A Role for Peroxymonocarbonate in the Stimulation of Biothiol

Peroxymonocarbonate (HCO4-) is an oxidant whose existence in equilibrium with hydrogen peroxide and bicarbonate has been known since the 1980s.Missing...
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Chem. Res. Toxicol. 2006, 19, 1475-1482

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A Role for Peroxymonocarbonate in the Stimulation of Biothiol Peroxidation by the Bicarbonate/Carbon Dioxide Pair Daniel F. Trindade, Giselle Cerchiaro, and Ohara Augusto* Departamento de Bioquı´mica, Instituto de Quı´mica, UniVersidade de Sa˜ o Paulo, CP 26077, CEP 05513-970, Sa˜ o Paulo, SP, Brazil ReceiVed June 28, 2006

Peroxymonocarbonate (HCO4-) is an oxidant whose existence in equilibrium with hydrogen peroxide and bicarbonate has been known since the 1980s. More recently, peroxymonocarbonate has been proposed to mediate oxidative processes stimulated by the bicarbonate/carbon dioxide pair. To better understand this emerging biological oxidant, we re-examined the kinetics of its formation from hydrogen peroxide and bicarbonate/carbon dioxide by 13C NMR. Also, we studied its role in the accelerating effects of bicarbonate on biothiol (GSH and BSA-cysSH) peroxidation by kinetics and product analysis. The rate constants for peroxymonocarbonate formation and decay were estimated and Keq values determined (pH 7.2, at 25 and 37 °C; in the absence and presence of BSA and liposomes of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine phosphatidylcholine). Noteworthy is the fact the rate constant for peroxymonocarbonate formation estimated here (k1 ∼10-2 M-1 s-1) was more than 1 order of magnitude higher than a previously reported value. Also, peroxymonocarbonate equilibrium was shown to be affected by BSA, liposomes, and a carbonic anhydrase mimetic. The Keq values determined in the absence and presence of BSA (0.35 and 0.48 M-1, respectively, at 37 °C) were employed to analyze the kinetics of BSAcysSH and GSH peroxidation in the presence of bicarbonate (2-25 mM). A good fit of experimental data with simulations indicated that peroxymonocarbonate is the main species responsible for biothiol peroxidation in the presence of bicarbonate. The results indicate that peroxymonocarbonate is a feasible biological oxidant, in addition to supporting emerging data that the main physiological buffer is redox active. Introduction Bicarbonate is an abundant anion whose serum and intracellular concentration of 25 and 14.7 mM, respectively, in equilibrium with around 1.3 mM carbon dioxide is essential to maintain physiological pH. In spite of its relevance, the bicarbonate/carbon dioxide buffer is rarely used in studies of enzymatic and nonenzymatic oxidations in Vitro. In addition, the bicarbonate/carbon dioxide pair is usually considered to be redox inactive, despite many studies demonstrating that it can stimulate biological oxidations, peroxidations, and nitrations (117). The mechanisms by which the bicarbonate/carbon dioxide pair accelerates biological oxidations remain under scrutiny. The better elucidated mechanism is the one by which the pair inhibits peroxynitrite-mediated two-electron oxidations, while increasing peroxynitrite-mediated one-electron oxidations and nitrations (18, 19). This occurs because carbon dioxide reacts rapidly with peroxynitrite (7) to produce 65% nitrate and 35% carbonate radical and nitrogen dioxide. Targets whose concentrations and rate constants of reaction with peroxynitrite cannot compete with carbon dioxide will be oxidized by nitrogen dioxide and carbonate radicals by one-electron mechanisms in substoichiometric yields. In the absence of carbon dioxide and at a pH around neutrality, these targets will be oxidized by two-electron mechanisms in yields close to stoichiometry (18, 19). The unequivocal EPR demonstration that the reaction between peroxynitrite and carbon dioxide produces the carbonate radical * Corresponding author. Phone: 55-11-3091-3873. Fax: 55-11-30912186. E-mail: [email protected].

