Superoxide Modulates the Oxidation and Nitrosation of Thiols by Nitric Oxide-derived Reactive Intermediates
CHEMICAL ASPECTS INVOLVED IN THE BALANCE BETWEEN OXIDATIVE AND NITROSATIVE STRESS*

(Received for publication, September 19, 1996, and in revised form, December 20, 1996)

David A. Wink Dagger §, John A. Cook Dagger , Sungmee Y. Kim Dagger , Yoram Vodovotz Dagger , Roberto Pacelli Dagger , Murali C. Krishna Dagger , Angelo Russo Dagger , James B. Mitchell Dagger , David Jourd'heuil , Allen M. Miles par and Matthew B. Grisham

From the Dagger  Tumor Biology Section, Radiation Biology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, the  Department of Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130, and the par  Department of Chemistry, Grambling State University, Grambling, Louisiana 71245

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by Obardot 2, while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or Obardot 2. A solution of glutathione (GSH), a model thiol-containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and Obardot 2 were nearly equivalent. The presence of superoxide dismutase reversed Obardot 2-mediated inhibition, while catalase had no effect. Increasing the flux of Obardot 2 yielded oxidized glutathione (GSSG), peaking when the flux of NO and Obardot 2 were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.


INTRODUCTION

The endogenously formed radicals, NO1 and superoxide (Obardot 2), are thought to play key roles in the regulation of a number of physiological and pathophysiological mechanisms. This interaction has been suggested to be pivotal in cytotoxic mechanisms involving the modulation of oxidative stress by NO. Thiols are critical sites of interaction for NO and Obardot 2 in biological systems, perhaps explaining why proteins and peptides containing thiols that undergo nitrosation as well as oxidation reactions might modulate cellular function. Reactions of chemical species derived from NO can result in the nitrosation of thiols to form S-nitrosothiol complexes (1, 2). Oxidation of thiols by oxidants, some of which are derived from NO (3), results in disulfide products, affecting cellular metabolism and physiological functions (4-7).

Recently, we have shown that oxidative and nitrosative chemistry resulting from a continuous chemical generator of NO, SPER/NO (a NONOate complex that spontaneously releases NO at physiological pH), in the presence of a source of Obardot 2, xanthine oxidase (XO), is modulated by the relative rate of formation of NO and superoxide (8, 9). On one hand, the nitrosative chemistry of amines is quenched by the presence of Obardot 2 (8), while increasing the flux of Obardot 2 increases oxidation mediated by peroxynitrite (9). On the other hand, oxidation mediated by peroxynitrite is quenched by excess formation of either NO or Obardot 2 (9). We have explored the oxidative and nitrosative chemistry resulting from the interaction of NO and Obardot 2 with thiols and demonstrate that there is a balance between fluxes of NO and Obardot 2 with respect to oxidative and nitrosative products of glutathione.


MATERIALS AND METHODS

Glutathione (GSH), hypoxanthine (HX), superoxide dismutase (SOD), catalase, and diethylenetriaminepentaacetic acid (DETAPAC) were purchased from Sigma. Stock solutions were prepared as follows: 50 mM HX was made in 0.08 mM NaOH, 10 mM DETAPAC in milliQ water, and 10 mM GSH in milliQ water. These stock solutions were diluted in PBS (pH 7.4) to the reported final concentration. XO was purchased from Boehringer Mannheim. DEA/NO was a generous gift from Dr. Joseph Saavedra. Stock solution of DEA/NO were prepared as described previously (10), and stock concentrations were determined by UV absorption at 250 nm, using an extinction coefficient of 8000 M-1 cm-1.

Reaction Method

A solution containing 0.5 mM HX and 50 µM DETAPAC in PBS was prepared and referred to as the HX solution. Typically, GSH or DAN was added to the solutions, followed by catalase and/or SOD, and when appropriate, XO was followed by DEA/NO. Samples were incubated for 1 h at 37 °C. At the end of this time DEA/NO was completely decomposed and 50 µM allopurinol was added to inhibit the activity of XO. The solutions were then analyzed as described below. Under these assay conditions, 1 milliunit/ml XO produced a flux of Obardot 2 and H2O2 of approximately 1 nmol/min each as described previously (9).

