One-electron reduction of S-nitrosothiols in aqueous medium

Free Radical Biology & Medicine 41 (2006) 1240 – 1246 www.elsevier.com/locate/freeradbiomed

Original Contribution

One-electron reduction of S-nitrosothiols in aqueous medium☆ V.M. Manoj a , H. Mohan b , U.K. Aravind a , C.T. Aravindakumar a,⁎ a

b

School of Chemical Sciences, Mahatma Gandhi University, Kottayam 686560, Kerala, India Radiation Chemistry and Chemical Dynamics Division, Bhabha Atomic Research Center, Mumbai, India Received 7 September 2005; revised 26 June 2006; accepted 29 June 2006 Available online 11 July 2006

Abstract One-electron reduction of S-nitrosothiols (RSNO) has been studied using radiolytically produced reducing entity, the hydrated electron (e−aq), in aqueous medium. Both kinetics of the reaction and the mechanistic aspects of the decomposition of S-nitroso derivatives of glutathione, Lcysteine, N-acetyl-L-cysteine, N-acetyl-D,L-penicillamine, N-acetylcysteamine, L-cysteine methyl ester, and D,L-penicillamine have been investigated at neutral and acidic pH. The second-order rate constants of the reaction of e−aq with RSNOs were determined using a pulse radiolysis technique and were found to be diffusion controlled (1010 dm3 mol− 1 s− 1) at neutral pH. The product analysis using HPLC, fluorimetry, and MS revealed the formation of thiol and nitric oxide as the major end products. It is therefore proposed that one-electron reduction of RSNO leads to the liberation of NO. There is no intermediacy of a thiyl radical as in the case of oxidation reactions of RSNOs. The radical anion of RSNO (RSN O−) is proposed as a possible intermediate. The overall reaction could be written as © 2006 Elsevier Inc. All rights reserved.

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Keywords: S-Nitrosothiols; Nitric oxide; One-electron reduction; Hydrated electron; Radiation chemical; Pulse radiolysis; Rate constants; Thiyl radical; N-centered radical

Introduction S-Nitrosothiol (RSNO), a molecule formed from the interaction of nitric oxide (NO) and thiol having a free S–H group under physiological conditions, is believed to act as a bioreservoir for NO [1,2]. The formation of RSNO in biological systems is understood as a result of the reaction of NO, produced from the conversion of L-arginine to L-citrulline by the action of nitric oxide synthase (NOS), with Abbreviations: RSNO, S-nitrosothiol; NO, nitric oxide; NOS, nitric oxide synthase; GSH, glutathione; CySH, L-cysteine; ACySH, N-acetyl-L-cysteine; PSH, D,L-penicillamine; APSH, N-acetyl-D,L-penicillamine; ACSH, N-acetylcysteamine; CMESH, L-cysteine methyl ester; DAN, 2,3-diaminonaphthalene; ACySNO, S-nitroso-N-acetyl-L-cysteine; APSNO, S-nitroso-N-acetyl-D,Lpenicillamine; NAPSNO, S-nitrosoacetylpenicillamine; CMESNO, S-nitrosocysteine methyl ester; ABTS2-, 2,2-azino-bis[3-ethylbenziazoline-6-sulfonic acid]; CME, cysteine methyl ester. ☆ Part of this work was presented at the 3rd International Conference on the Biology, Chemistry and Therapeutic Applications of Nitric Oxide, Nara, Japan, May 24–28, 2004. ⁎ Corresponding author. Fax: +91 481 2731009. E-mail address: [email protected] (C.T. Aravindakumar). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.06.025