(20) provided an excellent candidate to explain bicarbonate/ carbon dioxide pair-stimulated oxidations. Although less oxidizing than the hydroxyl radical (Eo)2.3 V, pH 7.0), the carbonate radical is a strong one-electron oxidant (Eo)1.8 V, pH 7.0) that does not add to biomolecules, in contrast to the former. Because the carbonate radical is more specific than the hydroxyl radical, it may increase the oxidation/nitration of particular biotargets (8, 14, 18). Accordingly, the carbonate radical has been proposed to be the major diffusible oxidant produced from the peroxidase activity of the enzyme Cu,Zn-SOD, which has been extensively studied in recent years because of its potential relationship with familial amyotrophic lateral sclerosis (7, 10-12, 21-23). The production of a diffusible carbonate radical during the peroxidase activity of Cu,Zn-SOD is supported by several lines of evidence, but its immediate precursor, whether it be bicarbonate (7, 21), carbon dioxide (11), or peroxymonocarbonate (22, 23), remains debatable. Peroxymonocarbonate (HCO4-) is an oxidant whose existence in equilibrium with hydrogen peroxide and bicarbonate has been known since the 1980s (eq 1) (24-26). Its relevance as a biological oxidant, however, has been evidenced only very recently. In the case of the peroxidase activity of Cu,Zn-SOD, the evidence was provided by crystallographic data, indicating a putative oxyanion-binding site in the structure of a mutant Cu,Zn-SOD associated with familial amyotrophic lateral sclerosis. It has been suggested that bicarbonate binding to this site is followed by hydrogen peroxide addition to produce an enzyme-bound peroxymonocarbonate (23). More recently, Richardson and co-workers and our group proposed peroxymono-

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carbonate formation to explain the accelerating effects of the bicarbonate/carbon dioxide pair upon hydrogen peroxidemediated oxidation of methionine (27) and BSAcys-SH (12), respectively. We also proposed peroxymonocarbonate as the precursor of the carbonate radical produced during xanthine oxidase turnover in the presence of the bicarbonate/carbon dioxide pair (28). In this case, peroxymonocarbonate formation and reduction to the carbonate radical (eq 2) was suggested to be facilitated by the many metal centers of xanthine oxidase.

H2O2 + HCO3- a HCO4- + H2O

(1)

HCO4- + e- + H+ f CO3•- + H2O

(2)

Taken together, the above studies suggested that peroxymonocarbonate may be an important biological oxidant derived from the bicarbonate/carbon dioxide pair (29). Thus, a better understanding of its formation and reactions in aqueous buffer is required. Here, we re-examined the kinetics of peroxymonocarbonate formation from hydrogen peroxide and bicarbonate/ carbon dioxide by 13C NMR under different conditions and examined its role in the acceleration of biothiol (GSH and BSAcysSH) peroxidation in the presence of bicarbonate.

Materials and Methods Materials. All reagents were purchased from Sigma, Aldrich, Merck, Fisher, or Alexis and were of analytical grade or better. All buffers were pretreated with Chelex-100 to remove metal ion contamination. All solutions were prepared with distilled water purified with a Millipore Milli-Q system. BSA1 (fraction V) from Merck was treated overnight with 10 times molar excess of 2-mercaptoethanol at 4 °C to reduce eventually oxidized thiol residues. Excess reductant was removed by dialysis against phosphate buffer (100 mM, pH 7.4) (12). BSA solutions prepared as described above were 2.0-2.5 mM and typically 0.7-0.9 BSAcys-SH/BSA. The free thiol group of BSA was blocked by incubation with 5 times molar excess of N-ethylmaleimide for 30 min at 25 °C. Excess N-ethylmaleimide was removed by dialysis against phosphate buffer (100 mM, pH 7.4) (12). Hydrogen peroxide concentration was determined spectrophotometrically (240 nm ) 43.6 M-1 cm-1) (30). Liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) (from Alexis) were prepared from 10 mM solutions of the lipid in chloroform. The solvent was removed under a steady stream of nitrogen gas in test tubes (35 × 95 mm) at room temperature; afterward, the test tubes containing the sample were maintained under vacuum for at least 8 h (31). Just before the 13C NMR experiments, the dry lipid was rehydrated by vortexing with 100 mM phosphate buffer at pH 7.2 to give a final concentration of 10 mM POPC. 13C NMR Spectra. Solutions of 13C-enriched sodium bicarbonate (99% 13C from ISOTEC) were prepared in phosphate buffer (100 mM) in the absence and presence of thiol-blocked BSA or POPC liposomes to give final concentrations of 0.2 M, 0.8 mM, and 0.5 mM, respectively, after the addition of all other incubation components. The required volume of D2O (99.9%) to give a final enrichment of 10% was always added and the pH adjusted to 7.2 ( 0.3 with HCl. Then, the required volume of concentrated hydrogen peroxide to give a final concentration of 2 M was added at zero time. The incubation mixture was then transferred to an NMR tube with an inserted standard tube (NE-5-CIC Wilmad) containing 13C-enriched sodium formate (98% 13C from ISOTEC) (0.5 M) and the 13C NMR spectrum recorded with time. All shifts 1 Abbreviations: BSA, bovine serum albumin; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid); DTPA, diethylenetriamine-N,N,N’,N’-pentaacetic acid; GSH, glutathione; NBD-Cl, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; NEM, N-ethylmaleimide; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