Determination of Decomposition of DEA/NO and Activity of XO

The rate of the decomposition of DEA/NO is typically determined by the loss of its characteristic absorbance at 250 nm. However, the HX solution strongly absorbs in this region and does not allow an accurate determination of the kinetics of decomposition. We have recently described a method used to determine the amount of NO released from an NO donor, which involves monitoring at 500 nm where HX solution does not absorb (11). In a solution containing 0.5 mM HX and 50 µM DETAPAC, 0.5 g/100 ml of sulfanilamide and 0.03 g of N-(1-naphthyl)ethylenediamine dihydrochloride (NEDD)/100 ml of PBS was dissolved as reported previously (11). Monitoring the appearance of the characteristic azo dye band at 500 nm, the rate constant for decay of DEA/NO was determined by a plot of ln(Abs - Absinf) versus time. Since the absorbance requires 2 molecules of NO for every one of azo dye formed, this rate was multiplied by two to get the rate of NO release under these conditions.

Detection of GSNO

GSNO was detected using the fluorescence method described by Cook et al. (12). 200 µl of the sample solution was placed in a 2-ml solution containing 500 µM DAN and 100 µM mercuric chloride in PBS. The samples were allowed to stand at room temperature for 30 min and then read on a Perkin-Elmer fluorometer LS50B with excitation at 375 nm and emission at 450 nm. To standardize the fluorescence reading from naphthaltriazole (NAT) with S-nitrosothiol, known amounts of GSNO (0-10 µM) were treated in the identical manner described above. A standard curve was generated correlating [GSNO] with fluorescence. GSNO concentrations from the experiments were extrapolated from standard curve.

HPLC Method for Determination of GSSG

1 µl of the sample of the incubation mixture was taken and 145 µl of 0.6% sulfosalicylic acid and 10 mM N-butylmaleimide. To this, 34 µl of borate (200 mM) was added to adjust the pH to 8.5. The solution was allowed to stand for 5 min, at which time 20 µl of 1 mM 7-methoxycoumarin-3-carboxylic acid, succimidyl ester (Molecular Probes, Eugene; OR) was added, and the solution was incubated for 15 min. 25 µl of the sample was injected onto a Millipore HPLC system with a NOvapacTM C-18 (3.9 × 150 mm) column (Waters Corp., Milford, MA). A gradient was run with buffer A containing 5% acetonitrile, 34 mM sodium perchlorate, 0.25% glacial acetic acid, and buffer B containing 80% acetonitrile, 34 mM sodium perchlorate, 0.25% glacial acetic acid. Fluorescence was determined by excitation at 345 and emission at 410 nm.

Electrochemical Determination of NO

The production of NO via the spontaneous decomposition of DEA/NO at 37 °C in the presence or absence of different fluxes of superoxide and hydrogen peroxide was determined electrochemically using an electrode specific for NO (World Precision Instruments, Sarasota, FL). DEA/NO was introduced into a thermostatted cell containing a 2.5-ml reaction volume to a final concentration of 20 and 50 µM in a solution containing 50 µM DETAPAC, 500 µM HX, and when appropriate, 10 milliunits/ml XO in PBS (pH 7.4) ± SOD (0.1 mg/ml). The current obtained was monitored as a function of time, and the electrode was standardized using acidified NaNO2.


RESULTS

Formation of GSNO and NAT from DEA/NO and XO

In the presence of 0.2 mM GSH in PBS, 20 and 50 µM DEA/NO yielded GSNO (Fig. 1, A and B). When HX/XO was added with increasing activity, a marked decrease in the formation of GSNO was observed: less than 0.2 µM GSNO was formed at 5 and 10 nmol/min XO activity when the concentrations of DEA/NO were 20 and 50 µM, respectively (Fig. 1). However, nitrosation of GSH surprisingly increased as XO activity was increased to 100 milliunits/ml (Fig. 1, A and B), a process unaffected by 20 µM catalase. The lack of effect of catalase suggests that Obardot 2 and not hydrogen peroxide attenuates nitrosation. Such results lessen the concerns of oxygen depletion by xanthine oxidase, since catalase will adequately reoxygenate the solution by converting hydrogen peroxide to oxygen.


Fig. 1. Effects of XO on nitrosation of GSH from DEA/NO. Various activities of XO (nmol/min) in the presence of 20 µM (A) and 50 µM (B) DEA/NO. PBS solutions containing 50 µM DETAPAC, 0.5 mM HX, and 0.2 mM GSH were incubated at 37 °C for 1 h. GSNO was determined as described under "Materials and Methods." All points were averages of three experiments.
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Next, N-nitrosation was examined as a function of increasing XO activity. A solution of 0.5 mM DAN and 20-50 µM DEA/NO in PBS results in the formation of NAT. Unlike the formation of GSNO, increasing XO activity had little effect on nitrosation until XO activity was 5-10 milliunits, at which point the yield of NAT was dramatically reduced (Fig. 2, A and B). Analogously to the formation of GSNO, nitrosation of DAN increased as the flux of Obardot 2 and H2O2 was increased to 100 nmol/min each, though not to the same extent as that observed prior to addition of XO (Fig. 2, A and B).