protein thiols under physiological conditions. Alternatively, its formation is predicted as a result of trans-nitrosation (direct exchange of NO+ between nitrosothiol and thiolate). Reports on the S-nitrosation process demonstrate that the principal target of cellular NO is the thiol group of Cys in peptides and proteins and that S-nitrosation conveys a large part of the regulatory influence of NO on cellular signal transduction [3]. Since the release of NO from RSNO, obviously, is of great importance, the various ways by which it degrades under physiological conditions are of current interest. Both oxidative and reductive conditions favor its degradation [4–12]. The bioactivity of RSNO depends mostly on the reducing environment within the cells [13]. For example, release of NO from GSNO is reported in the presence of endogenous cellular reductants and transition metals [14–16]. The reduction of GSNO might be catalyzed by superoxide dismutase and thioredoxin [16,17]. Reducing agents such as thiol [5,6], ascorbic acid [7–9], and superoxide [10–12] can induce the decomposition of RSNO. In the case of ascorbate and thiols, the reaction of the former with RSNO is metal-ion dependent while the latter is independent of metal-ion effect. Ascorbate-induced reaction enhances the bioactivity while

V.M. Manoj et al. / Free Radical Biology & Medicine 41 (2006) 1240–1246

thiols do not have any effect on the bioactivity [8]. At low concentrations, ascorbate acts as a reducing agent and the reaction is dependent on [Cu2+]. Ascorbate converts Cu2+ to Cu+ and subsequently the Cu+ induces the decomposition of RSNO to the corresponding disulfide (RSSR) and NO. But at high concentrations of ascorbate, the reaction is independent of Cu2+. In this case, a nucleophilic attack at nitroso nitrogen occurs and the final products are RSH and NO. 2RSNO þ ascorbate → 2RS− þ 2NO þ dehydroascorbic acid: ð1Þ In metal-induced decomposition, RS− converts Cu2+/Fe3+ to Cu+/Fe2+. The oxidative species thus formed converts RSNO to NO and RS−. Fe3þ =Cu2þ þ RS− → 1=2RSSR þ Cuþ =Fe2þ

ð2Þ

Fe2þ =Cuþ þ RSNO → RS− þ NO þ Cu2þ =Fe3þ :

ð3Þ

In the presence of reducing agents like thiol and ascorbic acid, Cu2+ is converted to Cu+ which in turn decomposes RSNO to RS− and NO. During superoxide-induced decomposition, the products were found to be nitrite, nitrate, and disulfide.

:

2GSNO þ O2  þ H2 O→GSSG þ NO−3 þ NO−2 þ 2Hþ :

ð4Þ

In all the observations with superoxide radicals, xanthine oxidase/xanthine systems were used to produce superoxide. A number of contradictory reports on the magnitude of the rate constants for the decomposition reaction induced by superoxide radical have been published [10–12]. However, a second-order rate constant of 300 dm3 mol− 1 s− 1 for this reaction using a radiation chemical technique has been determined and is very much lower than other reported values [12]. In order to understand the underlying mechanism of the reductive cleavage − of RSNOs, radiolytically produced hydrated electrons (eaq ) have been utilized. It is expected that understanding the chemistry of the reduction reaction is certainly important in elucidating the various steps in the reduction of RSNOs under biological conditions. It is also realized that not only the stable products but also the possible intermediates may have a potential role in different biological activities connected with RSNO. The radiation chemical method provides a clean environment without any interference from other reagents and hence the reduction can be studied under selective reducing conditions. Experimental procedures Materials Glutathione (GSH), L-cysteine (CySH), N-acetyl-L-cysteine (ACySH), D,L-penicillamine (PSH), N-acetyl-D,L-penicillamine (APSH), N-acetylcysteamine (ACSH), L-cysteine methyl ester (CMESH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (Aldrich), sodium nitrite, and ethylenediaminetetraacetic acid (Merck), 2,3-diaminonaphthalene (Alexis) were used without further purifications.