(δ, ppm) and integral values were based on those of the standard 13C-enriched sodium formate (0.5 M) (32). The employed instrument was a Bruker DRX 500 Fourier transform spectrometer (operating at 125.757 MHz for 13C NMR) equipped with a 5 mm dual probe and Xwinnmr 3.5 software. The employed parameters were pulse length, 90°; acquisition time, 1.00275 s; relation delay, 10 s (total recycling time 11.00275 s); spectral width, 32.679 MHz; digital resolution, 0.498 Hz/point; and temperature 25.0 ( 0.3 °C or 37.0 ( 0.3 °C. Exponential line broadening multiplication (LB ) 2.0 Hz) of the free induction decay was applied before Fourier transform measurements. The number of accumulated acquisitions (NS) was 8 for each spectrum. In some experiments, the incubations contained the Zn(His)2 complex that was obtained by mixing one equivalent of zinc chloride with two equivalents of L-histidine in water (33). Thiol Oxidation. Bicarbonate (2-25 mM) and hydrogen peroxide (1 mM) were mixed into phosphate buffer (0.1 M) containing DTPA (0.1 mM) at pH 7.4 at 37 °C, and the thiol solution (∼1 mM) was added to start the reaction. Aliquots were taken at various incubation times, and the reaction stopped by dilution with phosphate buffer (0.1 M, pH 8.2), containing catalase (50 units/ mL) and DTBN (1 mM) (7). After a further 25 min of incubation at room temperature, thiol concentration was determined spectrophotometrically by 5-thio-2-nitrobenzoate formation (412 nm ) 13.6 × 103 M-1 cm-1) (34). Trapping of BSA-Sulfenic Acid with NBD-Cl. Aliquots of the incubations were taken at various incubation times, and the reaction was stopped by dilution in a phosphate buffer (0.1 M, pH 7.4) containing catalase (50 units/mL) and NDB-Cl (0.1 mM) (two times molar excess over BSA). After 30 min of incubation at room temperature, the samples were filtered through centricon filters (10 KD cutoff) and washed two times with phosphate buffer (0.1 M) at pH 7.4. The re-suspended samples were analyzed by their UVvisible spectra in an upgraded Aminco SL 2000 spectrophotometer (7, 35, 36). HPLC Analysis of GSH Oxidation Products. Aliquots of the incubations were taken at various incubation times, and the reaction was stopped by dilution in a phosphate buffer (0.1 M, pH 7.4) containing catalase (50 units/mL). The samples were centrifuged (15.000g for 5 min) in a cellulose filter (5 KD cutoff) to remove catalase and submitted to HPLC analysis (37). The HPLC system (Shimadzu, Kyoto, Japan) consisted of a pump (LC-10AD), a guard cell (900 mV), a Rheodyme injector, and an electrochemical detector (ESA, Inc.) at 850 mV. Chromatographic separation was carried out with a Phenomenex C18 (250 × 3.0 mm) column at 45 °C that was eluted with phosphate buffer (H3PO4/NaOH, 20 mM, pH 2.7) containing octanesulphonic acid (50 µM) and acetonitrile (2%) at a flow rate of 1.0 mL/min. Identification and quantification of GSH and GSSG were performed by integration of the corresponding HPLC peaks and comparison of the areas obtained for standards under the same HPLC conditions. EPR Spin-Trapping Experiments. To examine the production of radicals during the peroxidation of GSH and BSA, EPR spintrapping experiments were performed with 5,5-dimethylpyrroline N-oxide (DMPO), which is an efficient trap for high and low molecular weight thiyl radicals (38). The incubation mixtures containing bicarbonate (25 mM) and hydrogen peroxide (1 mM) were mixed into a phosphate buffer (0.1 M) containing DTPA (0.1 mM) and the spin trap (100 mM) at pH 7.4 and 37 °C, and then the thiol solution was added. Aliquots were removed at various incubation times and examined by EPR. The spectra were recorded at room temperature on a Bruker ER 200 D-SRC upgraded to an EMX instrument and equipped with a high sensitivity cavity (4119HS). Kinetic Simulations. The Gepasi software, version 3.3, was used to simulate the kinetics of the peroxidation of biothiols in the presence of the bicarbonate/carbon dioxide pair (39, 40). Preequilibrium formation of peroxymonocarbonate, whose concentrations were maintained at steady-state, was assumed.

