A KINETIC-STUDY OF THE ONE-ELECTRON OXIDATION OF TROLOX-C BY THE HYDROPEROXIDASE ACTIVITY OF LIPOXYGENASE

The oxidation of Trolox C (a vitamin E analog) by the hydroperoxidase activity of lipoxygenase was studied. Trolox C was oxidized to its cor responding phenoxyl radical in the presence of hydrogen peroxide, evol ving through a ketodiene intermediate to the Trolox C quinone. The H2O 2/Trolox C quinone molar ratio was 1.0. The overall reaction followed an enzymatic-chemical second-order system and involved a substrate reg eneration mechanism. From the equations derived from this mechanism, t he dismutation constant of the Trolox C radical was evaluated by non-l inear regression as 4.10(5) M-1.s(-1). The accumulation curve of Trolo x C quinone was found to be linear, with no lag period, and dependent on enzyme concentration. No phenoxyl radical was detected when the rea ction was carried out in the presence of ascorbate. This synergistic r eaction between the Trolox C radical and ascorbate was quantitative an d depended on the respective concentrations of enzyme, Trolox C and hy drogen peroxide. The results presented in this paper suggest that the diferences observed in the kinetic behaviour of monophenols (one-elect ron donors) and diphenols (two-electron donors) stem from the fact tha t the latter evolve directly into ferric form without taking the slow pathway once the steady state is reached, whereas the monophenols are always forced take the slow way, even in the steady state. This peroxi dative oxidation of a vitamin E analog by the hydroperoxidase activity of lipoxygenase together with the oxidation produced by dioxygenase a ctivity suggests that lipoxygenase might be a key enzyme in destroying the lipophilic antioxidant barrier against the reactive oxygen specie s in membranes.