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Synthesis and preparation of RSNO solutions RSNO was synthesized from RSH using sodium nitrite/ HCl [18]. The purity of all these RSNOs was checked by HPLC. Millimolar solutions of RSNO were prepared in triply distilled water. EDTA (0.1 mM) was added to scavenge any traces of metal ions. The blank solutions containing EDTA were kept in dark for 24 h and were found to be stable. HCl and NaOH were used to adjust the pH of the solutions. Production of hydrated electrons using radiation chemical technique − γ-Rays were used to produce hydrated electron (eaq ) in aqueous solutions of RSNOs. Radiolysis of water produces the following species

ð5Þ The indirect effect of radiation, which involves the reaction of water-derived free radicals with selected solutes, is prominent in aqueous solutions containing low concentrations (mM) of solute molecules. 2-Methyl-2-propanol (0.2 mol dm− 3) was used to scavenge OH radicals. Under these conditions, the prominent − reactive species in N2-saturated aqueous solutions is eaq . The yields of various radicals and molecular products are normally expressed as G values which is defined as the number of molecules formed or destroyed per 100 eV absorption of radia− tion energy and in SI units, the yields are G( OH) ≈ G(eaq )≈ + G(H3O ) = 0.28, G( H) = 0.062, G(H2O2) = 0.072, and G(H2) = 0.047 μmol J− 1. γ-Irradiations were carried out using a 60Co-γ source. In the presence of 2-methyl-2-propanol, the reaction of − eaq can be selectively studied as it scavenges the OH (reaction (6)). The dose rate was determined using ceric sulfate dosimetry [19] and was about 75 Gy/min.

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:OH þ ðCH Þ COH→:CH ðCH Þ COH þ H O: 3 3

2

3 2

2

ð6Þ

End product analyses The decay of RSNOs were monitored using a UV-VIS spectrophotometer (Shimadzu UV 160A) and HPLC (Shimadzu LC-10 AS) with UV-VIS detector (Shimadzu SPD 10A) having a 25-cm, Nucleosil, 5C-18 column. A 50% mixture of disodium phosphate (1 × 10− 3 mol dm− 3) and sodium sulfate (10 × 10− 3 mol dm− 3) in water (pH 6) was used as the mobile phase. The flow rate was kept at 0.75 ml/min. The column was deaerated before each analysis. The products were monitored using HPLC and MS (Shimadzu GCMS-QP 5050). The samples collected from HPLC elute were extracted and injected directly into MS to record the mass spectra using selected ion monitoring (SIM) mode. Pulse radiolysis The pulse radiolysis technique was used to investigate the kinetics of the reaction by monitoring the decay of the hydrated

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− electron (eaq ) at 715 nm, and the details of the possible intermediates. This setup consists of a linear accelerator (LINAC) delivering 7 MeV electron pulses of 50 ns duration which is connected with an optical detection unit. The details of this setup have been published elsewhere [20].

Fluorescence spectrophotometer The fluorescence spectra were recorded by using an RF-5301 PC (Shimadzu) spectrofluorimeter. The light source used was 150 W xenon lamp with ozone resolving type lamp housing. The emission was focused into a monochromator and detected with a R928 photomultiplier tube. The excitation wavelength was set at 365 nm. Fluorescent detection of NO with 2,3-diaminonaphthalene (DAN) The fluorescence analyses were conducted by adding 0.5 ml of DAN solutions (10 mg of DAN was dissolved in 100 ml of 0.25 N hydrochloric acid) to 5 ml of the irradiated solutions. The mixture was shaken and allowed to stand for 15 min. 0.25 ml of 0.6 mol dm− 3 sodium hydroxide solution was then added and the fluorescence emission was immediately recorded [21,22].

Table 1 The second-order rate constants obtained for the reaction of e−aq with RSNOs at pH 7 RSNO

Rate/dm3 mol− 1 s− 1 (error value: 0.06–0.08)

S-Nitrosoglutathione (GSNO) S-Nitroso-L-cysteine (CySNO) S-Nitroso-N-acetyl-L-cysteine (ACySNO) S-Nitroso-N-acetyl-D,L-penicillamine (APSNO) S-Nitroso-N-acetylcysteamine (ACSNO) S-Nitroso-L-cysteine methyl ester (CMESNO) S-Nitroso-D,L-penicillamine (PSNO)

