Kinetics of Nitrosation of Thiols by Nitric Oxide in the Presence of Oxygen*

Nitrosothiols are powerful vasodilators. They act by releasing nitric oxide, which activates the heme protein guanylate cyclase. We have studied the kinetics of nitro- sothiol formation of glutathione, cysteine, N -acetylcys-teine, human serum albumin, and bovine serum albumin upon reaction with nitric oxide (NO) in the presence of oxygen. These studies have been made at low pH as well as at physiological pH. At pH 7.0, contrary to published reports, nitric oxide by itself does not react with thiols to yield nitrosothiol. However, formation of nitrosothiols is observed in the presence of oxygen. For all thiols studied, the rates of nitrosothiol formation were first order in O 2 concentration and second order in NO con- centration and at lower concentrations ( (cid:60) 5 m M thiol) also depended on thiol concentrations. Analysis of the kinetic data indicated that the rate-limiting step was the reaction of NO with oxygen. Analysis of the reaction products suggest that the main nitrosating species is N 2 O 3 : RSH (cid:49) N 2 O 3 3 RSNO (cid:49) NO 2 (cid:50) (cid:49) H (cid:49) . Rate constants for this reaction for glutathione and several other low molecular weight thiols are in the range of 3–1.5 (cid:51) 10 5 M (cid:50) 1 s (cid:50) 1 , and for human and bovine serum albumins 0.3 (cid:51) 10 5 M (cid:50) 1 s (cid:50) 1 and 0.06 (cid:51) 10 5 M (cid:50) 1 s (cid:50) 1 , respectively. 40 min. Kinetic measurements were made either on a Kontron 860 spectrophotometer in 4-ml matched quartz cuvettes fitted with septa at 29 °C or on a stopped-flow spectrophotometer with a thermostatted (20 °C) cell. Kinetic data were obtained in the form of (cid:39) 200 electronically digitized absorbance versus time data points. Spectra were recorded with the Kontron spectrophotometer using 1-cm-pathlength cuvettes. Only freshly prepared solutions were used in all experiments. HPLC studies were conducted using an anion exchange column (Aquapor AX-300, 4.6 (cid:51) 250 mm; Perkin-Elmer/ABI). The gradient was obtained with eluent A (10 m M sodium phosphate, pH 6.0) and eluent B (100 m M sodium sulfate in eluent A). After applying the sample (5 (cid:109) l), a linear change from 100% eluent A to 100% eluent B over 30 min was used. The flow rate was 1 ml per min, and detection was at 210 nm. N -Ethylmaleimide, a sulfhydryl-specific reagent, was added (6 m M ) to the solutions prior to HPLC analysis to block excess GSH and enable measurement of GSNO (Jocelyn, 1972).

Nitrosothiols are powerful vasodilators. They act by releasing nitric oxide, which activates the heme protein guanylate cyclase. We have studied the kinetics of nitrosothiol formation of glutathione, cysteine, N-acetylcysteine, human serum albumin, and bovine serum albumin upon reaction with nitric oxide (NO) in the presence of oxygen. These studies have been made at low pH as well as at physiological pH. At pH 7.0, contrary to published reports, nitric oxide by itself does not react with thiols to yield nitrosothiol. However, formation of nitrosothiols is observed in the presence of oxygen. For all thiols studied, the rates of nitrosothiol formation were first order in O 2 concentration and second order in NO concentration and at lower concentrations (<5 mM thiol) also depended on thiol concentrations. Analysis of the kinetic data indicated that the rate-limiting step was the reaction of NO with oxygen. Analysis of the reaction products suggest that the main nitrosating species is N 2 O 3 : RSH ؉ N 2 O 3 3 RSNO ؉ NO 2 ؊ ؉ H ؉ . Rate constants for this reaction for glutathione and several other low molecular weight thiols are in the range of 3-1.5 ؋ 10 5 M ؊1 s ؊1 , and for human and bovine serum albumins 0.3 ؋ 10 5 M ؊1 s ؊1 and 0.06 ؋ 10 5 M ؊1 s ؊1 , respectively. The data further indicate that the reaction rate of the nitrosating species N 2 O 3 with thiols is competitive with its rate of hydrolysis. At physiological concentrations nitrosoglutathione formation represents a significant metabolic fate of N 2 O 3 , and at glutathione concentrations of 5 mM or higher almost all of N 2 O 3 formed is consumed in nitrosation of glutathione. Implications of these results for in vivo nitrosation of thiols are discussed.
