Ozone Oxidizes Glutathione to a Sulfonic Acid - American Chemical

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Chem. Res. Toxicol. 2009, 22, 35–40

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Communication Ozone Oxidizes Glutathione to a Sulfonic Acid Shinichi Enami, M. R. Hoffmann, and A. J. Colussi* W. M. Keck Laboratories, California Institute of Technology, Pasadena, California 91125 ReceiVed August 7, 2008

Biosurfaces are universally covered with fluid microfilms containing reduced glutathione (GSH) and other antioxidants whose putative roles include the detoxification of ambient ozone (O3). It is generally believed that O3 accepts an electron from the thiolate GS2- function [pKa(GS-) ) 8.8] of GSH to produce thiyl GS•- radicals en route to the disulfide GSSG. Here, we report novel electrospray mass spectrometry experiments showing that sulfonates (GSO3-/GSO32-), not GSSG, are the exclusive final products on the surface of aqueous GSH microdroplets exposed to dilute O3(g) for ∼1 ms. The higher reactivity of the thiolate GS2- toward O3(g) over the thiol GS- is kinetically resolved in this time frame due to slow GS- acid dissociation. However, our experiments also show that O3 will be largely scavenged by the more reactive ascorbate coantioxidant in typical interfacial biofilms. The presence of GSSG and the absence of GSO3-/GSO32- in extracellular lining fluids are therefore evidence of GSH oxidation by species other than O3. Introduction Biosurfaces are universally protected from atmospheric ozone by extracellular fluid films (EF) containing antioxidants (AO) such as uric (UA), ascorbic (AH2) acids and reduced glutathione (GSH) (1-5). Ambient O3 cannot diffusively traverse these microfilms (in a few microseconds) (6) without being rapidly scavenged by AO into conceivably less nocive species, some of which may also transduce regulatory and defense signals (1, 7-12). The elucidation of mechanisms of oxidative stress and control requires the correct identification of such species (13). GSH is deemed to play a particularly important role in this context (8, 14, 15), both as an abundant scavenger of reactive oxygen species and as a reporter of cellular redox potential, because it is ultimately converted to the disulfide GSSG by endogenous one-electron oxidants such as O2•(15-18), which is recycled by GSH reductase (2, 19-23). Extracellular GSH is known to be biochemically, rather than diffusively, coupled to the underlying tissues because its levels may vastly exceed (at ∼400 µM, up to 100 times) those in the plasmatic pool (24). Thiols are versatile targets for most oxidants because they readily donate H-atoms to free radicals and accept O-atoms (8). More powerful oxidants can potentially convert, perhaps irreversibly, thiol (SH)/thiolate (S-) functionalities into higher S-oxidation states (14, 20, 25), thereby disrupting redox signaling via hitherto unidentified pathways (16, 19, 25). Because biosurfaces are multicomponent systems, kinetic competition among AO for reactive oxygen species along alternative reaction pathways is of the essence. Effective AO must scavenge exogenous oxidants X to prevent them from diffusing to the underlying tissues. For typical [AO] ∼ 0.1 mM in extracellular fluids (4, 24), and microsecond diffusional times * To whom correspondence should be addressed. E-mail: ajcoluss@ caltech.edu.

Figure 1. Mass spectra of the products of reaction of aqueous GSH with O3(g). A 1 mM concentration of GSH at pH 3.4 (upper) and 8.8 (lower) in the absence (blue)/presence (red) of 1260 ppmv O3(g) in 1 atm N2.

(6), the above condition invokes very fast reactions [k(AO + X) > 1 × 109 M-1 s-1 in bulk solution]. Similarly reactive

10.1021/tx800298j CCC: $40.75  2009 American Chemical Society Published on Web 12/12/2008

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Scheme 1. Mechanism of GSH Ozonolysis at the Air/Water Interface

AO at comparable concentrations could compete for X. Less reactive scavengers must be present in larger concentrations to be effective. Ozone is a thermodynamically powerful oxidant, a kinetically slow one-electron acceptor, and a reactive O-atom donor (26-29) that invariably induces airway inflammation and exacerbates asthma symptoms upon inhalation (30-33). Thus, chemistry alone dictates that the diffusionally controlled reaction of GSH(aq) with O3(aq) (22, 34-38) should proceed via (successive or simultaneous) O-atom transfer(s) into sulfenic, sulfinic, or sulfonic acids, rather than by electron transfer into thiyl plus ozonide radicals: GS2- + O3 f GS•- + O3•(21, 22, 34). Note in passing that the rapid decomposition of the ozonide radical, O3•-/HO3• f HO• + O2, implies that putative one-electron transfers would actually transform O3 into the more reactive HO• (26). Furthermore, O3(aq) and O3(g) are known to react at disparate rates with aqueous GSH (34, 35, 39-41). Because the relative reactivities of AH2, UA, and GSH toward O3(g) do not match those toward O3(aq) in bulk solution (34, 35, 38, 41), the gas/liquid interfaces relevant to EF conditions seem to represent unique reaction media (7, 35, 38-42). Using a novel technique that specifically monitors the composition of interfacial layers during fast gas/liquid reactions with millisecond time resolution (see the Experimental Procedures) (43, 44), we have found that among EF antioxidants only AH2 scavenges O3(g) below pH ∼4, whereupon it produces cytotoxic ozonides (45). Here, we report the rates and products of the interfacial ozonolysis of aqueous GSH in the 3.0 e pH e 10.8 range.

