Regiospecific nitrosation of N-terminal-blocked tryptophan derivatives by N2O3 at physiological pH

N(2)O(3) formed from nitric oxide in the presence of oxygen attacks thiols in proteins to yield S-nitrosothiols, which are believed to play a central role in NO signaling. In the present study we examined the N-nitrosation of N-terminal-blocked (N-blocked) tryptophan derivatives in the presence of N(2)O(3) generating systems, such as preformed nitric oxide and nitric oxide donor compounds in the presence of oxygen at pH 7.4. Under these conditions N-nitrosation of N-acetyltryptophan and lysine-tryptophan-lysine, respectively, was proven unequivocally by UV-visible spectroscopy as well as (15)N NMR spectrometry. Competition experiments performed with the known N(2)O(3) scavenger morpholine demonstrated that the selected tryptophan derivatives were nitrosated by N(2)O(3) with similar rate constants. It is further shown that the addition of ascorbate (vitamin C) induced the release of nitric oxide from N-acetyl-N-nitrosotryptophan as monitored polarographically with a NO electrode. Theoretical considerations strongly suggested that the reactivity of protein-bound tryptophan would be high enough to compete effectively with protein-bound cysteine for N(2)O(3). Our data demonstrate conclusively that N(2)O(3) nitrosates the secondary amine function (N(indole)) at the indole ring of N-blocked tryptophan with high reactivity at physiological pH values.