S-Nitrosothiols are potent vasodilators and inhibitors of platelet aggregation (Ignarro et al., 1981;Stamler et al., 1992;Butler and Williams, 1993). This activity is almost certainly dependent on the ability of the nitrosothiol to yield NO, a bioregulatory molecule of considerable current interest (Moncada et al., 1991). Although the mechanism of NO release is not certain, the overall reaction seems to be as follows: Combination reactions of NO with five coordinate ferro heme proteins are fast, and the NO dissociation reaction is slow (Moore and Gibson, 1976). GC(heme-NO)* represents the NOactivated form of the enzyme. Stamler et al. (1992) have reported that naturally produced nitric oxide circulates in plasma primarily complexed as Snitrosothiol species, principal among which is S-nitroso-serum albumin. Although nitrosothiols (both low M r and protein) have endothelium-derived relaxation factor or NO-like properties, the mechanism by which circulating or cellular RSNO is synthesized has not been described. At pH 7.0, contrary to the published reports, nitric oxide, by itself, does not react with thiols to yield nitrosothiol. However, formation of nitrosothiol observed in the presence of oxygen suggested that nitrosation proceeds via the formation of oxides of nitrogen (N 2 O 4 , N 2 O 3 , etc.).
In view of the fact that in aqueous solutions the rates of hydrolysis of these oxides are high, it is not known how much nitrosation of low M r thiols such as glutathione and protein thiols such as HSA 1 can be expected to proceed via this mechanism. The potential reactivity of NO with the sulfhydryl group is also of interest when one considers that during its diffusion from endothelial cells to the target molecule guanylate cyclase in neighboring muscle cells, NO will be exposed in the cytoplasm to mM concentrations of the tripeptide glutathione (Meister and Anderson, 1983). One wonders how NO is able to avoid reaction with such a large excess of an intracellular thiol. To answer some of these questions would require knowing the mechanism of nitrosation of thiols in aqueous solutions in the presence of oxygen and the relevant rate constants. In this article we present the results of our kinetic studies on the reaction of nitric oxide with several thiols in the presence of oxygen. In agreement with earlier studies it was observed that NO reacts with thiols via the rate-limiting step of nitric oxide's reaction with oxygen and formation of the nitrosating species N 2 O 3 (Wink et al., 1994). The reaction of N 2 O 3 with thiols is competitive with its reaction with water. The novel finding of the present study is that physiologically attainable concentrations (5 mM or higher) of glutathione compete successfully with water for reaction with N 2 O 3 . Nitrosation of HSA, on the other hand, is less significant by this reaction path.

MATERIALS AND METHODS
Reduced glutathione, cysteine-HCl, N-acetylcysteine, bis-Tris, Tris, sodium nitrite, N-ethylmaleimide, and human and bovine serum albumins (99%, fatty acid and globin free) were from Sigma. Since nitrosation of thiols can be sensitive to the presence of metal ions, deionized and glass-distilled water was used for washing all glassware and for the preparation of solutions. Spectral analysis and kinetic experiments were made in solutions both with and without 1 mM EDTA. Analytical grade sodium phosphate from Fisher was used in the present study. Like most other brands of sodium phosphate, it contained iron as a contaminant. In 0.1 M phosphate buffers used in the present study, iron contamination ranged between 0.6 and 1 M. Therefore, all buffers were made in 1 mM in EDTA. In the time domain of the kinetic experiments made in this study, only in the case of cysteine inclusion of EDTA affected the kinetics of the reaction.
Nitric oxide (99.0% pure) was purchased from Matheson. Two methods were used to purify NO: passing it through a column filled with fresh KOH pellets or bubbling it through a solution of 0.5 M NaOH. After extended periods of use, KOH pellets lost their effectiveness, probably due to surface accumulation of nitrite. Two source tanks of NO were used in experiments: a tank that had been received from the manufacturer within one year of use and one that had been received roughly a decade earlier; NO from both behaved the same.
