Formation of 6-nitrotryptophan in purified proteins by reactive nitrogen species: A possible new biomarker

Abstract

We analyzed products of tryptophan residues in two model proteins which were reacted with reactive nitrogen species. We modified human Cu, Zn-superoxide dismutase, which has a single tryptophan residue and no tyrosine residue, by using two reactive nitrogen species generating systems; peroxynitrite/CO₂ and myeroperoxidase/H₂O₂/NO₂⁻ systems. We identified 6-nitrotryptophan as a major nitration product along with other oxidized products as the reaction products of tryptophan residue by using LC-MS/MS and HPLC-photodiode array analyses of the tryptic peptides. We modified hen egg-white lysozyme as a model of a simple protein having both tryptophan and tyrosine residues by peroxynitrite/CO₂ system. The modified enzyme lost 89% of the enzymatic activity. Among six tryptophan residues in lysozyme, Trp62, Trp63, and Trp123 were nitrated to form 6-nitrotryptophan, along with the formation of 3-nitrotyrosine in all tyrosine residues. However, the efficiency of nitration was different for each residue. No oxidized product of tryptophan residue was observed in the modified lysozyme. In conclusion, we propose that 6-nitrotryptophan is a unique and major nitrated product of tryptophan residue in proteins reacted with reactive nitrogen species.