Covalent Adducts Arising from the Decomposition Products of Lipid Hydroperoxides in the Presence of Cytochrome c

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Abstract
Polyunsaturated fatty acids can be converted to lipid hydroperoxides through nonenzymatic and enzymatic pathways. The prototypic ω-6 lipid hydroperoxide 13-hydroperoxy-octadecadienoic acid (13-HPODE) homolytically decomposes to form highly reactive α,β-unsaturated aldehydes, such as 9,12-dioxo-10(E)-dodecenoic acid (DODE), 4-oxo-2(E)-nonenal (ONE), 4,5-epoxy-2(E)-decenal (EDE), and 4-hydroxy-2(E)-nonenal (HNE), that can form covalent adducts with DNA. Both 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal can also modify proteins to form products that can potentially serve as biomarkers of lipid hydroperoxide-mediated macromolecule damage. In this study, cytochrome c was used to identify and individually characterize the modification sites for each of these aldehydes and also determine the most abundant adduct formed following the decomposition of 13-HPODE. The adducts were characterized by ESI-TOF/MS analysis of the intact proteins and by a combination of ESI-ion-trap/MSn and quadrupole-TOF/MS/MS analysis of the tryptic and chymotryptic peptides. The major adducts included an HNE-His Michael adduct on H33, EDE-Lys adducts on K7 and K8, ONE-Lys ketoamide adducts on K5, K7, and K8, an apparent ONE-Lys Michael adduct on K5, and DODE-Lys carboxyl ketoamide adducts on K86 and K87. DODE was the most reactive aldehyde toward cytochrome c. The major adduct from this reaction was analogous to the most abundant adduct resulting from the decomposition of 13-HPODE in the presence of cytochrome c.