C. Batthyany et al., Direct evidence for apo B-100-mediated copper reduction: Studies with purified apo B-100 and detection of tryptophanyl radicals, ARCH BIOCH, 384(2), 2000, pp. 335-340
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.