Rj. Singh et al., SPIN-LABELING STUDY OF THE OXIDATIVE DAMAGE TO LOW-DENSITY-LIPOPROTEIN, Archives of biochemistry and biophysics, 320(1), 1995, pp. 155-161
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.