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Chem. Res. Toxicol. 1996, 9, 988-993
Kinetics of S-Nitrosation of Thiols in Nitric Oxide Solutions Manish Keshive,† Sukhjeet Singh,‡ John S. Wishnok,‡ Steven R. Tannenbaum,‡,§ and William M. Deen*,† Department of Chemical Engineering, Division of Toxicology, and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received February 27, 1996X
The S-nitroso adducts of nitric oxide (NO) may serve as carriers of NO and play a role in cell signaling and/or cytotoxicity. A quantitative understanding of the kinetics of S-nitrosothiol formation in solutions containing NO and O2 is important for understanding these roles of S-nitroso compounds in vivo. Rates of S-nitrosation in aqueous solutions were investigated for three thiols: glutathione, N-acetylcysteine, and N-acetylpenicillamine. Nitrous anhydride (N2O3), an intermediate in the formation of nitrite from NO and O2, is the most likely NO donor for N-nitrosation of amines as well as for S-nitrosation of thiols, at physiological pH. This motivated the use of a competitive kinetics approach, in which the rates of thiol nitrosation were compared with that of a secondary amine, morpholine. The kinetic studies were carried out with known amounts of NO and O2 in solutions containing one thiol (400 µM) and morpholine (200-5700 µM) in 0.01 M phosphate buffer at pH 7.4 and 23 °C. It was found that disulfide formation, transnitrosation reactions, and decomposition of the S-nitrosothiol products were all negligible under these conditions. The rate of formation of S-nitrosothiols was expressed as k7[N2O3][RSH], where RSH represents the thiol. The rate constant for S-nitrosation relative to that for N2O3 hydrolysis (k4) was found to be k7/k4 ) (4.15 ( 0.28) × 104, (2.11 ( 0.11) × 104, and (0.48 ( 0.04) × 104 M-1 for glutathione, N-acetylcysteine, and N-acetylpenicillamine, respectively. The overall (observed) rates of nitrosothiol formation reflect the fact that [N2O3] ∝ [NO]2[O2] and that [N2O3] also depends on [RSH] and the concentration of phosphate. Using a detailed kinetic model to account for these effects, the present results could be reconciled with apparently dissimilar findings reported previously by others.
Introduction Nitric oxide (NO) is a biological messenger molecule synthesized in mammals via the oxidation of L-arginine to citrulline by the enzyme NO synthase (1). Nitric oxide synthesis is observed in a wide variety of cell types, including macrophages, vascular endothelial cells, neurons, epithelial cells, and neutrophils. The actions of NO in the body are paradoxical, in that NO serves as a regulatory agent as well as a cytotoxic or mutagenic agent. Regulatory actions of NO include inhibition of platelet aggregation, vasodilation (blood pressure regulation), and neurotransmission (2). The mutagenic effects of NO are attributed to the intermediates formed during the reactions of NO with molecular oxygen and superoxide (O2-). The reaction of NO with O2, which proceeds ultimately to nitrite (NO2-), forms as an intermediate nitrous anhydride (N2O3), which can lead to cytotoxic effects. Peroxynitrite (ONOO-), an intermediate in the reaction of NO with O2- to form nitrate (NO3-), can exert mutagenic effects via the oxidation of DNA (3). Nitric oxide itself can react with heme (4) as well as non-heme iron (5). The high reactivity of NO with iron compounds, combined with their abundance in extracellular and intracellular fluids, has led to the argument that NO is * Address correspondence to Dr. William M. Deen, Department of Chemical Engineering, Room 66-509, Massachusetts Institute of Technology, Cambridge, MA 02139. Telephone: (617) 253-4535; FAX: (617) 258-8224; E-mail:
[email protected]. † Department of Chemical Engineering. ‡ Division of Toxicology. § Department of Chemistry. X Abstract published in Advance ACS Abstracts, July 15, 1996.