1.1 × 1010 2.4 × 1010 2.1 × 1010 3.5 × 1010 1.9 × 1010 1.5 × 1010 1.6 × 1010

These plots gave straight-line graphs with very good correlation coefficients (R ≥ 0.98). The rate constants are presented in Table 1. Ford et al. reported a second-order rate constant for the − with GSNO as 1.86 × 1010 dm3 mol− 1 s− 1 [11]. reaction of eaq It can be seen that the value in the present case matches very well with the earlier report. The values of the rate constants of other RSNOs are also in the same range. Reductive decomposition

− The reaction of the hydrated electron (eaq ) has been carried out in argon-saturated solutions of RSNOs containing 2methyl-2-propanol at pH 3 and 7. The second-order rate − constants of the reaction of eaq with the selected RSNOs were − determined by monitoring the decay of the eaq at 715 nm using the pulse radiolysis technique. The pseudo-first-order decay constants (kobs) were determined from the decay traces at 715 nm (Fig. 1). The second-order rate constants were then determined from the slope of kobs versus [RSNO] plots (Fig. 1).

− The decomposition reactions of RSNOs induced by eaq were investigated at neutral and acidic pH. Solutions containing RSNO and 2-methyl-2-propanol, saturated with Ar, were irradiated at different doses using γ-rays. The decay of RSNO solutions after irradiation was monitored using a UV-VIS spectrophotometer. A representative plot in the case of GSNO is shown in Fig. 2. It is observed that the absorption maximum at 334 nm, characteristic of the GSNO, has been decreased with increase in dose. This shows that the decay is dose dependent. The absorption maximum has almost vanished after about 2.5 kGy. Correspondingly a new peak at 272 nm is formed at pH 3. Similarly, a red-shifted spectrum around 270 nm is

Fig. 1. kobs versus concentration plot obtained for the reaction of e−aq with ACySNO (⋄), CySNO ( ), ACSNO (▴), and PSNO (⁎) using pulse radiolysis (similar plots were obtained in the case of GSNO, APSNO, and CMESNO). Inset: Decay traces of e−aq at 715 nm (a) in the absence of ACySNO and (b) in the presence of ACySNO (0.75 × 10− 4 mol dm− 3).

Fig. 2. Decay profile of the hydrated electron-induced degradation of GSNO (1 × 10− 3 mol dm− 3) at pH 7, after gamma irradiation at doses (i) 0 kGy, (ii) 0.419 kGy, and (iii) at 2.095 kGy, analyzed using UV/VIS spectrophotometer. Inset: (a) After gamma irradiation at doses (a) 0 kGy, (b) 0.419 kGy, and (c) 2.514 kGy, at pH3, and (b) absorption spectra of GSH at pH 3 (—) and at pH 7 (– – –).

Results Kinetics

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obtained after 2.5 kGy at pH 7. The isobestic point in the UV spectra suggests that the product having absorption maximum at 272 nm is formed from GSNO. The absorption spectra of glutathione (GSH) at pH 3 and 7 were also recorded and are given in the inset of Fig. 2. The pKa of GSH is 3.53, 8.66, and 9.12 [23]. The value 3.53 corresponds to the acid–base equilibrium of carboxylate anion (COOH ⇆ COO− + H+) and hence it is clear that the difference in the nature of the absorption spectra is due to its pKa at 3.53. A similar pattern of decay was obtained for other RSNOs as well. The HPLC analyses of the irradiated solutions were also carried out. The HPLC chromatogram obtained in the case of GSNO is shown in Fig. 3. The chromatogram revealed that GSH is the major product of the degradation of GSNO. In order to further confirm the formation of RSH from the degradation of RSNO, the product fraction obtained from the HPLC was separated in the case of S-nitroso-Nacetyl-L-cysteine (ACySNO). This is then extracted with chloroform and injected to MS and the analysis was carried out using SIM mode. The fragments obtained were 163 (ACySH), 118 (-CO2H), 75 (-CO2H,-CH3CO), 60 (-CO2H,-CH3CONH), 45 (CO2H). and 43 (CH3CO). The formation of parent thiol from the reductive reaction of RSNO is thus reconfirmed. Quantitative estimation of the dose-dependent decay of all the selected RSNOs has been carried out and a typical plot obtained in the case of S-nitroso-N-acetyl-D,L-penicillamine (APSNO) is shown in Fig. 4. From the initial decay, the G values for the decomposition of RSNOs were calculated and these values are close to − 0.28 μmol J− 1. The G(eaq ) is 0.28 μmol J− 1, which means that there is a quantitative decay of RSNO. The G values of RSH (formation) were also calculated from the initial formation of RSH (Fig. 4) and these are around 0.14 μmol J− 1 (Table 2). In addition to RSH, it is initially assumed that NO is liberated from this reaction. NO cannot be directly detected by HPLC or UV. It is reported that in aerated aqueous