Figure 2. Mass spectra of the aqueous GSSG in the absence/presence of O3(g). A 1 mM concentration of GSSG at pH 6.7 (upper) and 9.2 (lower) in the absence (blue)/presence (red) of 940 ppmv O3(g) in 1 atm N2.

become mechanically unstable because Coulomb repulsion eventually overtakes liquid cohesion, triggering the spontaneous shedding of their interfacial films into nanometer size droplets (53). This phenomenon repeats itself until ions are ultimately ejected from last-generation nanodroplets by the large electric fields thereby created (49). These gas-phase ions can then be deflected into the mass spectrometer by applying a suitable electric bias to its inlet port. This analytical technique therefore reports the composition of nanodroplets created from the interfacial layers of microdroplets that had just reacted with O3(g). Further experimental details and validation tests can be found in previous reports from our laboratory (43-46, 54). Because a few milliseconds elapse between droplet creation and product detection, the extent of secondary reactions

Experimental Procedures Our experiment recreates the reaction of O3(g) with aqueous GSH on microdroplets produced by spraying GSH solutions into dilute O3(g)/N2(g) mixtures at atmospheric pressure. The composition of the microdroplets interfacial layers is directly monitored by inline mass spectrometry (ESMS, HP-1100) of the electrostatically ejected anions after submillisecond reaction times (43, 44, 46-49). Aqueous solutions are pumped at 50 µL/min into the spraying chamber of the mass spectrometer through a grounded stainless steel needle surrounded by a coaxial sheath issuing nebulizer N2 gas. The large difference between the exit velocities of the liquid jet and nebulizer gas forces the liquid to fragment into fine droplets (50-52). After leaving the reaction zone, fast solvent evaporation leads to droplet shrinkage and concomitant surface charge crowding. Such droplets

Figure 3. Mass spectra of the products of reaction of aqueous GSH with H2O2. Ten minutes after mixing 1 mM GSH with 1.2 mM H2O2 at pH 7.8 (red). The blue trace corresponds to a reference 1 mM GSH solution at pH 8.8. The inset expands about m/z ) 300.

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Figure 4. (A) Mass spectral signals of aqueous GSH mono- and dianions as functions of O3(g) concentration. Normalized GS- (upper) and GS2(lower) mass spectral signals vs [O3(g)] at various pH values. [GSH]0 ) 1 mM. (B) Mass spectral signals of the products of GSH ozonation as functions of O3(g) concentration. GSO3- (upper) and GSO32- (lower) mass spectral signals vs [O3(g)] at various pH values. [GSH]0 ) 1 mM.

Figure 5. Initial GSH ozonation rates vs pH. Symbols are initial slopes γ from Figure 4A (lower) (see the text and Table S1 of the Supporting Information for details). The line is a best fit exponential growth curve.

involving the H2O2 and O2(1∆g) byproducts of O3(aq) decomposition is minimized. Typical instrumental parameters were as follows: drying gas temperature, 250 °C; nebulizer pressure, 2 atm; collector capillary voltage, +3.5 kV; and fragmentor voltage, 17 V. Solutions were prepared with MilliQ water. GSH (L-GSH reduced, >99%), GSSG (GSH oxidized, >90%), H2O2 (hydrogen peroxide, 30%), and AH2 (L-ascorbic acid, >99%) were obtained from SigmaAldrich. The solution pH was adjusted by adding NaOH and was measured with a calibrated pH meter.

Results Products of GSH Ozonation. Figure 1 shows mass spectra obtained by spraying 1.0 mM aqueous GSH solutions at pH