S0893-228x(96)00036-7 CCC: $12.00
stabilized by reactions with carrier molecules in vivo that prolong its half-life (6-8). It has been demonstrated that the intermediates generated during the reaction of NO with O2 at physiological pH readily react with secondary amines (9) and thiols (6-8, 10) to form N-nitrosamines and S-nitrosothiols, respectively. The nitrosation of morpholine (Mor),1 a secondary amine, proceeds via N2O3 at a rate which has been examined in detail by Lewis et al. (9). There is, however, relatively little information in the literature regarding the kinetics of the reactions of thiols with NO. Stamler et al. (6) suggested that NO generated in biological systems reacts in the presence of protein thiols to form S-nitrosoprotein derivatives. They also demonstrated that the concentrations of S-nitrosothiols in human plasma are about 3 orders of magnitude greater than that of free NO. This suggests that NO circulates in mammalian plasma complexed in S-nitrosothiol species. The S-nitroso adducts may serve as carriers of NO and/or may have cytotoxic effects. A quantitative understanding of the kinetics of S-nitrosothiol formation is thus essential in determining the role of S-nitroso compounds in vivo. The reaction kinetics for the nitrosation of cysteine (Cys) and glutathione (GSH) in aerobic NO solutions at 1 Abbreviations: Mor, morpholine; NMor, N-nitrosomorpholine; GSH, reduced glutathione; GSSG, glutathione disulfide; GSNO, Snitrosoglutathione; Cys, cysteine; N-Ac-Cys, N-acetylcysteine; N-AcCysNO, S-nitroso-N-acetylcysteine; N-Ac-Pen, N-acetylpenicillamine; SNAP, S-nitroso-N-acetylpenicillamine; ESI-MS, electrospray ionization-mass spectrometry.
© 1996 American Chemical Society
Kinetics of Nitrosothiol Formation
neutral pH have been studied, and a rate law for the formation of the S-nitroso adducts has been proposed by Wink et al. (10). The rate of formation of S-nitrosothiols was found to be second order in NO, first order in O2, and independent of the initial concentration of the thiol. The independence from the substrate (thiol) concentration, in particular, makes the form of this rate law unusual, but no explanation was offered for this finding. Thiols are readily oxidized to the disulfide form (11, 12), but it was not mentioned whether the disulfide was observed during the reactions. Very recently, Kharitonov et al. (13) investigated the kinetics of the nitrosation of thiols by NO in the presence of O2 and proposed rate laws for both high and low concentrations of thiols. In view of the biological importance of the kinetics of S-nitrosothiol formation, we carried out competitive kinetic studies to determine the rate constant for the reaction of various thiols with N2O3. In this approach, which differs from that employed in the previous studies of S-nitrosation cited above, the rate constant was estimated from the rate of nitrosothiol formation in the presence of morpholine. As will be discussed, this competitive kinetics approach avoided complications due to the effects of phosphate on the nitrosation of thiols, which we think must have been prominent in one of the previous studies (13). The reactor configuration developed by Lewis et al. (14) was used in these studies. The thiols examined were GSH, N-acetylcysteine (N-Ac-Cys), and N-acetylpenicillamine (N-Ac-Pen). All are biologically relevant: the level of GSH in cells can be as high as 10 mM (15), N-Ac-Cys provides an analog for the binding of NO to cysteine in serum albumin, and N-AcPen reacts with NO to form S-nitroso-N-acetylpenicillamine (SNAP), an important NO donor compound.
Materials and Methods Safety Considerations (Caution). All experiments were performed in a certified hood, due to the toxicity of NO. Precautions were also taken to avoid any skin contact with the potentially toxic nitrosothiols. Reagents. Nitric oxide was passed through a column containing 10 M NaOH to remove NOx impurities. Argon, after passing through an oxygen trap, was mixed with NO using controlled gas flowmeters (Porter Instrument Co., Hatfield, PA) to obtain the desired NO gas concentration. Buffer solutions at 0.01 M ionic strength and pH 7.4 were prepared from the acids and sodium salts of phosphate. Morpholine was obtained from Aldrich Chemical Co. (Milwaukee, WI). Nitrosomorpholine, glutathione, S-nitrosoglutathione, glutathione disulfide, N-acetylcysteine, S-nitroso-N-acetylcysteine, N-acetylpenicillamine, and S-nitroso-N-acetylpenicillamine were obtained from Sigma Chemical Co. (St. Louis, MO). All of the solutions were prepared using deionized water. Reactor. The reactor, a modified 200 mL stirred ultrafiltration cell (Amicon, Danvers, MA, Model 8200), was the same as that used previously for NO oxidation and nitrosation studies (9, 14). Briefly, a composite membrane (6.2 cm diameter) consisting of a 1 mm thick Teflon sheet laminated with a 0.13 mm thick polydimethylsiloxane sheet (Mempro, Troy, NY) was fitted to the base of the stirred cell for NO detection. Gas inlet and outlet ports, two ports for a flow loop connected to a spectrophotometer (for NMor and S-nitrosothiol measurements), and a thermometer were added. A 20 gauge hypodermic needle was inserted into the gas outlet port for gas purging and NO introduction and removal during the reaction. All of the samples were withdrawn from the reactor using a gas-tight hypodermic syringe. The studies were performed at room temperature (23 ( 1 °C).