Solutions of NO, O 2 , RSH, and RSH ϩ O 2 were prepared in deoxygenated buffers (0.1 M sodium phosphate, pH 7.0) in gas-tight syringes (Hamilton). To prepare anaerobic albumin solutions, a tube containing a weighed quantity of solid albumin was flushed with argon for 15 min and then filled with a required amount of deoxygenated buffer. Deoxygenated buffers in gas-tight syringes were prepared by bubbling high purity argon for Ͼ40 min.
Kinetic measurements were made either on a Kontron 860 spectrophotometer in 4-ml matched quartz cuvettes fitted with septa at 29°C or on a stopped-flow spectrophotometer with a thermostatted (20°C) cell. Kinetic data were obtained in the form of Ϸ200 electronically digitized absorbance versus time data points. Spectra were recorded with the Kontron spectrophotometer using 1-cm-pathlength cuvettes. Only freshly prepared solutions were used in all experiments.
HPLC studies were conducted using an anion exchange column (Aquapor AX-300, 4.6 ϫ 250 mm; Perkin-Elmer/ABI). The gradient was obtained with eluent A (10 mM sodium phosphate, pH 6.0) and eluent B (100 mM sodium sulfate in eluent A). After applying the sample (5 l), a linear change from 100% eluent A to 100% eluent B over 30 min was used. The flow rate was 1 ml per min, and detection was at 210 nm. N-Ethylmaleimide, a sulfhydryl-specific reagent, was added (6 mM) to the solutions prior to HPLC analysis to block excess GSH and enable measurement of GSNO (Jocelyn, 1972).

Effect of NO Purity on Low M r Nitrosothiol Formation
No significant S-nitrosothiol formation was detected when Cys, N-acetylcysteine, GSH, or human serum albumin was incubated with NO that had been purified by passage over fresh KOH pellets or by bubbling through aqueous NaOH. Incubations were conducted in a deaerated spectrophotometer cell (1-cm light pathlength), closed to air by a rubber septum, in 0.1 M sodium phosphate over the pH range 5-8 and with NO added either as a solution in a buffer or by direct bubbling. In every case, addition of purified NO to a solution of thiol extensively purged with argon did not result in a significant increase at the absorbance bands (330 -340 and 540 -560 nm) of the respective nitrosothiol. However, thiol oxidation to the disulfide did occur, as evidenced by the appearance of a band at Ϸ250 nm; oxidation of thiols by anaerobic nitric oxide producing disulfide and N 2 O is known (Pryor et al., 1982). This reaction, however, was much slower than the formation of nitrosothiol in the presence of oxygen. Results of control experiments indicated that for the concentration range of reactants used in this study, the reaction of NO with GSH in the absence of O 2 was 20 -100 times slower than the reaction of NO with GSH in the presence of oxygen.
When NO was used without alkali treatment, nitrosothiol formation was readily observed upon bubbling NO directly from the gas tank into the deoxygenated thiol solution. S-Nitrosoglutathione formation is demonstrated as an increase in the absorbance band at 335 nm in Fig. 1. Similarly, GSNO formation was readily observed when purified NO was bubbled through a solution of glutathione in the presence of oxygen. However, nitrosothiol formation was not observed when a solution of unpurified NO was prepared in deaerated phosphate buffer and added to the solution of thiol. Similarly, no RSNO formation was seen when solutions of unpurified NO were prepared in 0.1 M Tris, pH 7.0, prior to reaction with thiols. These results indicate that an oxidation product of NO present as a contaminant in unpurified NO is the nitrosating species and that it decomposes when aqueous solutions of NO are prepared prior to reaction with thiols.
