ESR spin-trapping studies on the reaction of Fe2+ ions with H2O2-reactive species in oxygen toxicity in biology.

Using ESR spin-trapping techniques with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we confirmed the 1:1 stoichiometry for the formation of hydroxyl radicals with Fe2+ in the Fenton reaction under experimental conditions wherein [H2O2] is 90 microM and [Fe2+] is very low, 1 microM or less. The stoichiometry decreased markedly as the Fe2+ concentration was increased. The efficiency of hydroxyl radical generation varied with the nature of the iron chelators used and increased in the order of phosphate alone approximately ADP less than EDTA less than diethylenetriaminepentaacetic acid (DETAPAC). The second order rate constant for the Fenton reaction was measured to be 2.0 x 10(4) M-1 s-1 for phosphate alone, 8.2 x 10(3) M-1 s-1 for ADP, 1.4 x 10(4) M-1 s-1 for EDTA, and 4.1 x 10(2) M-1 s-1 for DETAPAC. Measuring the radicals formed as spins trapped in the presence of ethanol, we estimated the amount of total oxidizing intermediates formed in the Fenton reaction, which we concluded consists of hydroxyl radicals and an iron species. The oxidizing species of iron which might be assigned as ferryl, FeO2+, or Fe(IV) = O was generated effectively in the presence of ADP even at low Fe2+ concentrations. In general, as the Fe2+ concentration was increased, the ferryl species predominated over the hydroxyl radical except for the case of Fe(II)-DETAPAC, which generated only hydroxyl radicals as the oxidizing species. Three possible pathways are proposed for the Fenton reaction, the dominant ones depending very much on the nature of the iron chelator being used.