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time was employed to obtain the values of Keq (eq 2 and 3) and kobs (Figure 2; Table 1). Because of the inherently long time required for scanning the NMR spectra, the few data points employed to determine kobs were those that reflected peroxymonocarbonte peak area increase with time (At), whereas the peak area at equilibrium was taken as the maximum concentration (A∞) (Figure 2). Because k1 and k-1 values were obtained from kobs and Keq values (eq 4) (42), they are likely to be underestimated (Table 1).

Keq ) Figure 1. Time-dependent 13C NMR spectra of incubations of 13Clabeled bicarbonate (0.2 M) and hydrogen peroxide (2 M) in a phosphate buffer (100 mM) at pH 7.2 ( 0.2 at 25 °C. Assignment of the formate (171.3 ppm), bicarbonate (160.5 ppm), and peroxymonocarbonate (158.9 ppm) peaks at pH 7.2 is in agreement with the literature as discussed in the text.

Figure 2. Time-dependent increase of the relative area of the 13C NMR peak of peroxymonocarbonate in the absence (9) and presence of thiolblocked BSA (BSA-cysSNEM) (0.8 mM) (0) at 25 °C. The incubations contained 13C-labeled bicarbonate (0.2 M) and hydrogen peroxide (2 M) in a phosphate buffer (100 mM) adjusted to pH 7.2 ( 0.3 at 25 °C. The inset shows the first-order plot employed to determine kobs for peroxymonocarbonate formation in the same incubation mixtures. The peroxymonocarbonate areas at equilibrium were taken as those at t∞. The shown HCO4-/HCO3- ratios specify the concentration ratio at each equilibrium condition.

Results Peroxymonocarbonate Equilibrium under Different Conditions. Our previous studies suggested that BSA was likely to influence peroxymonocarbonate formation in addition to being its target because of free thiol group oxidation (12). Thus, peroxymonocarbonate formation was followed in neutral aqueous solutions by 13C NMR (25, 26) in the absence and presence of thiol-blocked BSA (BSA-cysSNEM) (0.8 mM). Typical incubations contained 13C-labeled bicarbonate (0.2 M), hydrogen peroxide (2 M), 13C-labeled formate as the internal standard (0.5 M), and a phosphate buffer (100 mM) at pH 7.2 ( 0.3 at 25 °C, with or without thiol-blocked BSA (0.8 mM) (Figures 1 and 2). According to the literature, the formate peak appeared at 171.3 ppm (32), whereas those of bicarbonate and peroxymonocarbonate appeared at 160.5 and 158.9, respectively (25) (Figure 1). A time-dependent decrease and increase of bicarbonate and peroxymonocarbonate peaks, respectively, was observed up to 6-8 min. Thereafter, the peaks remained relatively stable, particularly in the incubations containing BSA-cysSNEM (Figure 2). The high hydrogen peroxide concentration is expected to oxidize most of the protein susceptible residues, which should be solvent-exposed methionine residues (41) in the case of thiol-blocked BSA. However, no gross structural protein alteration was noticeable by analysis of the CD spectra (data not shown). Integration of peroxymonocarbonate and bicarbonate peaks relative to those of the internal standard with

