FLUOROMETRIC DETECTION OF BIOLOGICAL S-NITROSOTHIOLS

A technique is presented for the quantitative detection of S-nitrosoth iols formed by model biological thiols, cysteine, glutathione, and ser um albumin. The technique is based on the detection of fluorescent com pound 1-[H]-naphthotriazole formed between 2,3-diaminonaphthalene and nitrous acid released from S-nitrosothiols by treatment with mercuric chloride in an acidic environment. Concentration of S-nitrosothiols is determined from the difference in fluorescent signal (excitation/emis sion wavelengths of 363 nm/450 nm, respectively) observed in the prese nce and absence of 0.18 mM HgCl2. The yield of the reaction between 2, 3-diaminonaphthalene and nitrous acid released from the S-NO bond by H gCl2 approaches 90-100% as documented by simultaneous assays of S-nitr osothiols by uv spectrophotometry and by Saville method, The assay can be applied to the analysis of mixtures containing excess of thiol and /or nitrite at neutral pH by pretreatment of samples with N-ethylmalei nimide and/or ammonium sulfamate, respectively. In analysis of S-nitro sothiols in protein-containing mixtures, HgCl2-mediated release of nit rous acid in the presence of 2,3-diaminonaphthalene is followed by neu tralization of samples and precipitation of protein with 0.5 M 5-sulfo salicylic acid. The fluorometric assay is carried out at an excitation wavelength of 380 nm to eliminate the background fluorescence of 5-su lfosalicylic acid observed at lower wavelengths. The technique offers simple and rapid determination of S-nitrosothiols in complex reaction mixtures with the detection limit at low nanomolar concentrations. (C) 1997 Academic Press.