A kinetic approach to the selection of a sensitive spin trapping system for the detection of hydroxyl radical

The spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) alone, as well as DMPO or N-tert-butyl-alpha-phenylnitrone (PBN) in the presence of excess dimethyl sulfoxide (Me2SO), have been used as spin trapping systems for the detection of hydroxyl radical. However, the instability of DMPO and many of its corresponding spin-trapped adducts has limited the usefulness of this spin trap, particularly in biological systems. Spin trapping of multiple free radicals by the PBN/Me2SO system may undermine the sensitivity of this method to detect small, yet biologically significant amounts of hydroxyl radical. The present study was undertaken to select a spin trapping system with greater sensitivity and selectivity toward.OH than DMPO, DMPO/Me2SO, or PBN/Me2SO. We report that alpha-hydroxyethyl radical, resulting from the reaction of photolytically generated.OH with excess ethanol is spin trapped by 4-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN) with a second-order rate constant nearly 10-fold greater than that for DMPO or PBN. In contrast to DMPO spin-trapped adducts, the alpha-hydroxyethyl radical adduct of 4-POBN, 4-POBN-CH(CH3)OH, is resistant to reduction by superoxide, even in the presence of cysteine. The efficiency of spin trapping and the marked stability of the resulting spin-trapped adduct confer a high degree of sensitivity and demonstrate the potential application of 4-POBN/ETOH toward the detection of hydroxyl radical in biological systems.