Results for glutathione are presented in Fig. 1. Curve A is for deoxygenated solution of GSH. After bubbling NO through it for 10 min, curve B is obtained; curve E is for solutions in which unpurified NO was bubbled through GSH solution for 6 min. A small absorbance change (curve B) around 350 nm, which is about 3 percent of ⌬A for curve E, is most likely due to RSNO formation from slow leakage of O 2 or incomplete deoxygenation of the starting solution. Because GSH is the preponderant low M r thiol in cells and because its nitroso derivative, GSNO, is very stable in solution (Park et al., 1993), the results of experiments with GSH are highlighted below. Similar results were obtained with N-acetylcysteine. Cysteine, however, behaved differently and is discussed separately.
Buffer Effect-With some buffers, longer living, buffer-derived nitrosating species may form. Bis-Tris (0.1 M, pH 7.0) buffered solutions of unpurified NO yielded measurable GSNO when freshly prepared and added to GSH. The nitrosating activity of this solution decreased to background level over a period of 6 h. This reactivity correlated with the appearance of multiple absorbance bands at 330 -370 nm, upon saturating deaerated bis-Tris with unpurified NO, and then with a timedependent decrease in intensity of these bands. When purified NO was used, neither the nitrosating activity nor the bands at 330 -370 nm were observed. Apparently, in the reaction of FIG. 1. Effect of NO purity on product formation from NO and GSH. Spectra were recorded of a deaerated solution of GSH (5 mM) before (A) and after (B) bubbling with purified NO for 10 min. In a second experiment, a deaerated GSH solution was bubbled with unpurified NO (C) for 2 min, 4 min (D), and 6 min (E). Solutions of GSH were prepared in 0.1 M sodium phosphate, pH 7.0.
unpurified NO with bis-Tris a nitrosated product forms, which is capable of nitrosating thiols and which is relatively stable. Due to these complications, bis-Tris buffer was not used further. All subsequent experiments were conducted in phosphate buffer.
Kinetic Studies-The reaction order with respect to each of the starting reactants, NO, O 2 , and GSH, was determined in single mixing stopped-flow experiments by varying the initial concentration of a single reactant while keeping the concentration of the others constant. In these experiments a solution containing GSH and O 2 was mixed with a solution of NO. The slope of ln(d[GSNO]/dt) initial versus ln[reactant] plots yielded the reaction order with respect to the reactant being varied.
Three sets of experiments were performed to determine the order of the reaction with respect to each of the reactants. 1) A solution of constant GSH (10.8 mM) and O 2 concentrations (0.72 mM) was mixed with nitric oxide solutions of varying concentrations (0.064 -0.44 mM). The slope of ln(d[GSNO]/dt) initial versus ln[NO] (Fig. 2) plot was 2, which demonstrates a second order concentration dependence of reaction rates on NO concentration. 2) Solutions with constant GSH concentrations (10.4 mM) and varying concentrations of O 2 (0.24 -1.2 mM) were mixed with a solution of constant nitric oxide concentration (0.2 mM). These data yielded first order dependence of reaction rates on O 2 concentration (Fig. 3). 3) Solutions of varying GSH concentrations (0.6 -60 mM) and constant O 2 concentration (0.72 mM) were mixed with a solution of constant NO concentration (0.2 mM). In Fig. 4, ln R initial is plotted as a function of ln [GSH]. It is clear that at lower GSH concentrations initial rates depend on GSH concentration. At higher GSH concentrations (Ն5 mM) initial rates become independent of GSH concentration.
Similar concentration dependence with respect to NO, O 2 , and thiol was observed for N-acetylcysteine.
On the basis of these kinetic observations and earlier studies by Wink et al. (1993Wink et al. ( , 1994, we can tentatively write the following reaction mechanism: Justification for the individual steps is provided under "Discussion."

