SPIN-LABELING STUDY OF THE OXIDATIVE DAMAGE TO LOW-DENSITY-LIPOPROTEIN

In this study, we have spin-labeled the lysine and cysteine residues o f low-density lipoprotein (LDL) using N-4-(2,2,6,6-tetramethylpiperidi nyl-1-oxyl-4-yl) maleimide (MAL-6) and 2,5,5-tetramethyl-3-pyrroline-1 -oxyl-3-carboxylate (SSL), respectively. The electron spin resonance ( ESR) spectrum of SSL bound to LDL indicated that the nitroxide moiety was relatively mobile. In contrast, the ESR spectrum of MAL-6 bound to LDL showed that the nitroxide moiety was rotationally restricted. Usi ng the continuous-wave power saturation technique in the presence of h ydrophobic and hydrophilic paramagnetic relaxing agents, we have deter mined that (i) approximately 60-70% of lysine-bound SSL is exposed to the aqueous phase, (ii) approximately 30-40% of SSL-LDL is buried in a hydrophobic region, and (iii) MAL-6 bound to LDL is localized predomi nantly in the hydrophobic region, During Cu2+-initiated oxidation of s pin-labeled LDL, nitroxide labels located in a hydrophobic environment were predominantly degraded. Nitroxide destruction was inhibited by b utylated hydroxytoluene, indicating the role of lipid peroxidation in this process. ESR data also showed that Cu2+ binding to lysine is esse ntial for LDL oxidation, The spin label methodology may be useful for the investigation of site-specific radical reactions in LDL. (C) 1995 Academic Press, Inc.