CONVENIENT COLORIMETRIC AND FLUOROMETRIC ASSAYS FOR S-NITROSOTHIOLS

S-nitrosothiols have been shown to affect a number of physiological: f unctions, Several techniques have been used to detect these species in biological systems, primarily by methods utilizing chemiluminescence, since the apparatus required far measurement of chemiluminescence are not readily available in mast laboratories, methods employing more co nventional techniques such as uv-vis and fluorescence spectroscopy may be of greater use, Herein, we report the development of colorimetric and fluorometric methods for the reliable quantitation of S-nitrosothi ols, Solutions containing sulfanilamide/N-(1-naphthyl)-ethylenediamine dihydrochloride or 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic ac id), when exposed to S-nitrosoglutathione (GSNO), S-nitrosocysteine, o r S-nitrosoacteylpenicillamine, resulted in no absorbance changes in t he range of 400-800 nm. Exposure to HgCl2 or Cu(acetate)(2) resulted i n release of nitric oxide (NO) from the S-nitrosothiols. The liberated NO reacted subsequently with oxygen and formed a chemical species whi ch reacted with either analysis solution, resulting in an increase in absorption between 400 and 8OO nm, A plot of RSNO versus absorbance wa s linear for both mercury(PI) and copper(IH) ions where the slope in t he presence of mercury ion was significantly greater than that for cop per ion, The Sensitivity was as low as 5 mu M RSNO using HgCl2. The fl uorometric method using 2,3-diaminonaphthalene as the scavenger of the NO/O-2 products gave a sensitivity of 50 nM for GSNO. In addition, S- nitrosylated proteins were quantitated using the fluorometric techniqu e, These methods provide accurate determination of low concentrations of S-nitro-sothiols, utilizing conventional spectroscopic techniques a vailable in most laboratories, (C) 1996 Academic Press, Lac.