Year: 2009
Type: article
Abstract: 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 4ketoamide on Lys residues was formed by 9,12-dioxo-10(E)-dodecenoic acid (KODA).By incubation of β-lactoglobulin (β-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 β-LG, the predominant protein modification detected is the Cys-ONE-Lys pyrrole cross-link.The cross-linking between Glutathione (GSH) or βalanylhistidine (carnosine) and proteins by ONE was investigated by SDS-PAGE and mass spectrometry.Under the reaction conditions we used, virtually every lysine of β-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.
Author: Xiaochun Zhu
Institution: Case Western Reserve University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2208521851