Direct evidence for apo B-100-mediated copper reduction: Studies with purified apo B-100 and detection of tryptophanyl radicals

Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by end ogenous components of low-density lipoprotein (LDL) represent critical step s in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated fro m cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has b een studied extensively, we developed a model to specifically analyze the p otential copper reducing activity of its protein moiety (apo B-100). Apo B- 100 was isolated after solubilization and extraction from size exclusion-HP LC purified LDL. We obtained, for the first time, direct evidence for apo B -100-mediated copper reduction in a process that involves protein-derived r adical formation. Kinetics of copper reduction by isolated apo B-100 was di fferent from that of LDL, mainly because apo B-100 showed a single phase-ex ponential kinetic, instead of the already described biphasic kinetics for L DL (namely alpha -tocopherol-dependent and independent phases). While at ea rly time points, the LDL copper reducing activity was higher due to the pre sence of alpha -tocopherol, at longer time points kinetics of copper reduct ion was similar in both LDL and apo B-100 samples. Electron paramagnetic re sonance studies of either LDL or apo B-100 incubated with Cu(II), in the pr esence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the for mation of protein-tryptophanyl radicals. Our results supports that apo B-10 0 plays a critical role in copper-dependent LDL oxidation, due to its lipid -independent-copper reductive ability. (C) 2000 Academic Press.