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 989 Nitric Oxide Analysis. The external side of the composite membrane was exposed to the high vacuum of a chemiluminescence detector (Thermedics Detection Inc., Woburn, MA, Model TEA-502) for monitoring of NO. The response time was ∼2 s, and the output of the detector was linear up to NO concentrations of at least 40 µM. The minimum aqueous NO concentration measurable by the chemiluminescence detector was estimated at ∼ 0.01 µM, based on a signal-to-noise ratio of 3 (14). N-Nitrosomorpholine and S-Nitrosothiol Analysis. The reactor had a flow loop consisting of 1/8 in. tubing (volume ∼8 mL). The aqueous solution in the reactor was continuously circulated by a pulseless pump (Cole Parmer, Chicago, IL, Models 000-305, 184-000) through this flow loop, into a 10 mm spectrophotometer flow cell (Shimadzu Model UV 160u). The concentrations of S-nitrosoglutathione (GSNO) and S-nitrosoN-acetylcysteine (N-Ac-CysNO) were measured at 335 nm and that of SNAP at 340 nm. The concentration of NMor was determined at 250 nm, with no interference from thiols, Snitrosothiols, or thiol disulfides. In each case the absorbance was linear in concentration. Competitive Kinetic Studies. Morpholine (200-5700 µM) and the thiol (400 µM) were added to 150 mL of 0.01 M phosphate buffer solution, and the pH was adjusted to ∼7.4 using NH4OH and H3PO4. The solution was added to the reactor, stirring initiated at 100 rpm, and recirculation begun through the spectrophotometer flow loop. Argon at a flow rate of 350 sccm was bubbled for 40 min through the solution to remove O2. A mixture of NO and Ar was then bubbled into the solution for at least 25 min to obtain the desired aqueous NO concentration. After the NO reached a steady-state aqueous concentration, the bubbling of the NO/Ar mixture was stopped, and the residual NO in the head space was removed by introducing Ar via the gas inlet for 1.5 min. A 21% O2, balance N2, mixture was then introduced through the gas inlet at a flow rate of 350 sccm. Diffusion of O2 into the aqueous phase initiated the oxidation of NO, producing the reactive intermediates responsible for the N- and S-nitrosations. The initial NO concentration in the solution (upon introduction of O2) was ∼30 µM. The reaction was allowed to proceed for at least 25 min, during which the NO and NMor concentrations were monitored continuously. The reaction protocol was then repeated, the only difference being that the concentrations of NO and S-nitrosothiol were monitored. The solution pH was measured at the end of each reaction. For each thiol, a control study was performed by carrying out the reaction protocol with 400 µM of the thiol in the buffer but no Mor, and monitoring the absorbance at 250 nm. This was done to check for any interference in the measurement of NMor absorbance at 250 nm. Electrospray Ionization-Mass Spectrometric (ESI-MS) Analysis. These experiments were done on a Hewlett Packard Model 5989B mass spectrophotometer in the negative ion mode. The instrument was tuned with special attention paid to the spray capillary voltages and offsets to prevent arcing. Kinetic experiments were carried out exactly as described above except that ammonium acetate (5 mM; pH approximately 7.4) was the buffer. To test for the formation of thiol disulfide, and to test for the constancy of the thiol concentration, samples were withdrawn from the reactor before and after the addition of O2. In a typical experiment, 100 µL of reaction solution was withdrawn with a gas-tight syringe and mixed with 100 µL of electrospray solvent and 20 µL of internal standard (1.9 mM succinic acid). Aliquots of the resulting solution were introduced into the mass spectrometer by flow injection into a stream of 50:50 water/methanol containing 1% acetic acid at a flow rate of 20 µL/min from a Harvard syringe pump. This solvent is more typical for positive-ion electrospray, but was used here because the analytes were more stable under these conditions. Spectra were taken over a 3-min period and averaged over the maximum abundance for the sample bolus, and the peak areas for the internal standard, the thiol, and the disulfide were recorded after background subtraction. Kinetic Model and Reaction Scheme. The oxidation kinetics of NO in aqueous solutions can be represented by the
990 Chem. Res. Toxicol., Vol. 9, No. 6, 1996
Keshive et al.
