Spin-labeling study of the oxidative damage to low-density Lipoprotein

Ravinder J. Singh, Jimmy B. Feix, Hassane S. McHaourab, Neil Hogg, B. Kalyanaraman

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)155-161
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume320
Issue number1
DOIs
StatePublished - Jun 20 1995

Keywords

  • ESR
  • Loop-gap resonator
  • Low-density lipoprotein
  • Spin labeling

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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