New insights into the detection of sulfur trioxide anion radical by spin trapping: radical trapping versus nucleophilic addition

Abstract

It has recently been demonstrated that (bi)sulfite (hydrated sulfur dioxide) reacts with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) via a nonradical, nucleophilic reaction, and further proposed that the radical adduct (DMPO/ SO 3 −) formation in biological systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical ( SO 3 −). Here, the one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase/H 2O 2 has been reinvestigated by ESR spin trapping with DMPO and oxygen uptake studies to obtain further evidence for the radical reaction mechanism. In the case of ESR experiments, the signal of the DMPO/ SO 3 − radical adduct was detected, and the initial rate of its formation was calculated. Support for the radical pathway via SO 3 − was obtained from the stoichiometry between the amount of consumed molecular oxygen and the amount of (bi)sulfite oxidized to sulfate (SO 4 2−). When DMPO was incubated with (bi)sulfite, oxygen consumption was completely inhibited owing to the efficiency of DMPO trapping. In the absence of DMPO, the initial rate of oxygen and H 2O 2 consumption was determined to be half of the initial rate of DMPO/ SO 3 − radical adduct formation as determined by ESR, demonstrating that DMPO forms the radical adduct by trapping the SO 3 − exclusively. We conclude that DMPO is not susceptible to artifacts arising from nonradical chemistry (nucleophilic addition) except when both (bi)sulfite and DMPO concentrations are at nonphysiological levels of at least 0.1 M and the incubations are for longer times.