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Fig. 4. HPLC analysis of the degradation of S-nitroso-N-acetyl-D,L-penicillamine (APSNO) (1 × 10− 3 mol dm− 3) at pH 7 and the formation of N-acetyl-D, L-penicillamine (APSH) in argon-saturated solution at pH 3 using γ-radiolysis.

solutions, NO is converted to nitrite (reaction 7) [24]. So, the degradation reaction was carried out in a partially oxygenated solution. ð7Þ The HPLC chromatogram obtained after this reaction is shown in the inset of Fig. 3. A nitrite peak was obtained along with a GSH peak which confirmed that NO is also formed along with GSH during the reduction reaction. A G value of the formation of nitrite is determined as 0.14 μmol J− 1. It is also reconfirmed that under Ar-saturated as well as partially oxygenated conditions, the GSNO undergoes a similar degradation pattern. The pH of the solution is expected to be low due to the formation of HNO2. After the irradiation, the pH of the solution under aerated conditions was found to be significantly low compared to that under argon-saturated

Fig. 3. The HPLC chromatogram after irradiation (1.398 kGy) of argon-saturated solutions of GSNO (1 × 10−3 mol dm−3) at pH 7 (a) nitrite, (b) GSH, and (c) GSNO. Inset: (i) Normalized fluorescent emission spectra of 1-(H)naphthotriazole, formed from the reaction of 2,3-diaminonaphthalene (DAN) with NO formed from the reduction reaction of S-nitrosoacetylpenicillamine (NAPSNO, 1 × 10− 3 mol dm− 3), excited at 365 nm. (1) 466 Gy, (2) 1398 Gy, and (3) 2330 Gy. (ii) After the irradiation of partially oxygenated solutions of GSNO (1 × 10− 3 mol dm− 3) at pH 7.

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Table 2 G values of the decomposition of RSNO and the formation of RSH from the reaction of e−aq with RSNO RSNO

G( −RSNO)/μmol J − 1

G(RSH)/μmol J − 1

pH ≈ 3

pH ≈ 7

pH ≈ 3

pH ≈ 7

GSNO ACySNO CySNO APSNO ACSNO CMESNO PSNO

0.28 0.29 0.28 0.28 0.29 0.28 0.28

0.28 0.28 0.29 0.28 0.28 0.28 0.28

0.14 0.15 0.15 0.14 0.14 0.14 0.15

0.15 0.14 0.15 0.14 0.14 0.15 0.14

conditions (Table 3). Similar observations were shown by other RSNOs as well. This confirms the formation of NO during the reductive decomposition. The NO production was reconfirmed using the DAN fluorescence method [21,22] with S-nitrosoacetylpenicillamine (NAPSNO) (Fig. 3). It can be seen that the fluorescence at 405 nm increased with dose which confirmed the formation of NO during the reduction reaction. In order to gain some details of the transient intermediates, pulse radiolysis experiments were carried out. At neutral pH, the transient absorption spectra in the range 300–600 nm showed no significant absorption (Fig. 5) in the case of S-nitrosocysteine methyl ester (CMESNO) other than a bleaching at 300– 400 nm, which is due to the decay of CMESNO. Pulse radiolysis experiments were also carried out for the reduction of CMESNO in the presence of low concentration of 2,2-azino-bis [3-ethylbenziazoline-6-sulfonic acid] (ABTS2−) at neutral pH. This experiment was used to investigate if thiyl radicals (RS ) were formed as an intermediate. If so, one can obtain a clear absorption buildup of ABTS − at 425 nm as demonstrated in the case of the reaction of OH with RSH [25]. However, no such buildup was obtained in the present case, indicating the absence of RS as the intermediate. Fig. 5 shows a comparison between the reaction of OH with cysteine methyl ester (CME) (where RS is the intermediate [26]) and with S-nitrosocysteine methyl ester at neutral pH in the presence of ABTS2− .