3.4 and 8.8 in the absence/presence of 1260 ppmv O3(g) in 1 atm N2. Signals at m/z ) 306 and 152.5 correspond to the monoanion GS- and dianion GS2- reactants, respectively. Figure S1 (see the Supporting Information) shows the complementary evolution of these signals between pH 2.7 and pH 9.8. We ascribe GS- to the carboxylate from the cysteinyl-glycine terminus (pKa2 ) 3.6) and GS2- to the conjugate base of the thiol group (pKa3 ) 8.8). The R-ammonium-glutamyl moiety remains part of a zwitterion throughout (Scheme 1). Because anion protonation generally proceeds at diffusionally controlled rates: kP (X- + H+) ∼ 1010 M-1 s-1 in H2O at 300 K, microreversibility requires that the conjugated acids XH dissociate with rate constants given by kD (s-1) ∼ 1010-pKa, leading to ∼0.1 µs and ∼50 ms half-lives for GSH f GS- + H+ and GS- f GS2- + H+ dissociations, respectively. Therefore, only the pool of preexisting GS2- can react with O3 in the ∼1 ms time frame of these experiments, whereas GS- remains in dynamic equilibrium with GSH throughout. O3 readily adds to GSH, yielding GSO3- (m/z ) 354) and GSO32- (m/z ) 176.5) species as exclusive products. GSH is several hundredfold more reactive at pH 8.8 than at pH 3.4 (Figure 1, upper panel) suggesting that O3(g) reacts with the thiolate rather than with the thiol. This is borne out by MS/MS spectra. Whereas GS- and GS2- split H2O (-18 Da) and SH2 (-34 Da) neutrals via collisionally induced dissociation (CID) (55), GSO3- and GSO32- species only lose H2O and yield m/z ) 336 and 167.5 daughter ions, respectively. Should the preceding considerations about acid dissociation rates apply at the interface, the nascent GSO3- sulfonic acid [cf. pKa (cysteic acid) ) 1.3)] should dissociate to GSO32- in less than a microsecond and remain undetectable under present conditions.

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Figure 6. Relative reactivities of GSH and ascorbate toward O3(g) as a function of pH. Normalized AH-, GS-, and/or GS2- mass spectral signals vs [O3(g)] at various pH values in 0.5 mM AH2 + 0.5 mM GSH solutions. -

GSO3 is therefore ascribed to the ammonium-sulfonate zwitterion (Scheme 1). Neither GSSG, which would have appeared as a mono-, di-, or trianion at m/z ) 611, 305, and 203, respectively, nor the putative sulfenic acid GSOH intermediate (20), were ever detected in these experiments in the range: 13.5 g [GSH2]0 (mM)/[O3(g)] (ppmv) g 6 × 10-5 [Henry’s law constant H ) 0.01 M atm-1 for O3(g) in H2O at 300 K] (46). Sulfonates are also the products of GSH ozonation in bulk solution. Electrospray mass spectra (ESMS) of samples taken from 5 mM aqueous GSH solutions (initially at pH 7.2) immediately after being sparged with O3(g) (∼2000 ppmv in 1 atm air) for 3 min confirmed the presence of GSO3- and GSO32- and the absence of GSSG among reaction products. Identical ESMS were obtained 24 h later, implying that GSO3and GSO32- are persistent sulfonates rather than fleeting trioxide GSOOO(2)- intermediates. The low pH ∼ 4.0 of the ozonized solutions is consistent with the conversion of weak thiol into strong sulfonic acids.

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Figure 2 shows mass spectra of GSSG solutions obtained in the absence and in the presence of 940 ppm O3(g). The lack of reactivity of the mono-, di-, and trianions of GSSG toward O3(g) under present conditions confirms that GSO3- and GSO32- are direct products of GSH ozonation, rather than secondary species derived from GSSG. We also found that at variance with ozonation, GSSG is indeed produced during the oxidation of GSH by H2O2 in bulk water (Figure 3), in accord with previous studies. Mechanism of GSH Ozonation. Figure 4 shows the dependence of reactants GS- and GS2- (Figure 4A) and products GSO3- and GSO32- (Figure 4B) on [O3(g)] at various pH values. Because (1) [GS-] actually accounts for ([GSH] + [GS-]), whereas [GS2-] remains uncoupled to [GS-] during these experiments (see above) and (2) only GS2- decays at pH ∼ 3, we infer that the dianion is the actual reactive species. We have shown elsewhere that initial slopes γ ) (∂[X]/∂[O3 (g)])[O3]f0 are proportional to reaction rate constants (45). The γ’s derived from Figure 4A (Table S1 of the Supporting Information) are plotted as a function of pH in Figure 5. It is apparent that the ability of GSH to scavenge O3(g) tracks the [GS2-]/[GS-] ratio (Figure S1 of the Supporting Information) and that, in contrast with AH2 and UA, GSH becomes inactive below pH ∼ 5 (45, 54). The possible participation of OH radicals in these processes is precluded by the observation that neither the identity of products nor the reactivity of GSH change upon addition of up to 1.1 M tert-butanol to GSH solutions (Figure S2 of the Supporting Information). O3(g) Scavenging by GSH/Ascorbic Acid Mixtures. Figure 6 shows the decay of AH2 and GSH in equimolar (0.5 mM AH2 + 0.5 mM GSH) microdroplets at bulk pH 3.3, 6.4, and 9.8 as functions of [O3(g)]. Not only is AH2 more reactive than GSH in all cases, but it remains active at pH 3.3 (Figure 6). Thus, at pH 6.4, 50% of AH2 and GSH is oxidized upon exposure to 1 and 1200 ppmv O3(g), respectively, for ∼1 ms (Figure 6). The ratio of initial rates at pH ∼ 7.3 [γ(AH2 + O3)/ γ(GSH + O3)] ∼ 30 indicates that less than 3% of impinging O3 will be scavenged by GSH in equimolar (AH2 + GSH) solutions. The much larger reactivity of AH2 vs GSH toward O3(g) at the air/water interface under all conditions is at variance with reported rate constants for these reactions in bulk solution (34, 39, 41).

