Effect of probucol on the physical properties of low-density lipoproteins oxidized by copper

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Abstract
Human plasma low-density lipoproteins (LDL) were incubated with 10 .mu.M probucol for 1 h at 37.degree. C. Probucol incorporation into the LDL was complete as judged by filtration through a 0.2-.mu.m filter, ultracentrifugation, and gel filtration. LDL with and without probucol were incubated for up to 24 h with 5 .mu.M Cu2+ at 37.degree. C. Copper oxidation increased the content of random structure in the LDL protein from 30% to 36% at the expense of .beta.-structure (which decreased from 22% to 16%) without a change in .alpha.-helical content as measured by circular dichroism spectroscopy. This loss of .beta.-structure was prevented by the presence of probucol in the LDL during the copper incubation. Probucol reduced the rate of increase of fluorescence during copper oxidation at 37.degree. C. After 6 h, the fluorescence intensity at 360-nm excitation and 430-nm emission was 30% less in probucol-containing samples. Probucol had no effect on the circular dichroic spectrum of LDL and only minimal effects (< 5%) on the fluorescence emission spectrum at wavelengths below 500 nm. Two fluorescence peaks, with emission at 420 nm and excitation at 340 and 360 nm, are resolved in three-dimensional fluorescence spectra of oxidized LDL. Probucol reduces the intensity of both peaks equally. The binding of a highly reactive heparin (HRH) fraction to LDL was measured by titration of LDL with HRH in the presence of fluoresceinamine-labeled HRH. The decrease in fluorescence anisotropy of the labeled HRH is proportional to the concentration of bound HRH. Copper oxidation of LDL reduced the number of binding sites for HRH from 3.5 to 0.8 and increased the Kd of binding from 1.2 to 9.9 .mu.M. Probucol completely protected the LDL so that incubation with Cu2+ for 24 h at 37.degree. C had no effect on HRH binding to the probucol-LDL. Similarly, probucol completely prevented the 40% decrease in reactive amino groups on the surface of LDL observed after 24 h of incubation with Cu2+. These data support the hypothesis that probucol reduces the rate of lipid oxidation and prevents lipoprotein surface modifications associated with oxidation of LDL.