The Chemistry of the S-Nitrosoglutathione/Glutathione
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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14428–14433, December 1996 Biochemistry The chemistry of the S-nitrosoglutathioneyglutathione system (nitric oxideyglutathionyl radicalysulfenamideytransnitrosationyN-nitrosomorpholine) S. P. SINGH*, J. S. WISHNOK*, M. KESHIVE†,W.M.DEEN†, AND S. R. TANNENBAUM*‡§ *Division of Toxicology, †Department of Chemical Engineering, and ‡Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 Communicated by Alexander M. Klibanov, Massachusetts Institute of Technology, Cambridge, MA, October 15, 1996 (received for review July 23, 1996) ABSTRACT S-Nitrosothiols have generated considerable concentrations as high as 10 mM (10). It is involved in the cell’s interest due to their ability to act as nitric oxide (NO) donors antioxidant defense, xenobiotic detoxification, and amino acid and due to their possible involvement in bioregulatory sys- transport (11). GSNO is also believed to be involved in many tems—e.g., NO transfer reactions. Elucidation of the reaction physiologic and pathophysiologic processes. It has recently pathways involved in the modification of the thiol group by been shown that exposure of human neutrophils to NO results S-nitrosothiols is important for understanding the role of in the conversion of intracellular GSH to GSNO and that S-nitroso compounds in vivo. The modification of glutathione intracellular GSNO activates the hexose monophosphate shunt (GSH) in the presence of S-nitrosoglutathione (GSNO) was of the cells (12). Since GSH concentration is millimolar in cells examined as a model reaction. Incubation of GSNO (1 mM) and NO concentration is less than micromolar, it follows that with GSH at various concentrations (1–10 mM) in phosphate the concentration of GSNO will always be small compared that buffer (pH 7.4) yielded oxidized glutathione, nitrite, nitrous of GSH. oxide, and ammonia as end products. The product yields were We report here that, in addition to release of NO from dependent on the concentrations of GSH and oxygen. Tran- GSNO in the presence of GSH, an important pathway for sient signals corresponding to GSH conjugates, which in- GSNO decomposition is reaction with GSH to form glutathi- creased by one mass unit when the reaction was carried out one conjugates similar to those reported for the reaction of with 15N-labeled GSNO, were identified by electrospray ion- GSH with other electrophilic metabolites (13–16). Our data ization mass spectrometry. When morpholine was present in further show that the major end product from this transfor- the reaction system, N-nitrosomorpholine was formed. In- mation is oxidized GSH, GSSG. The amount formed is creasing concentrations of either phosphate or GSH led to dependent on the availability of oxygen in the system, sug- lower yields of N-nitrosomorpholine. The inhibitory effect of gesting the possible involvement of radicals. phosphate may be due to reaction with the nitrosating agent, nitrous anhydride (N2O3), formed by oxidation of NO. This MATERIALS AND METHODS supports the release of NO during the reaction of GSNO with GSH. The products noted above account quantitatively for Materials. Thiazolidine-4-carboxylic acid, GSH, N- virtually all of the GSNO nitrogen consumed during the nitrosomorpholine (NMOR), and GSSG were from Sigma. reaction, and it is now possible to construct a complete set of Sodium nitrite (15N-labeled) was from MSD isotopes. Mor- pathways for the complex transformations arising from pholine (MOR) was from Aldrich. GSNO and GS15NO were GSNO 1 GSH. prepared according to Hart (17) by acid-catalyzed nitrosation of GSH; these compounds were quantitated by measuring the 21 21 S-Nitrosothiols, RSNO, with certain exceptions, are unstable absorbance at 334 nm (A334 5 800 M zcm ). N-Nitroso- in aqueous solution. For example, S-nitrosoglutathione N9,N9,N-trimethylurea (NTMU) was synthesized by nitrosat- (GSNO) undergoes decomposition over hours, whereas S- ing trimethylurea (Alfa) at pH 2.5 in the presence of acetic acid nitrosocysteine has a half-life of less than 2 min. The initial step and was quantitated by measuring the absorbance at 374 nm 21 21 in the decomposition of RSNO is believed to be homolytic (A374 5 88 M zcm ) (18). N-Acetylthiazolidine-4-carboxylic cleavage of the SON bond to give nitric oxide (NO) and a thiyl acid was prepared by addition of acetic anhydride to a hot radical (1, 2). These compounds are involved in many bio- solution of thiazolidine-4-carboxylic acid in acetic acid (19). regulatory functions, including vasodilation and inhibition of Nitrous oxide (N2O) and NO were from Matheson, and platelet aggregation. The existence of more stable transport isobutane was from Med Tech (Medford, MA). forms of NO has been postulated in view of the short half-life Experiments. The reactions were conducted in 0.01 M or of