Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins
Research output: Contribution to journal › Journal article › Research › peer-review
Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure and function. Due to its low oxidation potential, the indole ring of tryptophan (Trp) is a major target, with this resulting in the formation of indolyl radicals (Trp•). These undergo multiple reactions including ring opening and dimerization which can result in protein aggregation. The factors that govern Trp• dimerization, the rate constants for these reactions and the exact nature of the products are not fully elucidated. In this study, second-order rate constants were determined for Trp• dimerization in Trp-containing peptides to be 2-6 × 108M-1s-1 by pulse radiolysis. Peptide charge and molecular mass correlated negatively with these rate constants. Exposure of Trp-containing peptides to steady-state radiolysis in the presence of NaN3 resulted in consumption of the parent peptide, and detection by LC-MS of up to 4 different isomeric Trp-Trp cross-links. Similar species were detected with other oxidants, including CO3•- (from the HCO3- -dependent peroxidase activity of bovine superoxide dismutase) and peroxynitrous acid (ONOOH) in the presence or absence of HCO3-. Trp-Trp species were also isolated and detected after alkaline hydrolysis of the oxidized peptides and proteins. These studies demonstrate that Trp• formed on peptides and proteins undergo rapid recombination reactions to form Trp-Trp cross-linked species. These products may serve as markers of radical-mediated protein damage, and represent an additional pathway to protein aggregation in cellular dysfunction and disease.
|Journal||Free Radical Biology & Medicine|
|Number of pages||11|
|Publication status||Published - Dec 2017|