Kinetics of Nitrosothiol Formation by Cysteine
The reaction time course for the reaction of NO with cysteine in the presence of O 2 was complex and very different than for GSH described above. Initial rates, d[Cys-NO]/dt, were dependent only on NO concentration in a first order manner, although the presence of O 2 was essential for the reaction to take place. Later in the reaction, reaction rates depended both on NO and O 2 concentrations, approaching second and first order dependence on [NO] and [O 2 ], respectively. This behavior was observed in 0.1 M phosphate buffer of pH 7.0 and not in Tris or dilute phosphate buffers. Atomic absorption spectra analysis of the buffer solution indicated micromolar amounts of iron impurity; a solution of cysteine in water did not show this impurity. These observations suggest the involvement of an ironmediated redox system responsible for the initial first order (in The approximate value of k 8 was 1.5 s Ϫ1 . However, it has not been possible to obtain quantitatively consistent results over a large enough concentration range of the reactants to give some confidence to the rate law and a reaction mechanism that may be postulated. Observations such as the effect of buffers on the reaction rates are difficult to explain even qualitatively. McAninly et al. (1993) made similar observations in their study of nitrosothiol decomposition and attributed these effects to the presence of metal ion contaminants. The effect disappeared on inclusion of EDTA in the reaction mixture. The reaction of cysteine with NO in the presence of O 2 therefore was studied in detail only in the presence of 50 mM EDTA in 0.1 M phosphate buffer. Under these conditions the reaction follows the same kinetics as those observed for the formation of GSNO and N-acetylcysteine-NO.

Nitrosothiol Formation by HSA and BSA
For both of these proteins nitrosothiol formation could be observed only in the presence of oxygen, indicating that the kinetics of nitrosothiol formation is qualitatively similar to that observed for the low M r thiols. In the "Discussion" we point out that nitrosothiol formation is competitive with the hydrolysis of the nitrosating reagent N 2 O 3 (see Reactions 6 and 7). Therefore, high concentrations of HSA and BSA (Ն8 mM) were needed to observe measurable absorbance changes due to nitrosothiol formation. Such solutions are very viscous and pose mixing difficulties in stopped-flow experiments. For these reasons kinetic studies with HSA and BSA were not made. However, estimates of rate constants for the reaction of these proteins with N 2 O 3 could be made from static spectrophotometric measurements. The data (plots of [NO] added versus [RSNO] formed) for BSA and HSA along with those for other thiols are shown in Fig. 5 and are described in the next section.

Relationship between NO Added and RSNO Formed in the Reaction
In Fig. 5 is shown a plot of [NO] added to an air-equilibrated solution of thiols versus [RSNO] formed when a solution of NO was rapidly mixed with the thiol solution on a stopped-flow spectrophotometer. In the case of protein thiols, the reaction was slower, and therefore similar experiments were made on a Kontron spectrophotometer. Concentration of thiols was much greater than NO concentration. Concentration of nitroso derivatives formed in the reaction was calculated from their published extinction coefficients (Park et al., 1993;Stamler et al., 1992;Byler et al., 1983). A linear relationship between [NO] added and [RSNO] formed was observed in mixing experiments. Wink et al. (1994) have already shown that in the presence of oxygen nitrosation of thiols proceeds more rapidly than of amines and tyrosine. We therefore can write the following: The coefficient m represents the fraction of total NO used in nitrosothiol formation. The following can be shown: m ϭ k 7 ϫ ͓RSH͔/2͑k 6 ϫ ͓H 2 O͔ ϩ k 7 ϫ ͓RSH͔͒ (Eq. 3)

Product Identification Studies
These studies were made for two reasons: 1) to ascertain the formation NO 2 Ϫ in Reaction 7 and 2) to rule out any significant formation of GSSG in the reaction of NO with GSH in the presence of oxygen. We have already provided a kinetic argument against this possibility. Fig. 6 shows HPLC chromatograms of the following: A, a solution of GSH (5 mM) ϩ O 2 (0.4 mM) mixed with NO (0.5 mM); B, GSH (5 mM) in air-equilibrated buffer; C, buffer blank ϩ 1 mM NEM; and D, GSNO ϩ GSSG ϩ NaNO 3 ϩ NaNO 2 . Addition of the sulfhydryl-specific reagent NEM prior to HPLC analysis to yield the GS adduct of NEM had two effects: it blocked excess GSH and enabled measurement of GSNO, which otherwise elutes with GSH in this HPLC protocol (Jocelyn, 1972). The solutions were applied to the column within 5 min of mixing. In A, we observe the formation of GSNO (peak 3) and NO 2 Ϫ (peak 5) but no significant NO 3 Ϫ (peak 5). The peak corresponding to GSSG has approximately the same area under it as in control GSH solution (B, peak 4).