Fig. 2. Effects of XO activity on the nitrosation of DAN from DEA/NO. Various activities of XO were incubated with 20 µM (A) and 50 µM (B) DEA/NO. PBS solutions containing 50 µM DETAPAC, 0.5 mM HX, and 0.5 mM DAN were incubated at 37 °C for 1 h. GSNO was determined as described under "Materials and Methods." All points were averages of three experiments. The fluorescence correspond to 46 per µM.
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Increasing amounts of SOD, and not catalase, increased the nitrosation of GSH resulting from 50 µM DEA/NO in the presence of 20 milliunits of XO (Fig. 3A)., suggesting that Obardot 2 and not hydrogen peroxide may attenuate nitrosation. Nitrosation of DAN was inversely proportional to the flux of Obardot 2 in this case as well (Fig. 3B). Since SOD would not affect the rate of oxygen consumption by XO but the availability of Obardot 2, reduction of GSNO and NAT formation by XO does not result from oxygen depletion but rather implicates Obardot 2 as being the responsible species for reducing nitrosation.


Fig. 3. Effect of SOD on the formation of GSNO and NAT. Solutions containing 20 mU XO in the presence of various amounts of SOD were exposed to 50 µM DEA/NO. Solutions containing either 50 µM DETAPAC, 0.5 mM HX, and 0.2 mM GSH in PBS (A) or 0.2 mM DAN (B) were incubated at 37 °C for 1 h. GSNO was determined described under "Materials and Methods." Formation of triazole was determined directly. All points were averages of three experiments.
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Formation of GSSG

Peroxynitrite can oxidize GSH to GSSG. The nearly diffusion controlled reaction between NO and Obardot 2 to form peroxynitrite (13) suggests this reaction could affect the redox status of intracellular and extracellular thiols. GSH can be oxidized in the presence of hydrogen peroxide (3); therefore, catalase was present in these experiments to prevent this side reaction, since the nitrosation of GSH was not effected by the presence of catalase. When 20 or 50 µM DEA/NO was exposed to a solution of HX containing 0.2 mM GSH, variation of XO activity increased formation of GSSG (Fig. 4). The levels of GSH decreased in both cases.


Fig. 4. GSSG formed in the presence of DEA/NO and GSH from XO. Solutions contained either 20 or 50 µM DEA/NO with various activities of XO. Solution contained 50 µM DETAPAC, 0.5 mM HX, catalase, and 0.2 mM GSH in PBS. Allopurinol was added to the end of each run.
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Detection of NO

An electrode sensitive for NO was used to determine the role of this diatomic molecule in the reactions described above. 50 µM DEA/NO was introduced to an aqueous solution as described under "Materials and Methods." The concentration of NO initially rose to ~6 µM and then fell to near zero (Fig. 5). This result is consistent with NO being released by DEA/NO, followed by consumption via the auto-oxidation reaction. When 10 milliunits of XO were present, NO was undetectable by this method. When 0.1 mg/ml SOD was present with 50 µM DEA/NO and 10 milliunits of XO, NO was detected, suggesting that Obardot 2 was responsible for the decrease in NO released from DEA/NO.


Fig. 5. Determination of the flux of NO from DEA/NO in the presence and absence of XO ± SOD (0.1 mg/ml). 50 µM DEA/NO was introduced to a solution containing 50 µM DETAPAC and 0.5 mM HX in PBS (A). To the same solution, 10 milliunits of XO were added without (B) or with 0.1 mg/ml SOD. NO was detected with an electrode sensitive for this molecule as described under "Materials and Methods."
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DISCUSSION

The formation of reactive intermediates derived from oxygen and NO has been invoked in mechanisms for the degradation of biomacromolecules, with attendant pathophysiological consequences. The near diffusion-controlled reaction between NO and Obardot 2 to form peroxynitrite (13) has suggested that this RNOS may be central in the toxic and regulatory mechanisms of NO and oxygen radicals (14, 15). One class of reaction products of NO is the S-nitrosothiols, which are formed in vivo and which have been suggested to regulate a variety of physiological functions (7, 16). It has been proposed that proteins modified by NO to contain S-nitrosothiol groups may regulate physiological functions such as vascular tension (16-18) as well as modulating the transcription of genes. Conversely, S-nitrosation of some proteins inhibits key enzymes (19, 20), including those which contain zinc finger motifs (19, 21, 22). Furthermore, NO via RNOS reacts with metallothionein to form an S-nitrosothiol (21), which either represents a detoxification pathway (23) or results in liberation of toxic metals such as cadmium (24).