authentic NO in vivo (3). Low molecular weight thiols such 0.05 M potassium phosphate buffer (pH 7.4) at room temper- as cysteine, glutathione (GSH), and penicillamine are prime ature in vials equipped with rubber septa. The pH of GSH and candidates for such carrier molecules, and they can form GSNO solutions in the appropriate buffers was adjusted to 7.4 S-nitrosothiols on reaction with oxides of nitrogen (4). It has with diluted KOH. When needed, MORytrimethylurea (1 mM been assumed that the biological effects of these compounds each) solutions were prepared similarly and added to the are due to the spontaneous release of NO; however, this GSHyGSNO solution. A fresh solution of GSNO was prepared hypothesis is not supported by currently available data (5–7). immediately before each experiment, and the concentration Although a few studies have been carried out in an attempt was confirmed spectrophotometrically at 334 nm. Typically, to determine the reaction products and to deduce the mech- the GSNO concentration was fixed at 1 mM and the GSH anism of the modification of the thiol group by S-nitrosothiols, concentration was varied between 1 and 10 mM. For reactions the experiments were purely qualitative and no clear mecha- under argon or nitrogen, the vials, containing the appropriate nistic picture has emerged (8, 9). In this report, we describe the reaction of GSNO with GSH, a tripeptide with intracelluclar Abbreviations: GSH, glutathione; GSSG, oxidized GSH; GSNO, S-nitrosoglutathione; MOR, morpholine; NMOR, N-nitrosomorpho- line; NTMU, N-nitroso-N9,N9,N-trimethylurea; ESI-MS, electrospray The publication costs of this article were defrayed in part by page charge ionization mass spectrometry. payment. This article must therefore be hereby marked ‘‘advertisement’’ in §To whom reprint requests should be addressed. e-mail: srt@ accordance with 18 U.S.C. §1734 solely to indicate this fact. mitvma.mit.edu. 14428 Downloaded by guest on September 26, 2021 Biochemistry: Singh et al. Proc. Natl. Acad. Sci. USA 93 (1996) 14429 buffer, were deaerated for 30 min prior to use. Concentrated HPLC Analysis. HPLC studies, for quantification of GSSG, solutions of GSNO that were not deaerated were added in were conducted using a reversed-phase column (Supelcosil small amounts ('50 ml) along with deaerated GSH solutions LC-18-DB, 25 cm 3 4.6 mm). The gradient was obtained with by using a Hamilton syringe. For experiments in which NMOR eluent A (0.1% phosphoric acid) and eluent B (acetonitrile). was measured, the MOR solution was added to the buffer After injecting the sample (20 ml in 1 ml of 0.1% phosphoric solution prior to deaerating. The vials were then stored in acid) along with a fixed amount of an internal standard argon- or nitrogen-filled balloons for 24 or 40 h. Internal (N-acetylthiazolidine-4-carboxylic acid), a linear change from standard was added to the vial at the end of the reaction and the 100% A to 80% A over 20 min was carried out. The GSSG area solutions were then analyzed for the compounds of interest. was normalized to the area of the internal standard. The flow Electrospray Ionization Mass Spectrometry (ESI-MS). rate was 1 mlymin, and the detector absorbance was at 215 nm. These experiments were done on a Hewlett Packard model 5989B mass spectrophotometer in the negative ion mode. The reactions were conducted in ammonium acetate (1 mM; pH RESULTS '7.5) buffer at room temperature or at 48C, and the reaction Nitrite Formation. The half-life of GSNO was dependent on mixture was analyzed at the end of 2 h. Typically, 50 mlof the concentrations of GSNO and GSH. As measured by the reaction solution was added to 100 ml of the electrospray decrease in absorbance at 334 nm, GSNO (1 mM) was stable solvent [50:50 waterymethanol containing 1% acetic acid or for at least 90 min in the absence of GSH. Decomposition of 50:40:10 (volyvol) methanolywateryacetonitrile containing GSNO was observed upon addition of GSH (0.5–10 mM). The 1% ammonium hydroxide]. A Harvard syringe pump was used effect was dependent on the GSH concentration (Fig. 1). This to introduce the electrospray solvent into the mass spectrom- is in contrast to previously reported chemical stabilization of eter at 20 mlymin and the samples were flow-injected into the S-nitrosothiols in the presence of excess thiol (22). In the solvent stream by means of a Valco injector with a 10-ml absence of oxygen, the rate of GSNO decomposition in the sample loop. Under these conditions, the residence time of the presence of GSH decreased by a factor of 2 (data not shown). samples in the mass spectrometer was about 0.6 min. Spectra GSNO (1 mM) was incubated with various amounts of GSH were averaged over the maximum central portion '0.3 min) of (1–10 mM) in aerated or deaerated phosphate buffer, and the the resulting total-ion plot. reaction mixtures were analyzed for nitrite and nitrate. Nitrite Determination of Nitrite and Nitrate.