FIG. 5. Dependence of nitrosothiol yield on initial NO concentration.
A, the low M r thiols in 0.1 M phosphate, pH 7.0, were mixed with NO in the presence of O 2 , and the yield of nitrosothiol was calculated based on the total increase in A 335 using published extinction coefficients for each nitrosothiol. Measurements were made in a stopped-flow spectrophotometer. Data for albumins are included for comparison; details are given in B. All concentrations are in mM. B, same as A for human and bovine serum albumin.
At GSH concentrations 5 mM or higher, the rates of GSNO formation are independent of GSH concentration, indicating that k 7 [GSH] ӷ k 6 [H 2 O]. Under these conditions the rate Equation 4 simplifies to the following: Using Equation 4 and the stoichiometric relationship 1 ⁄4⌬[NO] ϭ ⌬[O 2 ], k 4 can be calculated from a reaction time course using the algorithm described earlier (Kharitonov et al., 1994). Over the entire range of concentrations of NO, O 2 , and thiols used in the present study, least squares analysis of reaction time courses (each consisting of more than 200 data points) yielded k 4 ϭ 7.0 Ϯ 1 ϫ 10 6 (S.D.) M Ϫ2 s Ϫ1 . This is a global average of k 4 for the three thiols used in this study. Fig. 7 shows typical observed and calculated reaction time courses for glutathione. This value of k 4 should be compared with the value of k 4 ϭ 6.3 ϫ 10 6 M Ϫ2 s Ϫ1 reported by us earlier for the auto-oxidation of NO (Kharitonov et al., 1994).

Nitrosothiol Formation at Low pH
These studies were undertaken in order to determine rate constants for nitrosation of protein thiols (BSA and HSA) via their reaction with nitrous acid. Knowledge of these rate constants will allow us to estimate the extent to which this reaction pathway may lead to nitrosation of BSA and HSA under physiological conditions. Nitrosothiol formation by glutathione, N-acetylcysteine, and cysteine at low pH has been studied earlier (Williams, 1988). It was shown that the overall reaction is third order: d[RSNO]/dt ϭ k ot [RSH][HNO 2 ][H ϩ ], where k ot is the overall third order rate constant. However, there are no data for BSA and HSA. Kinetic studies of low pH nitrosothiol formation by HSA and BSA were made in phosphate buffer of pH 2.0, using sodium nitrite. At a constant pH, the reaction rates were first order with respect to both the thiol (or the thiol protein) and sodium nitrite. In the presence of excess nitrite, reactions become pseudo first order in thiol concentration. The reaction time courses under these conditions were used to calculate the pseudo first order constant (k pf ) defined by the equation: d[RSNO]/dt ϭ k pf [RSH]. This was achieved by making a least square fit of the data to a single exponential equation. The constant thus obtained, if divided by nitrite and proton concentrations, yields k ot , the overall third order rate constant listed in Table I. In the same table, the data for glutathione obtained in this study is compared with the literature values.

DISCUSSION
Reactions 4 -7 explain all qualitative and quantitative observations described under "Results." First and second order dependence of reaction rates with respect to NO and O 2 is explained by Reaction 4 and substantiated by data in Figs. 2 and 3. Dependence of reaction rates (d[GSNO]/dt) on thiol concentration arises from competition between Reactions 6 and 7. This should be obvious from the rate Equation 4 and is made further clear by Fig. 8, in which initial reaction rates corrected for hydrolysis of N 2 O 3 by Reaction 6 are plotted against thiol concentration (i.e. initial reaction rates are divided by the factor m). Rate Equation 4 predicts that 1) at high thiol concentrations the reaction rates should be independent of thiol concentration and 2) at low thiol concentrations the reaction rates should approach first order dependence on thiol concentration. Both of these predictions are consistent with the data in Fig. 4. These observations indicate that the rate-limiting step in Reactions 4 -7 is Reaction 4.