following reaction scheme (14, 16-18): k1
2NO + O2 98 2NO2
(1)
k2
NO + \ {k } N2O3
(2)
3
k4
N2O3 + H2O 98 2NO2- + 2H+
(3)
The other reactions needed to interpret the rates of nitrosation are k5
N2O3 + Pi 98 PiNO + NO2
-
k6
N2O3 + Mor 98 NMor + NO2- + H+ k7
N2O3 + RSH 98 RSNO + NO2- + H+
(4) (5) (6)
Equation 4 represents the reaction of N2O3 with the inorganic phosphate (Pi) in the buffer. (The active form of phosphate could be H2PO4- and/or HPO42-; all phosphate concentrations are expressed here as total phosphate at pH 7.4.) This reaction has the effect of enhancing the hydrolysis of N2O3, represented otherwise by eq 3 (9). Equations 5 and 6 represent the nitrosation reactions of Mor and the thiol (RSH), respectively. All of the rate constants in eqs 1-6 are known (at least approximately) except for k7, the rate constant for the reaction of N2O3 with the thiol. The following kinetic analysis, applicable to a solution containing Mor and one thiol, was used to estimate the unknown rate constant, k7. Order-of-magnitude estimates of the rate constants k1 through k4 (19, 20) suggest that NO2 and N2O3 were present in the system in very small amounts. This justifies the use of pseudo-steady-state approximations for those species,
d[NO2] ) 2k1[NO]2[O2] - k2[NO][NO2] + k3[N2O3] = 0 dt
(7)
d[N2O3] ) k2[NO][NO2] - (k3 + k4)[N2O3] - (k5[Pi] + dt k6[Mor0] + k7[RSH])[N2O3] = 0 (8) where [Mor0] is the concentration of neutral (unprotonated) Mor. Using eqs 7 and 8, the pseudo-steady-state concentration of N2O3 is
[N2O3] )
2k1[NO]2[O2] k4 + k5[Pi] + k6[Mor0] + k7[RSH]
(9)
The rates of formation of NMor and RSNO are
d[NMor] ) k6[Mor0][N2O3] dt
(10)
d[RSNO] ) k7[RSH][N2O3] dt
(11)
where eq 9 is used to evaluate [N2O3]. Combining eqs 10 and 11 we obtain
d[RSNO] k7 [RSH] ) d[NMor] k6 [Mor0]
(12)
Integration of eq 12, with the assumption that [RSH] and [Mor0] are essentially constant during the reaction, gives
k7 k6 ∆[RSNO] [Mor0] ∆[RSNO] [Mor0] or k7 ) k6 ) k4 k4 ∆[NMor] [RSH] ∆[NMor] [RSH]
(13)
where ∆[RSNO] and ∆[NMor] represent the changes in the respective concentrations during the reaction (i.e., after O2 addition). The second form of eq 13 is preferred because the rate of N-nitrosation of Mor relative to the rate of N2O3 hydrolysis (k6/k4) is known more precisely than are the absolute rate constants for either reaction (9). Accordingly, although estimates are given also for the absolute value of k7, the rate constants for thiol nitrosation are reported here primarily as the ratio, k7/k4.
