluorescence intensity, and transformed to a stable diamagnetic O-alkoxyamine, a high-fluorescence compound. The fluorescence increment was proportional to the concentration of the hydroxyl radical, and then to the concentration of peroxynitrite. NTEMPO therefore was demonstrated to be capable of detecting hydroxyl radicals generated from peroxynitrite, and the method was proved to be simple and sensitive. The hydroxyl radical-mediated reactivities of peroxynitrite to several amino acids, such as tyrosine, phenylalanine and histidine, were then evaluated by the spin-labeling fluorophore NTEMPO at pH 7.4 and 37°C. The obtained data were in good agreement with the reference values, respectively.