Discussion The products of the ozonation of amino acids, peptides, and proteins have been analyzed in the past using enzymatic/ colorimetric methods (22, 35) and, more recently, by means of liquid chromatography with spectrophotometric detection (57). It should be emphasized, however, that the reverse-phase columns typically used to analyze neutral species (such as GSH/ GSSG) in slightly acidic (0.1% formic acid) mobile phases may be inadequate for ionic species, such as the sulfonates generated in the ozonation of GSH (57). Furthermore, because GSH reductase does not reduce sulfonates back to GSH, previous enzymatic analyses based on the back conversion of GSSG to GSH are silent about the formation of sulfonates in the ozonation of GSH (22, 24, 35). Direct infusion electrospray ionization mass spectrometry have revealed, for example, that the products of Tyr, His, Met, Phe, and Trp ozonolysis involve multiple O-atom additions (58, 59), in contrast with earlier studies. Most extracellular fluids contain various AO that can potentially scavenge ambient ozone. However, only the most reactive species count, and then only those present at comparable concentrations. Reactive but sparingly soluble or water-insoluble

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substrates, such as PUFA’s and lipidic surface membranes, may not act as primary O3(g) scavengers. The results of Figure 6, together with the condition [GSH] ∼ [AH2] ∼ 100 µM in typical epithelial lining fluids (4), account for the absence of GSH sulfonates in bronchoalveolar lavages. They are also consistent with documented reactivity patterns, because O3 engages in outer-sphere one-electron transfers [E0(O3/O3-)aq ) 1.02 V] with reactants that readily donate one but not two electrons and otherwise lack O-atom acceptor sites (28). Thus, whereas O3 can oxidize GS2- to thiyl radicals [E0(GS•-/ GS2-) ) 0.92 V] (60), the thiolate will rather add O-atoms to produce GSO1-32species (28). Two-electron oxidizers, such as HOCl, may lead to GSH hyperoxidation (20, 61) or to GSSG, such as the much less reactive H2O2 (Figure 3) (8, 18). Another important consideration relates to the fact that O3 reactions in aqueous media generate secondary oxidants, such as H2O2, •OH, and O2(1∆g) (26, 62), that eventually build up and participate in the oxidation process upon protracted O3 exposure (57, 63). Our experiments, which last ∼1 ms vs ∼103 s in conventional studies (22, 24, 35), minimize the development of secondary chemistry (1, 37). The possibility that the sulfonate is the last stage of successive O-atom transfers proceeding via the sulfenic acid GSOH-, which at sufficiently high [GSH]/ [O3] ratios could have been diverted to GSSG, is not realized in our experiments (see above) (14, 15). It appears that unprotected by a protein microenvironment, sulfenic acids exposed at the air/water interface readily undergo further oxidation. As mentioned before, kinetic information is critical to assess whether extracellular GSH will significantly scavenge ozone (8). Serious and unexplained inconsistencies remain among reported rate constants for AH2, UA, and GSH reactions with O3 (35, 38-41). Pryor et al. reported reaction rate constants k (M-1 s-1) ) 6.0 × 107, 1.4 × 106, and 7.0 × 108 for of AH2, UA, and GSH at pH ∼7 with O3(aq), respectively, measured by stopped-flow techniques (34, 39). In contrast, Kermani et al. obtained k (M-1 s-1) ) 5.5 × 104, 5.8 × 104, and 57.5 M-0.75 s-1, respectively, by sparging aqueous solutions with ozonated air through a sintered glass diffuser (41). Our studies (45, 54) lead to a reactivity ranking AH2 ∼ UA . GSH at the air/water interface that is in accord with that observed in the interaction of O3(g) with quiescent antioxidant solutions at pH ∼ 7 (35, 38, 41). In summary, we have determined that O3(g) oxidizes the thiolate function of GSH to a sulfonate rather than a disulfide and that this process is fully inhibited by the presence of comparable ascorbate concentrations. Acknowledgment. This project was financially supported by the National Science Foundation (ATM-0534990). S.E. is grateful to the Japan Society for the Promotion of Science Research Fellowship for Young Scientists. Supporting Information Available: Additional data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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