Covalent crosslinks within or between proteins play a key role in determining the structure and function of proteins. Some of these are formed intentionally by either enzymatic or molecular reactions and are critical to normal physiological function. Others are generated as a consequence of exposure to oxidants (radicals, excited states or two-electron species) and other endogenous or external stimuli, or as a result of the actions of a number of enzymes (e.g., oxidases and peroxidases). Increasing evidence indicates that the accumulation of unwanted crosslinks, as is seen in ageing and multiple pathologies, has adverse effects on biological function. In this article, we review the spectrum of crosslinks, both reducible and non-reducible, currently known to be formed on proteins; the mechanisms of their formation; and experimental approaches to the detection, identification and characterization of these species.

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The oxidation of tryptophan (Trp) residues, mediated by peroxyl radicals (ROO.), follows a complex mechanism involving free radical intermediates, and short chain reactions. The reactivity of Trp towards ROO. should be strongly affected by its inclusion in peptides and proteins. To examine the latter, we investigated (by fluorescence) the kinetic of the consumption of free, peptide-and protein-Trp residues towards AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride)-derived free radicals. Interestingly, the initial consumption rates (R-i) were only slightly influenced by the inclusion of Trp in small peptides and proteins (human serum albumin and human superoxide dismutase). Depending on the Trp concentration, the R-i versus Trp concentration ([Trp]) plots showed three regions. At low Trp concentrations (1-10 mu M), a linear dependence was observed between R-i and [Trp]; at intermediate Trp concentrations (10-50 mu M), the values of R-i were nearly constant; and at high Trp concentrations (50 mu M to 1 mM), a slower increase of R-i than expected for chain reactions. Similar behavior was detected for all three systems (free Trp, and Trp in peptides and proteins). For the first time we are showing that alkoxyl radicals, formed from self-reaction of ROO., are responsible of the Trp oxidation at low concentrations, while at high Trp concentrations, a mixture of peroxyl and alkoxyl radicals are involved in the oxidation of Trp residuesFondecyt 1141142 3140307 Novo Nordisk Foundation NNF13OC000429