The study of structural accessibility of free thiol groups in human low-density lipoproteins.
The experimental evidence for the apolipoprotein B100 (apoB) domain structuring in low-density lipoprotein (LDL) was investigated focusing on the accessibility of free thiol groups. Three different spectroscopic methods were combined with the biochemical perturbations of LDL particle. The spectrophotometric method was adapted for LDL and the exposure of free thiols was analyzed in the native LDL and LDL exposed to sequential denaturation. The results indicate that 24-h denaturation does not expose all free thiols in LDL. Using thiol-specific spin labeling and electron paramagnetic resonance spectroscopy (EPR), different populations of labeled thiols were resolved. The comparison of the EPR spectra of native LDL and LDL with selectively blocked thiol groups revealed significant difference in the respective hyperfine splittings. The phenomenon can arise due to different polarity and/or mobility of the nitroxides in the microenvironments of spin label binding sites of these two LDL samples. The results indicate that nine thiol groups in apoB are distributed in different domains of LDL: two are more exposed, two are buried deeply in the lipid matrix of the particle and the rest are located in hydrophobic parts of this extremely complex protein-lipid assembly. These observations provide experimental support for the emerging theoretical models of apoB.
Kveder
Marina
M
Laboratory for Magnetic Resonances, Ruder Bosković Institute, Bijenicka 54, 10000, Zagreb, Croatia. kveder@rudjer.irb.hr <kveder@rudjer.irb.hr>
Krisko
Anita
A
Pifat
Greta
G
Steinhoff
Heinz-Jürgen
HJ
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008077:Lipoproteins, LDL / Q000737:chemistry*; D011489:Protein Denaturation; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000737:chemistry*
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D013113:Spin Labels; D013438:Sulfhydryl Compounds