Nitration and nitrosation of N-acetyl- -tryptophan and tryptophan residues in proteins by various reactive nitrogen species

Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tryptophan residues are especially susceptible to attack by reactive nitrogen species. We carried out experiments on the reactions of peroxynitrite and other reactive nitrogen species with N-acetyl- -tryptophan under various conditions. Four major products were identified as 1-nitroso-N-acetyl- -tryptophan, 1-nitro-N-acetyl- -tryptophan, 6-nitro-N-acetyl- -tryptophan, and N-acetyl-N′-formyl- -kynurenine on the basis of their mass and UV spectra. The reactions with SIN-1 (a peroxynitrite generator), Angeliʹs salt (a nitroxyl donor), and spermine NONOate (a nitric oxide donor) generated the nitroso derivative but not the nitro derivatives. A myeloperoxidase–H2O2–NO2− system generated the nitro derivatives but not the nitroso derivative. Under physiological conditions 6-nitro-N-acetyl- -tryptophan was stable, whereas the 1-nitroso and 1-nitro derivatives decomposed with half-lives of 1.5 and 18 h, respectively. After treatment with various reactive nitrogen species, bovine serum albumin was enzymatically hydrolyzed and analyzed for 6-nitro- -tryptophan and 3-nitro- -tyrosine by HPLC with electrochemical detection. Levels of 6-nitro- -tryptophan and 3-nitro- -tyrosine were similar in the nitrated protein. 6-Nitro- -tryptophan in proteins can be measured as an additional biomarker of protein nitration.