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Discussion − The quantitative decay of G(−RSNO ≈ G(eaq )) as well as the 10 3 −1 −1 high rate constants (10 dm mol s ) are clear indications − of the high affinity of eaq with these compounds. The rate constants are slightly higher compared to the rate constants of − the reaction of eaq with the corresponding sulfhydrils [27,28]. For example, the rate constants in the case of cysteine (8.5 × 109

Table 3 Observed pH changes of the GSNO solution with dose Dose/kGy

0 0.419 2.514

Fig. 5. Transient absorption spectra obtained in argon-saturated solutions of Snitrosocysteine methyl ester (CMESNO) (1 × 10− 3 mol dm− 3) at pH 7 at 4 μs after the pulse at neutral pH (dose per pulse = 15.4 Gy). Inset: Comparison of the reaction of e−aq with (CMESNO) and of OH with cysteine methyl ester (CME) (1 × 10− 3 mol dm− 3) at pH 7 in the presence of ABTS2− (5 × 10− 5 mol dm− 3). (a) The ABTS − buildup at 425 nm obtained from the reaction of OH with CME and (b) absorption trace obtained at 425 nm for the reaction of e−aq with CMESNO in the presence of ABTS2−.

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dm3 mol− 1 s− 1), and penicillamine (5.1 × 109 dm3 mol− 1 s− 1) are smaller compared to the S-nitroso derivatives of cysteine (2.4 × 1010 dm3 mol− 1 s− 1) and penicillamine (1.6 × 1010 dm3 − mol− 1 s− 1). In the case of sulfhydryls, the reaction with eaq follows two pathways, a rapid dissociative electron capture reaction producing R and H2S, and the formation of RS and H2 [27,28]. In the present case –SH is replaced by –S–N _ O and hence the reaction mechanism is obviously different from the previous case. The product analyses have revealed that NO and the respective thiol are the major products. The possibility of the formation of a thiyl radical intermediate can be ruled out from the pulse radiolysis studies where no indication of the absorption spectrum of CMES or the formation of ABTS − from the reaction of CMES with ABTS2− was obtained (Fig. 5). The inset of Fig. 5 clearly showed a buildup of absorption of ABTS − at 425 nm due to the electron transfer reaction of the thiyl radical (which is formed from the reaction of hydroxyl radical ( OH) with CMESH, reaction (8)) to ABTS − (reaction (9)). This buildup is complete within 20 μs and no such − buildup of absorption is observed when the reaction of eaq was 2− carried out in the presence of ABTS .

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:

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:

CMESH þ OH → CMES þ H2 O

:

:

CMES þ ABTS2 → CMESþ þ ABTS  :

Ar saturated

Aerated

6.9 6.7 6.9

7.1 6.1 5.3

ð9Þ

Thiyl radical is the major intermediate in the oxidative degradation of RSNO induced by hydroxyl radical [4,29]. It can − with RSNO does not be thus concluded that the reaction of eaq proceed through a thiyl radical intermediate. It is therefore proposed that a radical anion [RSNO] − is initially formed as an intermediate (reaction (4)) for which no clear experimental evidence is available. It is also probable that this species is highly unstable. Hence the radical anion is likely to get dissociated to thiolate anion (RS−) and NO. As the pKa values of low molecular weight thiols corresponding to the thiol/

