Nitrosation of N ‐Terminally Blocked Tryptophan and Tryptophan‐Containing Peptides by Peroxynitrite

Abstract Tryptophan is known to be a major target of oxidative stress and to take part in electron transfer. In proteins, its fluorescence is extinguished after treatment with oxidative agents, like peroxynitrite (ONOO − /ONOOH)—the product of the reaction of NO . and superoxide anion (O 2 .− ) radicals. The main reactions of N ‐blocked tryptophan derivatives (melatonin or N ‐acetyl‐ L ‐tryptophan) exposed to peroxynitrite at physiological pH are oxidation to formylkynuramine or formylkynurenine, respectively, and nitrosation, which leads to substituted 1‐nitrosoindoles. Here we show that peroxynitrite‐induced nitrosation is specific to N ‐blocked L ‐tryptophan derivatives and is not obtained with free L ‐tryptophan. Such a nitrosation can be evaluated by using 4,5‐diaminofluorescein (DAF‐2), which is converted to the fluorescent triazolofluorescein by NO . donors and nitrosating agents. N ‐acetyl‐ L ‐tryptophan was shown to be twice as efficient as melatonin in transferring NO from peroxynitrite to DAF‐2. DAF‐2 responses were then used to assess the ability of a series of L ‐tryptophan‐containing peptides to give transient N ‐nitrosoindoles upon treatment with peroxynitrite. Many peptides proved not to be susceptible to nitrosation under these conditions. However, the N‐terminally blocked peptide of endothelin‐1 (Ac–Asp–Ile–Ile–Trp) reacted in a very similar fashion to melatonin; this shows that tryptophan residue nitrosation could occur when it was exposed to peroxynitrite.