Characterization of protein modification by products of lipid peroxidation.

摘要

In this dissertation, we show that the apparent 4-oxo-2-nonenal (ONE)-Lys Michael adducts (m+154) actually represent the isomeric 4-ketoamides. The same type of 4-ketoamide on Lys residues was formed by 9,12-dioxo-10( E)-dodecenoic acid (KODA). By incubation of beta-lactoglobulin (beta-LG) with 4-hydroxy-2-nonenal (HNE) and ONE, HNE was found to form Cys/His/Lys/N-terminal amine Michael and Lys/N-terminal amine Schiff base adducts by mass spectrometry. ONE-Lys 4-ketoamide, ONE-Lys pyrrolinone and Cys/His-ONE-Lys/N-terminal amine pyrrole cross-links were detected after both 10 min and 24 h incubations. ONE-Lys Schiff base and ONE-His/Lys/Cys Michael adducts were observed at 10 min. When the ONE concentration is less than stoichiometric with beta-LG, the predominant protein modification detected is the Cys-ONE-Lys pyrrole cross-link. The cross-linking between Glutathione (GSH) or beta-alanylhistidine (carnosine) and proteins by ONE was investigated by SDS-PAGE and mass spectrometry. Under the reaction conditions we used, virtually every lysine of beta-LG was found to be cross-linked to GSH to some degree. Using cytochrome c and ribonuclease A (RNase), we showed that ONE becomes more protein-reactive in the presence of GSH. His and Lys surrogates PHis and PLys were synthesized, both of which have detection limits of 30 fmol with a signal-to-noise ratio of 5:1 by MALDI-TOF-MS. By incubation of PHis or PLys with HNE, ONE, alkenals or a mixture of linoleic acid (d0:d 5 = 1:1), Fe(II) and ascorbic acid, besides the previously reported known adducts, several novel adducts were detected by MALDI-TOF-MS and LC-ESI-MS. When the beta-LG was incubated with LA or 17,17,18,18,18-d 5-(9Z,12Z)-octadeca-9,12-dienoic acid (d5-LA), Fe(II), and ascorbic acid, a novel 2-octenoic acid-His Michael adduct was discovered by LC-ESI-MS. In addition, many of the adducts found represent those previously observed when treating protein with the pure electrophilic modifier. The same modifications by the "mirror-image" carboxy terminus of LA were also identified. 2(E), 4( E)-Decadienal (DDE) was found to modify different proteins through different ways. beta-LG is intensively cross-linked by DDE but cytochrome c and RNase are resistant to the DDE cross-linking. DDE majorly form Lys Schiff base adducts with cytochrome c and RNase. However, in addition to the these adducts, DDE forms Cys Michael and Lys pyridinium adducts with beta-LG.