Oxidative modifications of apoB-100 by exposure of low density lipoproteins to HOCL in vitro.
Although the products of oxidation of the lipid components of LDL have been studied extensively, much less is known about the specific products of oxidative modification of the apoprotein. We reacted native LDL and LDL that had been treated with HOCl with 2,4-dinitrophenylhydrazine (DNPH), delipidated and trypsinized the protein, and analyzed the products by HPLC. Although tryptic digests of native LDL and LDL oxidized by limited quantities of HOCl showed similar patterns by HPLC with detection at 220 nm, oxidized LDL showed several discrete peaks at 365 nm, which is characteristic of hydrazones formed with aldehydes and ketones, commonly termed protein carbonyls. Native LDl showed no peaks in the chromatograms at 365 nm. Peptides absorbing at 365 nm were isolated by HPLC and characterized. In most cases, the probable sites of modification on the peptides could be implied by failure of an anticipated amino acid to appear in the expected sequence. Of the 14 peptides isolated and characterized to date, eight peptides contained Cys residues. In other peptides, Lys, Trp, and Met were identified as amino acid residues apparently modified by HOCl treatment of LDL. Thirteen of the peptides identified are from trypsin-releasable peptides located on the surface of unoxidized native LDL. Our studies suggest a selective process of modification of apoB-100 by HOCl and the approaches used in the present studies should be useful for the characterization of the mechanisms of oxidation of this and other proteins.
Yang
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CY
Department of Medicine, Baylor College of Medicine, Methodist Hospital, Houston, TX 77030, USA.
Gu
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ZW
Yang
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Yang
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Gotto
A M
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Smith
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CV
D000595:Amino Acid Sequence; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000097:blood / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008077:Lipoproteins, LDL / Q000097:blood / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D010659:Phenylhydrazines / Q000378:metabolism; D012973:Sodium Hypochlorite / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D010659:Phenylhydrazines; C099252:oxidized low density lipoprotein; C004787:2,4-dinitrophenylhydrazine; D012973:Sodium Hypochlorite; D014357:Trypsin