Spin-labeling study of the oxidative damage to low-density lipoprotein.
In this study, we have spin-labeled the lysine and cysteine residues of low-density lipoprotein (LDL) using N-4-(2,2,6,6-tetramethylpiperidinyl-1-oxyl-4-yl) maleimide (MAL-6) and succinimidyl-2,2,5,5-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. Using the continuous-wave power saturation technique in the presence of hydrophobic and hydrophilic paramagnetic relaxing agents, we have determined 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 predominantly in the hydrophobic region. During Cu(2+)-initiated oxidation of spin-labeled LDL, nitroxide labels located in a hydrophobic environment were predominantly degraded. Nitroxide destruction was inhibited by butylated hydroxytoluene, indicating the role of lipid peroxidation in this process. ESR data also showed that Cu2+ binding to lysine is essential for LDL oxidation. The spin label methodology may be useful for the investigation of site-specific radical reactions in LDL.
Singh
R J
RJ
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee 53226-0509, USA.
Feix
J B
JB
Mchaourab
H S
HS
Hogg
N
N
Kalyanaraman
B
B
D001055:Apolipoproteins B / Q000737:chemistry; D001665:Binding Sites; D003497:Cyclic N-Oxides; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006801:Humans; D066298:In Vitro Techniques; D015227:Lipid Peroxidation; D008077:Lipoproteins, LDL / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008872:Microwaves; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013113:Spin Labels
D001055:Apolipoproteins B; D003497:Cyclic N-Oxides; D005609:Free Radicals; D008077:Lipoproteins, LDL; D013113:Spin Labels; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl; C094286:succinimidyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylate; D008239:Lysine; D003545:Cysteine