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Analytical biochemistry

Convenient colorimetric and fluorometric assays for S-nitrosothiols.


PMID 8660604

Abstract

S-nitrosothiols have been shown to affect a number of physiological functions. Several techniques have been used to detect these species in biological systems, primarily by methods utilizing chemiluminescence. Since the apparatus required for measurement of chemiluminescence are not readily available in most laboratories, methods employing more conventional 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-nitrosothiols. Solutions containing sulfanilamide/N-(1-naphthyl)- ethylenediamine dihydrochloride or 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid), when exposed to S-nitrosoglutathione (GSNO), S-nitrosocysteine, or S-nitrosoacteylpenicillamine, resulted in no absorbance changes in the range of 400-800 nm. Exposure to HgCl2 or Cu(acetate)2 resulted in release of nitric oxide (NO) from the S-nitrosothiols. The liberated NO reacted subsequently with oxygen and formed a chemical species which reacted with either analysis solution, resulting in an increase in absorption between 400 and 800 nm. A plot of RSNO versus absorbance was linear for both mercury(II) and copper(II) ions where the slope in the presence of mercury ion was significantly greater than that for copper ion. The sensitivity was as low as 5 microM RSNO using HgCl2. The fluorometric method using 2, 3-diaminonaphthalene as the scavenger of the NOsolidusO2 products gave a sensitivity of 50 nM for GSNO. In addition, S-nitrosylated proteins were quantitated using the fluorometric technique. These methods provide accurate determination of low concentrations of S-nitrosothiols, utilizing conventional spectroscopic techniques available in most laboratories.