Evidence for a concerted reaction between lipid hydroperoxides and polypeptides.

The events accompanying oxidative modification of low density lipoprotein (LDL) are multiple and complex, and the precise mechanisms remain to be determined. In the present studies, we examined a simple system in which we first prepared large amounts of lipid hydroperoxides (from linoleic acid or from phospholipids containing linoleic acid) by using soybean lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12). Linoleoyl hydroperoxide was then incubated with polypeptides in the absence of metal ions. We observed the generation of fluorescent products with a spectrum like that of oxidized LDL. The generation of fluorescent products from incubation of polypeptides with linoleoyl hydroperoxide was manyfold greater than that generated on incubation with preformed 4-hydroxynonenal at the same concentration. Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) had no effect on the generation of fluorescent products. Incubation of linoleoyl hydroperoxide with cytochrome c (cyt c) under the same conditions led to progressive reduction of cyt c at a rate determined by the initial linoleoyl hydroperoxide concentration. This reduction was not significantly inhibited by probucol but was inhibited, although never completely, by superoxide dismutase. Even at 100 micrograms/ml, superoxide dismutase inhibited by only 65%. From these results, we are led to suggest a concerted reaction between the peroxy radical and free amino groups of polypeptides or phosphatidylethanolamine to generate fluorescent adducts. During oxidation of LDL or of cell membranes, this mechanism may occur side by side with the conventional Schiff base mechanism.