Results Thiol Concentration and Disulfide Formation. The reaction scheme and kinetic analysis given above assume that the thiol concentration remained essentially constant during the course of the reaction and that disulfide formation was negligible. To check these assumptions, samples were withdrawn from the reactor before and after O2 addition during each experiment. The analysis of these samples by ESI-MS showed insignificant changes in the concentration of each thiol. Disulfides were detectable in small amounts, but the concentrations were always similar in the pairs of samples. This indicates that the disulfide was present as an impurity in the thiol, as opposed to being formed during the reaction period. Morpholine Concentration and pH. The concentration of unprotonated morpholine, the substrate for nitrosation, is related to the total morpholine concentration by
[Mor0] )
[Mor] 1 + 10pK-pH
(14)
where the pK at 25 °C is 8.5 for morpholine (9). The total morpholine concentration did not change appreciably in any experiment, because negligible amounts were converted to NMor (5 mM), namely:
d[RSNO] ) k[NO]2[O2] dt
(18)
where k ) (7.0 ( 0.1) × 106 M-2 s-1. (What is shown here as k is given as k4 in their eq 5.) Their derivation contained an algebraic error, however; the correct coefficient in eq 18 should be k/2, or 3.5 × 106 M-2 s-1. This corrected value is essentially the same as our value of 4.2 × 106 M-2 s-1. To summarize the situation for high thiol concentrations, the present results are in agreement with those of previous studies (10, 13) not only on the form of the rate law but also (essentially) on the numerical value of the rate coefficient. It seems clear that, for high thiol concentrations, the rate of nitrosothiol formation is controlled entirely by the availability of NO and O2. Consequently, measurements of nitrosothiol formation at thiol concentrations exceeding several millimolar will not ordinarily yield rate data specific to individual thiols. The values of the N2O3-thiol rate constant (k7) obtained from the present data are 6.6 × 107 M-1 s-1 for GSH and 3.4 × 107 M-1 s-1 for N-Ac-Cys. These values are 2 orders of magnitude larger than those reported by Kharitonov et al. (13) for the same compounds, 2.9 × 105 M-1 s-1 and 1.6 × 105 M-1 s-1 for GSH and N-Ac-Cys, respectively. This discrepancy is explained by the fact that those authors used a 0.1 M phosphate buffer in their nitrosation experiments, but their kinetic analysis did not account for the reactivity of phosphate with N2O3. In other words, they did not consider the reaction given here as eq 4. As shown by Lewis et al. (9), phosphate has a scavenging effect on N2O3; similar effects were seen with Cl-, but not with several other anions tested. Consequently, neglecting the effect of phosphate in the kinetic analysis for nitrosothiol formation will overestimate the concentration of N2O3, and therefore underestimate k7. The expected magnitude of the error in the rate constant is calculated by examining the rate of Snitrosothiol formation in the presence of phosphate, but without morpholine. Proceeding as before, the rate is predicted to be
(
or
)
2k1[NO]2[O2] d[RSNO] ) k7[RSH] dt k4 + k5[Pi] + k7[RSH]
(19)
d[RSNO]/dt 2
4k1[NO] [O2]
)
(
)
k7[RSH] 1 2 k4 + k5[Pi] + k7[RSH]
(20)
where m (similar to Kharitonov et al.) represents the fraction of NO converted to S-nitrosothiol. (The fraction of NO converted to NO2- is 1 - m.) Equation 20 is rearranged to give
{
k7 k5 2m ) 1 + [Pi] k4 (1 - 2m)[RSH] k4
}
(21)
Ignoring phosphate has the effect of setting the last term in braces equal to unity. The value of k5/k4 at 25 °C is 400 M-1 (9). For a phosphate concentration of 0.1 M, k5[Pi]/k4 ) 40, so that the correction factor is estimated as 1 + 40 ) 41. Multiplying the rate constants of Kharitonov et al. (13) by this factor, the corrected values of k7 are 1.2 × 107 M-1 s-1 and 0.6 × 107 M-1 s-1 for GSH and N-Ac-Cys, respectively. These values are of the same order of magnitude as those of the present study. Further support for this interpretation is given by the data plotted in Figure 4 of Kharitonov et al. (13), which show that, for [GSH] >5 mM, the rate of GSNO formation is independent of [GSH]. As discussed earlier, this will be true only if k7[GSH] . k4. This relationship holds at [GSH] ) 5 mM for the corrected value of k7, but not for their reported value. Using the corrected value, k7[GSH] ) 6 × 104 . 1.6 × 103 ) k4. In summary, it appears that our data and those of Kharitonov et al. (13) are basically consistent and that the large discrepancy in the calculated rate constants is due mainly to the omission of the phosphate effect from their kinetic analysis. The kinetic analysis used here assumes that the S-nitrosation reaction involves the protonated (uncharged) thiol, rather than the dissociated (anionic) species. Thus, as has been done in studies of S-nitrosation at pH 2-3 (22, 23), the rate expressions are written in terms of [RSH] rather than [RS-]. There is essentially no dissociated thiol under those acidic conditions, and also very little at physiological pH. For example, the pK of GSH at 25 °C is 8.75 (24), so that at pH 7.4 only 4% is in the dissociated form. Nonetheless, participation of the dissociated forms of the thiols at physiological pH cannot be excluded, and it would be necessary to examine the kinetics over a range of pH to more definitely establish the identity of the reactants. It is inappropriate to compare the present rate constants with the acidcatalyzed values for GSH (23) and N-Ac-Pen (22), because the nitrosating agent under those conditions is thought to be NO+ (or the equivalent) rather than N2O3. Several reactions that were omitted from the present kinetic scheme may be relevant under other conditions. The decomposition reaction of S-nitrosothiols to release NO and form disulfides can be important if experiments are carried out under basic conditions (11, 24). Under sufficiently acidic conditions, HNO2 becomes a prominent species, the scavenging of N2O3 by phosphate and chloride is suppressed, and certain anions are able to catalyze N-nitrosation (9, 21). Transnitrosation reactions between S-nitrosothiols and other thiols (25) may play an important role in regulating the concentrations of these species in intracellular and extracellular fluids. The relative amounts of various thiols and S-nitrosothiols present in a given milieu will dictate the equilibrium concentrations
Kinetics of Nitrosothiol Formation
and eventually the regulatory or cytotoxic effects of NO or the nitroso adducts. Additional studies are thus required to completely understand the eventual fate of the S-nitrosothiol species formed in the body.