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pH

ð8Þ

V.M. Manoj et al. / Free Radical Biology & Medicine 41 (2006) 1240–1246

thiolate equilibrium (−SH ⇆ −S− + H+) are greater than 9 (for example; pKa of GSH is 9.12 and for CysH is 10.78) [23], the thiolate is immediately protonated to the corresponding thiol at pH 7 as well as at pH 3. This can be understood from the UVVIS spectrum of the fully decayed GSNO at pH 3 as well as at pH 7 and the comparison of the spectrum of GSH at pH 3 and 7 (Fig. 2). The difference in the UV-VIS spectrum at pH 7 and at pH 3 (Fig. 2) is attributed to the existence of a high percentage of the acid group and a low percentage of the carboxylate group [30]. The proposed mechanism of the reaction can be, thus, summarized as in reactions (10)–(13). The reduction reactions of RSNOs initiated by Cu+ [5] as well as formate radicals (CO2 −) [11] follow a similar reaction pattern where the major products are NO and thiol. Electrochemical reduction also leads to the formation of NO and RS−, which subsequently gets protonated to form thiols [31].

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: þ :NO

RSNO þ e−aq → RSN O−

ð10Þ

RSN O− → RS−

ð11Þ

:

RS− þ Hþ ⇆ RSH:

ð12Þ

Therefore, the overall reaction can be written as ð13Þ It can also be assumed that in the reduction reaction of RSNO such as the reduction by ascorbic acid, formate radical, and thiol, leading to the formation of RSH and NO, the intermediate entity would be a radical anion of the type RSN O−.

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theoretically confirmed using a kinetic model and it has also led to the conclusion that higher oxides of nitrogen (viz. NO2 and N2O3) do not play a significant role in an intracellular Snitrosation reaction [33]. Recent in vitro studies [35] on the Snitrosation of the nucleotide binding protein, Ras, also predicted the importance of the radical intermediate in the enhanced guanine nucleotide exchange (GNE). The lifetime of the proposed intermediate radical in the present study, RSN O−, is expected to be very low and it will be interesting to understand more on its stabilization under in vivo conditions. It is, however, not clear under what circumstances the radical anion gets stabilized and this merits further investigation. Furthermore, it is interesting to note that the one-electron reduction reaction presented in this study and the photochemical liberation of NO from RSNO [36,37] are NO-releasing reactions. However, the reaction mechanism is very different where the photochemical degradation always proceeds via a homolytic cleavage with the formation of thiyl radical as the intermediate [31]. Another important point concerning the use of S-nitrosothiol-based drugs is that any reduction reaction such as the reaction with ascorbate may interfere with the sitespecific release of nitric oxide from the drugs. It is reported that ascorbate promotes NO production from GSNO in blood plasma [15]. Such competition reactions must be thoroughly understood and it may help in the design of any kind of NOreleasing drugs.

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Acknowledgments V.M.M. is thankful to CSIR, New Delhi, for a Senior Research Fellowship. Part of the financial support for this work is from BRNS, Government of India. We are also thankful to RRII, Kottayam, for providing the gamma source.

Conclusion References Being a bioreservoir of NO, the reduction reaction of RSNOs leading to the release of NO is of high importance. It can be understood that any reductive degradation of RSNO leading to the liberation of NO is a result of a one-electron reduction reaction. The proposed intermediate radical, RSN O−, is an intermediate entity in the S-nitrosation of thiolate groups at physiological concentrations of NO as suggested by Gow et al. [32] in a slightly different form. The subsequent electron transfer to an electron acceptor such as oxygen would ultimately lead to the formation of the RSNO. The fact that in the presence of oxygen in the present study, we did not observe any change in the degradation profile of GSNO compared to that in the absence of oxygen, it can be understood that the degradation of RSNO to RSH and NO at neutral and acidic pH is not a reversible reaction. As the principal target of the cellular NO is the thiol group of Cys in peptides and proteins, the S-nitrosation of the thiolate moiety is extremely important. Since the reaction of NO with thiols is proven as an electrophilic attack of NO on thiolate anion [33,34], it can be assumed that a radical anion of RSNO (i.e., RSN O−), as proposed in this work, will be formed as an intermediate (RS− + NO → RSN O−). The mechanism suggested by Gow et al. [32] (in a slightly different form) was

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