TY - JOUR
T1 - Spin-labeling study of the oxidative damage to low-density Lipoprotein
AU - Singh, Ravinder J.
AU - Feix, Jimmy B.
AU - McHaourab, Hassane S.
AU - Hogg, Neil
AU - Kalyanaraman, B.
PY - 1995/6/20
Y1 - 1995/6/20
N2 - 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 Cu2+-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.
AB - 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 Cu2+-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.
KW - ESR
KW - Loop-gap resonator
KW - Low-density lipoprotein
KW - Spin labeling
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U2 - 10.1006/abbi.1995.1354
DO - 10.1006/abbi.1995.1354
M3 - Article
C2 - 7793976
AN - SCOPUS:0029013080
SN - 0003-9861
VL - 320
SP - 155
EP - 161
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -