Identification of modified tryptophan residues in apolipoprotein B-100 derived from copper ion-oxidized low-density lipoprotein

Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of th e lipid components of LDL have been studied extensively, less is known abou t the oxidation products of the apoprotein, apolipoprotein B-100. To identi fy the specific oxidative modifications, we oxidized LDL in the presence of Cu2+, treated with DNPH, precipitated and delipidated the protein, digeste d the protein with trypsin, and analyzed the peptides by high-performance l iquid chromatography. We isolated nine peptides that exhibited measurable a bsorbance at 365 nm, which is characteristic of hydrazones derived from DNP H and is not observed in peptides derived from unoxidized LDL. Unexpectedly , we obtained the same peptides with absorbance at 365 nm in Cu2+-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectromet ry indicated that the peptides isolated from the Cu2+-oxidized LDL all cont ained kynurenine residues in place of Trp residues found in the native apop rotein. The product profile we observed in Cu2+-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperox idase in vitro, and the preferential oxidation of Trp to kynurenine in Cu2-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseas es.