cytochrome-c-t and 13-hydroperoxy-9-11-octadecadienoic-acid

The hydroperoxide of linoleic acid (13-HPODE) degrades to 9,12-dioxo-10(E)-dodecenoic acid (DODE), which readily modifies proteins. This study identified the major proteins in MCF7 cells modified by DODE. To reduce false positives, three methods were used to identify DODE-modified proteins. First, cells were treated with a synthetically biotinylated 13-HPODE (13-HPODE-biotin). Modified proteins were enriched by neutravidin affinity and identified by two-dimensional liquid chromatography--tandem mass spectrometry (2D LC-MS/MS). Second, cells were treated with native 13-HPODE. Protein carbonyls were biotinylated with an aldehyde reactive probe, and modified proteins were enriched by neutravidin affinity and identified by 2D LC-MS/MS. Third, using a newly developed DODE antibody, DODE-modified proteins were located by 2D sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot and identified by in-gel digestion and LC-MS/MS. Analysis of the proteins characterized by all three methods revealed a significant overlap and identified 32 primary proteins modified by DODE in MCF7 cells. These results demonstrated the feasibility for the cellular formation of DODE protein-carbonyl adducts that may be future indicators of oxidative stress.

Topics: Cell Line, Tumor; Chromatography, Liquid; Cytochromes c; Fatty Acids, Monounsaturated; Humans; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Proteins; Tandem Mass Spectrometry

Polyunsaturated fatty acids can be converted to lipid hydroperoxides through nonenzymatic and enzymatic pathways. The prototypic omega-6 lipid hydroperoxide 13-hydroperoxy-octadecadienoic acid (13-HPODE) homolytically decomposes to form highly reactive alpha,beta-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.

Topics: Cytochromes c; DNA Adducts; DNA Damage; Linoleic Acids; Lipid Peroxides; Myoglobin; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Methemoglobin (MetHb), oxyhemoglobin (oxyHb), metmyoglobin (metMb), and oxymyoglobin (oxyMb) catalyze formation of the 7-carboxyheptyl and pentyl radicals from 13-hydroperoxy-9,11-octadecadienoic acid. The relative HPLC-ESR peak height of the pentyl radical to the 7-carboxyheptyl radical was found to depend on the oxygen concentration in the reaction mixture. Under aerobic conditions, the 7-carboxyheptyl radical was predominant for the reaction mixture with ferrous ions (or cytochrome c, metHb, or metMb). On the other hand, under anaerobic conditions, the pentyl radical was predominant for the reaction mixture with ferrous ions (or cytochrome c), but the 7-carboxyheptyl radical was still predominant for the reaction mixture with metHb (or metMb), suggesting that metHb (or metMb) catalyzes the reaction through a mechanism different from that in the case of ferrous ions (or cytochrome c). In order to explain the above results, a mechanism, in which molecular oxygen is not involved, is proposed for the formation of the 7-carboxyheptyl radical in the reaction mixture of 13-HPODE with metHb (or metMb) under anaerobic conditions.

Topics: Aerobiosis; Anaerobiosis; Animals; Catalysis; Cattle; Cytochromes c; Electron Spin Resonance Spectroscopy; Ferric Compounds; Ferrous Compounds; Free Radicals; Hemin; Heptanoic Acids; Horses; Linoleic Acids; Lipid Peroxides; Methemoglobin; Metmyoglobin; Muscle, Skeletal; Myoglobin; Oxyhemoglobins