In Reactions 4 -7 there are two potential nitrosating agents: N 2 O 4 , a dimer of NO 2 , and N 2 O 3 . For electrophilic nitrosation, these two reagents should act as sources of NO ϩ . In the case of N 2 O 4 , this would lead to the formation of NO 3 Ϫ . Nitrosation via N 2 O 3 would form NO 2 Ϫ . The HPLC data support the latter possibility.
In Table II  about four orders of magnitude greater than k 6 for the hydrolysis of N 2 O 3 . For the two protein thiols k 7 is much lower than for the low M r thiols. This is most likely due to the inaccessibility of the SH group in the protein matrix.
A similar trend is present in the rate constants for nitrosation at low pH. As compared to low M r thiols for both HSA and BSA the rate constant (k ot ) is lower by a factor of more than 100. The rate constants listed in Table I are overall third order constants. Simple calculations show that nitrosation by this reaction path at pH values of physiological interest should be insignificant.
Glutathione is the most important intracellular thiol. Its intracellular concentration in mammalian cells varies over a wide range (0.5-10 mM, Meister and Anderson (1983)). Blood plasma concentrations are in the micromolar range. From the data in Fig. 4, it is clear that in cells with GSH concentration 5 mM or more almost all of N 2 O 3 formed from the reaction of naturally occurring NO will be used up for the nitrosation of intracellular glutathione. However, this discussion should not lead to the conclusion that all of NO formed in vivo will be consumed to form RSNO. Nitric oxide can react with various molecules found in biological systems. Those most often considered in this regard are oxygen, thiols, amines, tyrosine, free radicals, and ferric and ferro heme proteins. Kinetic studies of Wink et al. (1993Wink et al. ( , 1994 indicate that the third order reaction of nitric oxide with oxygen is slow and acts as the rate-limiting step in nitrosation reactions via the formation of N 2 O 3 . Reactions of NO with O 2 Ϫ and ferro and ferric heme proteins, on the other hand, are very fast (Padmaja and Huie, 1993;Beckman and Crow, 1993;Cassoly and Gibson, 1975;Sharma et al., 1983Sharma et al., , 1987. Therefore, in vivo only a small fraction of naturally produced NO will be able to react with oxygen to produce N 2 O 3 . The novel finding of the present study is that almost all of N 2 O 3 thus formed is consumed in GSNO formation in cells in which GSH concentrations are 5 mM or higher. At lower concentration of GSH, nitrosation of GSH by N 2 O 3 will become progressively less significant. In the case of the two protein thiols HSA and BSA, the situation is quite different. The rate constant for nitrosation is about 1 ⁄10 of that for the low molecular weight thiols, and their plasma concentration rarely exceeds 1 mM. Therefore, it seems that nitrosation of these two proteins via reaction with N 2 O 3 may not be as significant. For protein thiols, alternative reaction mechanisms of nitrosation may be of more physiological significance than the reaction with N 2 O 3 . Keaney et al. (1993) have suggested the possibility of HSA nitrosation by reaction with the nitrosonium ion, NO ϩ . Metal-catalyzed formation of NO ϩ observed by us is consistent with this proposition. We have also observed that the reaction of NO with ferric hemeproteins can also be a potential source of nitrosonium ion formation as shown: Hb ϩ ϩ NO 3 Hb ϩ NO ϩ REACTION 10 Brackman and Smit (1965) have reported nitrosation via the mediation of copper salts and NO ϩ formation. Nitrosation of thiols and other reactive groups by metal ion-mediated formation of NO ϩ is likely to be considerably faster than via the reactions of NO with oxygen.
To summarize, in aqueous solutions of thiols, NO and O 2 nitrosation of the thiol proceeds via the rate-limiting step of formation of oxides of nitrogen. The nitrosating intermediate seems to be N 2 O 3 . Its reaction with thiols is competitive with the rate of its hydrolysis. Considerations based on the rate constants and in vivo concentrations of glutathione and HSA suggest that in the case of glutathione, significant nitrosation can take place by its reaction with N 2 O 3 particularly in cells with high GSH concentration (Ն5 mM). This reaction path seems to be less significant for the nitrosation of HSA.