SUBSECOND TIME-RESOLVED SPIN-TRAPPING FOLLOWED BY STOPPED-FLOW EPR OF FENTON REACTION-PRODUCTS

Rapid spin-trapping kinetics on a subsecond time scale were observed from hydroxyl radicals created by the Fenton reaction where Fe(II)-EDTA and H2O2 were the reagents. Hydroxyl radical was trapped by the commonly used spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to give the spin adduct DMPO-OH. The application of recent loop-gap resonator technology to stopped-flow EPR has given us the time resolution on 10-muL samples to follow rapid kinetics that were formerly only inferred from scavenger competition studies or optically monitored stopped-flow. The sub-100-ms time resolution has helped us focus straightforwardly on initial rates of trapped hydroxyl production and has put limits on any delay before radical onset. We have followed trapped hydroxyl radical production, which is completed within 1 s upon the mixing of Fe(II)-EDTA and H2O2 at approximately 100 muM concentrations. The initial rate of trapped hydroxyl radical production was linearly dependent on both the initial Fe(II)-EDTA and the initial H2O2 concentrations. Thus, as estimated from experimental initial rates of DMPO-OH production, the production of hydroxyl radical that could be explicitly trapped showed a second-order reaction between Fe(II)-EDTA and H2O2 whose second-order rate constant was 3.2 x 10(3) M-1 s-1. Beyond the first 100 ms, transient DMPO-OH formation, which indicated the availability of trappable hydroxyl radical, was limited in time. Empirical fits to the overall time course of DMPO-OH production suggested total second-order rates for the Fenton reaction of greater-than-or-equal-to 10(4) M-1 s-1. An explanation for the difference between this larger rate and the smaller 3.2 x 10(3) M-1 s-1 rate is that the reaction yielding explicitly trapped hydroxyl radical is only one of several that consume H2O2 and Fe(II)-EDTA reactants. Preliminary time-resolved spin-trapping studies were done on a radical created by the Fenton reaction from ethanol to determine that the kinetic behavior of this radical does not simply parallel the production of trapped hydroxyl radical.