In an attempt to examine the mechanisms by which such diverse actions are mediated, we have begun to examine the chemistry resulting from the interaction of NO and Obardot 2 with respect to nitrosation and oxidation (8, 9). Previously, we showed that the nitrosation of DAN was suppressed by the presence of Obardot 2 (8). As seen in Figs. 1 and 3, increasing concentrations of Obardot 2 dramatically reduced the nitrosation of both GSH and DAN. As seen in Fig. 3, SOD reversed this inhibition, suggesting that the reduction in nitrosation is due to the presence of Obardot 2. Since NO or resultant intermediates from NO donor complexes do not inhibit the activity of XO (25), this inhibition of nitrosation by Obardot 2 can be explained by the following mechanism. In the absence of XO, nitrosation is mediated by intermediates formed in the NO/O2 reaction (i.e. N2O3) (Equations 1-3).
4<UP>NO</UP>+<UP>O</UP><SUB>2</SUB> → 2<UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB> (Eq. 1)
<UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB>+<UP>GSH</UP> → <UP>GSNO</UP>+<UP>HNO</UP><SUB>2</SUB> (Eq. 2)
<UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB>+<UP>DAN</UP> → <UP>NAT</UP>+<UP>HNO</UP><SUB>2</SUB> (Eq. 3)
As superoxide concentration increases, NO reacts with it at nearly diffusion control to form ONOO- (Equation 4), which in turn can isomerize to nitrate upon protonation (Equation 5).
<UP>NO</UP>+<UP>O&cjs1138;<SUB>2</SUB> →</UP> <UP>ONOO<SUP>−</SUP></UP> (Eq. 4)
<UP>H</UP><SUP><UP>+</UP></SUP>+<UP>ONOO<SUP>−</SUP></UP> ⇔ <UP>HOONO</UP> (Eq. 5)
<UP>HOONO</UP> → <UP>HNO</UP><SUB>3</SUB> (Eq. 6)
This diversion of NO from the RNOS in the auto-oxidation reaction (Equation 1) may explain the inhibition of nitrosation, since ONOO- itself is not a nitrosating agent (8, 26).

The relative fluxes of NO and Obardot 2 are an important aspect of the foregoing chemical reactions. As described above, maximal oxidation and minimal nitrosation occurred when the activity of XO was 5-20 milliunits. Since the rate of formation of NO from DEA/NO was 5 × 10-3 s-1, the flux of NO generated by 20 and 50 µM DEA/NO would be predicted to be initially approximately 10 and 25 µM per min. This suggests that the maximal oxidation and minimal nitrosation occurs when the flux of NO and Obardot 2 were nearly equivalent. These results are consistent with our previous finding that maximal oxidation of dihydrorhodamine occurred when the rate of formation of Obardot 2 was nearly equivalent to that of NO (9).

The effects of XO on nitrosation of GSH and DAN in Figs. 1 and 3 were distinctly different, suggesting a difference in the effects on these two substrates of increasing the flux of Obardot 2. As XO increased from 1 to 10 milliunits, the formation of GSNO decreased, yet nitrosation of DAN did not decrease with increasing XO until XO activity was 5-10 milliunits. One reason for this discrepancy is the formation of ONOO-, which reacts sequentially with another 2 mol of NO to form N2O3, via the intermediate formation of NO2 (Equations 7 and 8) (27, 28), nitrosating DAN or GSH (Equations 1 and 2).
<UP>HOONO</UP>+<UP>NO</UP> → <UP>NO</UP><SUB>2</SUB>+<UP>NO</UP><SUP><UP>−</UP></SUP><SUB>2</SUB> (Eq. 7)
<UP>NO</UP><SUB>2</SUB>+<UP>NO</UP> → <UP>N</UP><SUB>2</SUB><UP>O</UP><SUB>3</SUB> (Eq. 8)
Radi et al. showed that GSH reacts with OONO-, the chemical species thought to be responsible for oxidation and nitration chemistry (3).
<UP>ONOO</UP><SUP><UP>−</UP></SUP>+<UP>GSH</UP> → 1/2 <UP>GSSG</UP> (Eq. 9)
The rate constant for ONOO- ion reacting with cysteine at pH 7.4 is 5900 M-1 s-1. Since cysteine reacts with ONOO-, the most likely explanation for the difference between N-nitrosation and S-nitrosation is that GSH intercepts ONOO- prior to protonation (Equation 9), thereby preventing the formation of NO2 and N2O3 via Equations 7 and 8. However, in the case of DAN, the nitrosation reaction switches from the intermediate formed from the NO/O2 reaction (Equation 1) to those formed through the reaction with ONOO- (7, 8) under lower activity of XO (1-5 milliunits). Therefore, no net change in nitrosation should occur until the flux of NO and Obardot 2 is one to one, at which point not enough NO is available to react with ONOO- to form a nitrosating agent before ONOO- reacts with thiols.