Acknowledgment. This work was supported by a grant from the National Cancer Institute (PO1-CA26731).
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Chem. Res. Toxicol., Vol. 9, No. 6, 1996 993 utility and toxicological implications. J. Org. Chem. 47, 156-159. (12) Park, J. W., Billman, G. E., and Means, G. E. (1993) Transnitrosation as a predominant mechanism in the hypotensive effect of S-nitrosoglutathione. Biochem. Mol. Biol. Int. 5, 885-891. (13) Kharitonov, V. G., Sundquist, A. R., and Sharma, V. S. (1995) Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J. Biol. Chem. 270, 28158-28164. (14) Lewis, R. S., and Deen, W. M. (1994) Kinetics of the reaction of nitric oxide with oxygen in aqueous solutions. Chem. Res. Toxicol. 7, 568-574. (15) Sies, H., Brigelius, R., and Akerboom, T. P. M. (1983) Intrahepatic glutathione status. In Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects (Larsson, A. L., Orrenius, S., Holmgren, A., and Mannervik, B., Eds.) pp 51-64, Raven Press, New York. (16) Wink, D. A., Darbyshire, J. F., Nims, R. W., Saavedra, J. E., and Ford, P. C. (1993) Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/ O2 reaction. Chem. Res. Toxicol. 6, 23-27. (17) Awad, H. H., and Stanbury, D. M. (1993) Autoxidation of NO in aqueous solution. Int. J. Chem. Kinet. 25, 375-381. (18) Ignarro, L. J., Fukuto, J. M., Griscavage, J. M., Rogers, N. E., and Byrns, R. E. (1993) Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from L-arginine. Proc. Natl. Acad. Sci. U.S.A. 90, 8103-8107. (19) Pogrebnaya, V. L., Usov, A. P., Baranov, A. V., Nesterenko, A. I., and Bez’yazychnyi, P. I. (1975) Liquid-phase oxidation of nitric oxide by oxygen. Zh. Prikl. Khim. 48, 954-958. (20) Schwartz, S. E. (1983) Trace atmospheric constituents: properties, transformations, and fates. In Advances in Environmental Science and Technology (Nriagu, J. O., Ed.) p 91, Wiley, New York. (21) Licht, W. R., Tannenbaum, S. R., and Deen, W. M. (1988) Use of ascorbic acid to inhibit nitrosation: kinetic and mass transfer considerations for an in vitro system. Carcinogenesis 9, 365-372. (22) Aldred, S. E., Williams, D. L. H., and Garley, M. (1982) Kinetics and mechanism of the nitrosation of alcohols, carbohydrates, and a thiol. J. Chem. Soc., Perkin Trans. 2 777-782. (23) Williams, D. L. H. (1985) S-nitrosation and the reactions of S-nitroso compounds. Chem. Soc. Rev. 14, 171-196. (24) Arnelle, D. R., and Stamler, J. S. (1995) NO+, NO•, and NOdonation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation. Arch. Biochem. Biophy. 318, 279-285. (25) Pietraforte, D., Mallozzi, C., Scorza, G., and Minetti, M. (1995) Role of thiols in the targeting of S-nitroso thiols to red blood cells. Biochemistry 34, 7177-7185.
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