Direct Observation of Trapping and Release of Nitric Oxide by Glutathione and Cysteine with Electron Paramagnetic Resonance Spectroscopy Fwu-Shan Sheu,* Wen Zhu, †‡ and P. C. W. Fung † *Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; and † Division of Medical Physics, Department of Medicine, and ‡ Department of Physics, The University of Hong Kong, Pokfulam, Hong Kong, China ABSTRACT While the biosynthesis of nitric oxide (NO) is well established, one of the key issues that remains to be solved is whether NO participates in the biological responses right after generation through biosynthesis or there is a “secret passage” via which NO itself is trapped, transported, and released to exert its functions. It has been shown that NO reacts with thiol-containing biomolecules (RSH), like cysteine (Cys), glutathione (GSH), etc., to form S-nitrosothiols (RSNOs), which then release nitrogen compounds, including NO. The direct observation of trapping of NO and its release by RSNO has not been well documented, as most of the detection techniques measure the content of NO as well as nitrite and nitrate. Here we use spin-trapping electron paramagnetic resonance (EPR) technique to measure NO content directly in the reaction time course of samples of GSH and Cys ( mM) mixed with NO ( M) in the presence of metal ion chelator, which pertains to physiological conditions. We demonstrate that NO is readily trapped by these thiols in less than 10 min and 70 –90% is released afterward. These data imply that 10 –30% of the reaction product of NO does not exist in the free radical form. The NO release versus time curves are slightly pH dependent in the presence of metal ion chelator. Because GSH and Cys exist in high molar concentrations in blood and in mammalian cells, the trapping and release passage of NO by these thiols may provide a mechanism for temporal and spatial sequestration of NO to overcome its concentration gradient-dependent diffusion, so as to exert its multiple biological effects by reacting with various targets through regeneration. INTRODUCTION The endogenously formed free radical NO has been estab- lished as a novel messenger in numerous cellular processes of both physiological and pathological responses (Furchgott and Zawadzki, 1980; Ignarro, 1989; Moncada et al., 1991; Bredt and Snyder, 1994; Nathan and Xie, 1994; Stamler, 1994). Some of these responses have long been postulated to result from a redox-mediated reaction of NO with the thiol-containing biomolecules through thiol/nitrosothiol ex- change, leading to disulfide formation and regeneration of NO at the site of action (Girard and Potier, 1993). In an aerobic environment, NO reacts first with oxygen to form higher oxides like N 2 O 3 , which would then react with the thiol groups of low-molecular-weight peptides and proteins to form S-nitrosothiols (RSNOs) (Kharitonov et al., 1995). There is evidence that RSNOs are potent vasodilators and inhibitors of platelet aggregation (Ignarro and Gruetter, 1980; Ignarro et al., 1981; Kelm and Schrader, 1990; Stam- ler et al., 1992b; Butler and Williams, 1993). On the other hand, it is well established that NO is an endothelium- derived relaxing factor (EDRF) producing vasorelaxation (Palmer et al., 1987; Ignarro et al., 1987). Obviously, the activity of RSNOs is very likely dependent on their ability to regenerate NO, and this suggests the possibility that nitrosated peptides and proteins act as NO transporters and congeners (Minamiyama et al., 1996; Scorza et al., 1997; Ewing et al., 1997). Although the detailed mechanisms of NO release are complicated by the catalytic effect of both trace transition metal ions and the reducing agents thiols and ascorbate (Scorza et al., 1997; McAninly et al., 1993; R. J. Singh et al., 1996), the overall reaction seems to be as follows: 2RSNO 3 RSSR + 2NO NO released in this reaction can then react with either other thiol-containing proteins or the heme group of guanylate cyclase to exert its biological activity. In our recent study of the redox reaction between NO and thiol-containing bi- omolecules, using a chemically modified electrode (CME) for sensitive, selective, and quantitative detection of the oxidation currents of both NO and thiols of biomolecules (Miao et al., 1999), we observed that CME detects Cys at +450 mV, while the oxidation of NO does not occur at this potential but can be optimally detected at +700 mV. The result thus provides a selective control to examine the oxidation of Cys by NO at +450 mV without interference from NO itself. With this CME as a detector, we found that at the completion of the oxidation reaction of Cys by NO as indicated by the decrease in current at +450 mV, the oxidation current at +700 mV remains almost the same as in the initial stage before the reaction. These results are Received for publication 22 September 1999 and in final form 6 December 1999. Address correspondence to Prof. P. C. W. Fung, Division of Medical Physics, Department of Medicine, The University of Hong Kong, Profes- sorial Block, 4th Floor, Queen Mary Hospital, Pokfulam Road, Hong Kong. Tel: 852-2855-3356; Fax: 852-2816-2863, E-mail: hrspfcw@ hkucc.hku.hk. © 2000 by the Biophysical Society 0006-3495/00/03/1216/11 $2.00 1216 Biophysical Journal Volume 78 March 2000 1216 –1226