6 - Formation and Detection of Tryptophan Crosslinks

Abstract

Oxidation, aggregation and accumulation of damaged proteins are key determinants of the shelf life and activity of protein-based medicines and foodstuffs, result in diminished agricultural yields, and are associated with multiple human diseases. Protein aggregation can occur both reversibly (via disulfides from Cys residues) and irreversibly via cross-linking of Tyr phenoxyl radicals. Recent studies have also provided evidence for dimerization of Trp radicals. This may be a major aggregation pathway due to the ease of oxidation (low reduction potential) of the indole ring of Trp. Hypothesis: that Trp radicals rapidly dimerize, generating cross-linked species. Results: Trp radicals generated by pulse radiolysis of free amino acid and peptides, at pH 7.4, undergo dimerization with k ~ 2 – 6 x 108 M-1s-1. These reactions are rapid compared to O 2 addition (k 2 O 2 /CO 3 2– system which generates CO 3 •– . Hydrolysis methods have been developed to allow detection and quantification of these species in complex systems. Conclusions: These studies demonstrate that indolyl radicals formed on Trp residues undergo rapid dimerization to give multiple cross-linked products. These reactions are a feasible mechanism for aggregation and degradation of proteins, and could contribute to protein dysfunction and the accumulation of damaged proteins in multiple diseases.