ANALYTICAL BIOCHEMISTRY 238, 150–158 (1996) ARTICLE NO. 0268 Convenient Colorimetric and Fluorometric Assays for S-Nitrosothiols John A. Cook,* Sungmee Y. Kim,* Diane Teague,* Murali C. Krishna,* Roberto Pacelli,* James B. Mitchell,* Yoram Vodovotz,* Raymond W. Nims,‡ Danae Christodoulou,‡ Allen M. Miles,§ Matthew B. Grisham,§ and David A. Wink* ,1 *Tumor Biology Section, Radiation Biology Branch, National Cancer Institute, Bethesda, Maryland 20892; ‡Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702 and Department of Chemistry, Grambling State University, Grambling, Louisiana 71241; and §Department of Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130 Received December 8, 1995 tion, S-nitrosylated proteins were quantitated using the fluorometric technique. These methods provide ac- S-nitrosothiols have been shown to affect a number of physiological functions. Several techniques have curate determination of low concentrations of S-nitro- sothiols, utilizing conventional spectroscopic tech- been used to detect these species in biological systems, primarily by methods utilizing chemiluminescence. niques available in most laboratories.  1996 Academic Press, Inc. 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 spectros- S-nitrosothiols (RSNO) have been suggested to medi- copy may be of greater use. Herein, we report the de- ate certain aspects of signal transduction (1). Ignarro et velopment of colorimetric and fluorometric methods al. showed that these adducts stimulated the conversion for the reliable quantitation of S-nitrosothiols. of GTP to cGMP in guanylate cyclase (2), suggesting they Solutions containing sulfanilamide/N-(1-naphthyl)- were key intermediates in the action of various nitrova- ethylenediamine dihydrochloride or 2,2 -azinobis (3- sodilating compounds such as sodium nitroprusside and ethylbenzthiazoline-6-sulfonic acid), when exposed to nitroglycerin (3, 4). Although endothelial-derived relax- S-nitrosoglutathione (GSNO), S-nitrosocysteine, or S- ation factor (EDRF) was reported to be NO (5, 6), a later nitrosoacteylpenicillamine, resulted in no absorbance report suggested that S-nitrosopeptides could modulate changes in the range of 400 – 800 nm. Exposure to HgCl 2 vascular tone (7). Stamler and co-workers have suggested or Cu(acetate) 2 resulted in release of nitric oxide (NO) that S-nitrosothiol proteins might serve as a circulating from the S-nitrosothiols. The liberated NO reacted sub- source of NO (8). Further studies have revealed that S- sequently with oxygen and formed a chemical species nitrosoproteins are formed during various phases of the which reacted with either analysis solution, resulting immune response (9). in an increase in absorption between 400 and 800 nm. A plot of RSNO versus absorbance was linear for both The formation of S-nitroso adducts in vivo has mercury(II) and copper(II) ions where the slope in the prompted the investigation of their chemistry. S-nitro- presence of mercury ion was significantly greater than sothiols can be formed from nitrosating agents derived that for copper ion. The sensitivity was as low as 5 mM from acidic nitrite (10). Pryor et al. showed that S- RSNO using HgCl 2 . The fluorometric method using 2,3- nitrosothiols cannot be formed directly from NO, but diaminonaphthalene as the scavenger of the NO/O 2 rather through the formation of reactive nitrogen oxide products gave a sensitivity of 50 nM for GSNO. In addi- species (RNOS) 2 (11). Formation of S-nitrosothiol pep- 2 Abbreviations used: ABTS, 2,2 -azinobis(3-ethylbenzthiazoline- 1 To whom correspondence should be addressed at Radiation Biol- ogy Branch, National Institutes of Health/National Cancer Institute, 6-sulfonic acid); BSA, bovine serum albumin; BSAS-NO, S-nitrosyl- ated bovine serum albumin; CYS, cysteine; DAN, 2,3-diaminonaph- Building 10, Room B3-B69, Bethesda, MD 20892. Fax: (301) 480- 2238. thalene; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; HSA, 150 0003-2697/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.