These data suggest that nitrosative and oxidative stresses are in a delicate balance, which depends on the relative fluxes of NO and Obardot 2. At a 1:1 ratio of formation of these radicals, the formation of OONO- is favored, which then results in maximal metal free oxidation and hydroxylation (9). Nitrosation occurs when the fluxes of NO and Obardot 2 are not balanced. These competing processes suggest a mechanism whereby nitrosative and oxidative stresses may be created, with effects in vivo. The formation of nitrosylated thiols versus disulfide adducts could be important in the regulation of gene transcription as well as the modulation of ion channels (7). Furthermore, GSH may be critical in attenuating oxidative and nitrosative stresses mediated by RNOS. Formation of Obardot 2 and subsequent conversion of NO to ONOO- in the presence of GSH may in fact be a detoxification reaction, since GSH scavenges ONOO- anion prior to the formation of the potent oxidant HOONO. Such a mechanism would prevent the formation of both N2O3 and oxidation mediated by ONOO-. Finally, SOD may attenuate nitrosation and oxidation reactions, since this enzyme reversed the inhibition of nitrosation observed at activities of XO (Fig. 3).

One of the intriguing aspects of this work is that the fluxes of NO in these experiments are as low as 1-5 µM, yet can bring about either nitrosative or oxidative chemistry. In Fig. 5, the 50 µM DEA/NO generated less than 6 µM NO for about 30 min. The presence of Obardot 2 vitally eliminated the detection of NO (Fig. 5). This result suggests that concentrations of NO could be in the nanomolar range, at which point nitrosation and oxidation by RNOS can occur. Although the production of RNOS is well controlled in vivo, their production could be dysregulated at sites near a cellular or organellar source of Obardot 2 in vivo. Superoxide would not be expected to migrate far from its source, while NO can migrate over many cell lengths (29). When NO enters such a site of superoxide formation, we hypothesize that a gradient of concentrations would be established, with attendant high fluxes of nitrosation. At similar fluxes, ONOO- could be formed and react chemically. Unlike Obardot 2, ONOO- can migrate and then either be consumed by intracellular thiols such as GSH or react with NO or superoxide to form NO2 (Scheme 1). Additionally, extracellular SOD bound to the endothelium may be critical in attenuating Obardot 2 in the extracellular space. Our data suggest that SOD may also modulate nitrosation, thereby attenuating oxidative and nitrosative stresses.


Scheme 1. Peroxynitrite reactions. Formation and reactions of peroxynitrite with NO and GSH.
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FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Radiation Biology Branch, National Cancer Institute, Bldg. 10, Rm. B3-B69, Bethesda, MD 20892. Tel.: 301-496-7511; Fax: 301-480-2238; E-mail: wink{at}box-w.nih.gov.
1   The abbreviations used are: NO, nitric oxide; DAN, 2,3-diaminonaphthalene; DEA/NO, Et2NN(O)NONa; DETAPAC, diethylenetriaminepentaacetic acid; HX, hypoxanthine; GSH, glutathione (reduced); GSSG, glutathione (oxidized); NAT, 2,3-naphthaltriazole; NEDD, N-(1-naphthyl)ethylenediamine dihydrochloride; PBS, phosphate-buffered saline; RNOS, reactive nitrogen oxide species; GSNO, S-nitrosoglutathione; SPER/NO, H2N(CH2)3NH2(CH2)4N[N(O)NO](CH2)3NH2; SOD, superoxide dismutase; XO, xanthine oxidase; HPLC, high performance liquid chromatography.

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