Fluorometric detection of biological S-nitrosothiols.

A technique is presented for the quantitative detection of S-nitrosothiols formed by model biological thiols, cysteine, glutathione, and serum albumin. The technique is based on the detection of fluorescent compound 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/emission wavelengths of 363 nm/450 nm, respectively) observed in the presence 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 HgCl2 approaches 90-100% as documented by simultaneous assays of S-nitrosothiols 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-ethylmaleinimide and/or ammonium sulfamate, respectively. In analysis of S-nitrosothiols in protein-containing mixtures, HgCl2-mediated release of nitrous acid in the presence of 2,3-diaminonaphthalene is followed by neutralization of samples and precipitation of protein with 0.5 M 5-sulfosalicylic acid. The fluorometric assay is carried out at an excitation wavelength of 380 nm to eliminate the background fluorescence of 5-sulfosalicylic 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.