Nitrosation of tryptophan residue(s) in serum albumin and model dipeptides. Biochemical characterization and bioactivity.
Nitrosation of bovine serum albumin with acidified NaNO2 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 carboxymethyl-bovine 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 EC50 values of 1.1 +/- 0. 3 and 3.5 +/- 0.9 microM, 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.-like biological effects suggests the existence of a (membrane-associated) transnitrosation system that facilitates delivery of -NO to its specific biologic target(s).
Zhang
Y Y
YY
Whitaker Cardiovascular Institute, Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118-2394, USA.
Xu
A M
AM
Nomen
M
M
Walsh
M
M
Keaney
J F
JF
Loscalzo
J
J
D000818:Animals; D001011:Aorta; D002417:Cattle; D002942:Circular Dichroism; D003545:Cysteine; D004151:Dipeptides / Q000737:chemistry* / Q000494:pharmacology*; D005978:Glutathione; D006801:Humans; D008163:Luminescent Measurements; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D009603:Nitroso Compounds / Q000494:pharmacology; D010782:Photolysis; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D012977:Sodium Nitrite; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis; D014364:Tryptophan; D014665:Vasodilator Agents / Q000494:pharmacology
D004151:Dipeptides; D009603:Nitroso Compounds; D010975:Platelet Aggregation Inhibitors; D013438:Sulfhydryl Compounds; D014665:Vasodilator Agents; D012710:Serum Albumin, Bovine; D014364:Tryptophan; D005978:Glutathione; D003545:Cysteine; D012977:Sodium Nitrite