Nitrosation of Tryptophan Residue(s) in Serum Albumin and Model Dipeptides

Abstract

Nitrosation of bovine serum albumin with acidified NaNO 2 was compared to that of carboxymethyl-bovine serum albumin in which the thiol group is covalently blocked. Differential ultraviolet-visible (UV-Vis) spectroscopy and a modified Saville assay indicated that a non-cysteine residue(s) in carboxymethyl-bovine serum albumin was nitrosated. The nitrosated carboxymethylbovine serum albumin exhibited similar vasorelaxation activity as that observed with nitrosated bovine serum albumin. Identification of the nitrosated non-cysteine residue(s) was studied using 16 model dipeptides, each of which contained a glycyl residue and a variable residue. Using photolysis-chemiluminescence analysis, modified Saville assay, differential UV-Vis spectroscopy, and bioassays, L-glycyl-L-tryptophan (Gly-Trp) was found to be the only dipeptide that underwent significant nitrosation under these conditions. Liquid chromatography-UV-Vis spectroscopy-mass spectrometry showed that the NO group was attached to the indole nitrogen of tryptophan. Nitrosated Gly-Trp exhibited dose-dependent vasorelaxation and platelet inhibiting activity with apparent EC 50 values of 1.1 ؎ 0.3 and 3.5 ؎ 0.9 M, respectively. Because N-nitroso-Gly-Trp does not release NO radical via spontaneous homolytic N-NO bond fission nor freely diffuse through cellular membranes, the ability of this compound to induce NO ⅐-like biological effects suggests the existence of a (membraneassociated) transnitrosation system that facilitates delivery of-NO to its specific biologic target(s). The nitrosation reaction introduces the nitroso group (-NO) 1 into an organic molecule, leading to the formation of C-nitroso, N-nitroso, O-nitroso, or S-nitroso derivatives of the parent molecule. Commonly used nitrosating agents include HNO 2 , N 2 O 3 , and N 2 O 4 (for review, see Ref. 1), but NO 2 has also been suggested to be effective in this regard (2). Nitrosation reactions may be relevant to the metabolism and

Formation of S-Nitrosothiols from Regiospecific Reaction of Thiols with N-Nitrosotryptophan Derivatives

“…In 1996 Zhang et al (32) studied the biological activity of N-nitrosated Gly-Trp and carboxymethyl bovine serum albumin (CM-BSA), a modified derivative in which the thiol group is covalently blocked, in comparison with GSNO. Because these data were a bit distorted by the nitroso content of the applied nitroso compounds, i.e.…”

“…Interestingly, the corrected data clearly demonstrate that the efficacy of the Gly-NO-Trp-induced vasorelaxation and the inhibition of platelet aggregation was nearly identical to that of GSNO. Because Gly-NO-Trp does not spontaneously diffuse through cellular membranes, Zhang et al (32) suggested that extracellular N-nitrosocompounds transfer the nitroso function via a transnitrosation reaction to a NO-carrier "located on or associated with the plasma membrane" (32). Assuming that a protein-bound cysteine is the NO carrier, which can indeed be highly effective in transporting nitric oxide through cellular membranes (23,24), the small peptide Gly-NO-Trp may induce such a process.…”

“…Thus, a reason other than a N-nitroso CM-BSA-dependent transnitrosation of the membrane-bound cysteine must be found to explain the observed capabilities (Table IV). It should be noted that the experiments of Zhang et al (32) were performed either in the presence of phenylephrine, as in the case of the vasorelaxation activity experiments, or (presumably) in the presence of ascorbate, as in the case of the platelet aggregation experiments. Because catecholamines 3 and ascorbate (10) release nitric oxide from N-nitrosotryptophan compounds, the N-nitroso CM-BSA-dependent effects of vasodilatation and anti-coagulation were obviously thiol-independent.…”

Structural Features of Proteins Responsible for Resistance of Tryptophan Residues to Nitrosylation

“…In micellar oxidative nitrosation when the nitrosating agent is formed from NO and O 2 within hydrophobic phases, the carbon atoms of tryptophan residues embedded into the hydrophobic phase are supposed to be attacked much more efficiently than the nitrogen atom in a more hydrophilic surrounding. Moreover, it is well known that nitrosation of acylated derivatives of tryptophan proceeds via the C 3 [C(G)] atom and that the intramolecular rearrangement ON-C 3 → ON-N 1 is probably a rate-determining stage (16)(17)(18). The equilibrium constant for the formation of N -acetyl-N 1 -nitrosotryptophan in water was found to be 850 M −1 (18).…”

“…However, the nitroso-tryptophan fraction in proteins is usually small. NOW was detected in in vitro experiments upon nitrosation of serum albumin, and its functional activity as EDRF was demonstrated (16). The reaction of NOW denitrosation was previously studied using acetyl-N 1 -nitrosotryptophan (17,18).…”