[HCO4-] [H2O2] [HCO3-]

kobs ) k1 [H2O2] + k-1 ) k1 [H2O2] + k1/Keq

(3) (4)

In the absence of thiol-blocked BSA, the obtained value of Keq was in agreement with that reported by Richardson and coworkers (26), but the values of k1 and k-1 were 1 order of magnitude higher. This discrepancy is most likely due to the fact that our incubations did not contain ethanol and that their pH was controlled (7.2 ( 0.3). This was confirmed by parallel measurements of the pH during the incubations (data not shown) and by the position of bicarbonate and peroxymonocarbonate peaks in the NMR spectrum (25) (Figure 1). Indeed, ethanol competes with hydrogen peroxide for bicarbonate (26), whereas alkaline pH has been previously reported to decrease the rate of peroxymonocarbonate formation (25). The presence of BSA-cysSNEM affected peroxymonocarbonate equilibrium (Table 1). It decreased k1 and k-1 values at 25 °C and increased the Keq value (Table 1). Thus, BSA displaced the equilibrium toward peroxymonocarbonate and stabilized it, as also attested to by the constancy of the equilibrium concentration of peroxymonocarbonate in the protein-containing incubations (Figure 2). After hydrogen peroxide addition, bubbles were abundant in buffer-containing solutions but hardly seen in protein-containing solutions. Thus, BSA-cysSNEM is likely to influence the equilibrium between dissolved and gaseous carbon dioxide. However, other possibilities cannot be excluded because hydrogen peroxide, bicarbonate, and peroxymonocarbonate (eqs 2 and 3) are in equilibrium with several other species (eqs 5-7) (43, 44) that are invisible to 13C NMR. Indeed, our 13C NMR spectra only showed bicarbonate and peroxymonocarbonate peaks consistently.

CO2 (g) a CO2 (d) + H2O a H2CO3 a HCO3- + H+ pKa 6.4/6.1(25/37 °C) (5) HCO3- a CO32- + H+ pKa 10.3

(6)

H2O2 a H+ + HO2- pKa 11.7

(7)

That the peroxymonocarbonate equilibrium is complex was further attested to by following peroxymonocarbonate formation at 37 °C (Figure 3). In the absence of BSA-cysSNEM, k1 and k-1 decreased, whereas Keq increased at 37 °C compared with that at 25 °C (Table 1). Again, this may reflect lower concentrations of dissolved carbon dioxide because of the temperature dependence of bicarbonate/carbon dioxide pKa (eq 5). In the presence of BSA, the equilibrium was attained faster at 37 (∼4 min) (Figure 3) than at 25 °C (∼6 min) (Figure 2). It was possible to determine the Keq (Table 1) but not kobs because equilibrium was attained at the time the second NMR spectrum was scanned (Figure 3). Nevertheless, at 37 °C,

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Table 1. Values of Rate Constants and Keq Determined for Peroxymonocarbonate Equilibrium at pH 7.2 Under Different Conditionsa conditions

k1 (M-1 s-1)

k-1 (s-1)

Keq (M-1)

25 °C 25 °C, BSA-cysSNEM 25 °C, POPC liposomes 25 °C, Zn(His)2 37 °C 37 °C, BSA-cysSNEM 37 °C, POPC liposomes

(3.1 ( 0.3) × 10-3 (1.4 ( 0.2) × 10-3 (2.0 ( 0.2) × 10-3 ∼9 × 10-3b (2.4 ( 0.3) × 10-3 ∼3.6 × 10-3c ∼3.6 × 10-3c

(9.9 ( 0.3) × 10-3 (3.6 ( 0.1) × 10-3 (5.1 ( 0.2) × 10-3 ∼2.6 × 10-2b (6.9 ( 0.3) × 10-3 ∼7.5 × 10-3c ∼7.5 × 10-3c

0.31 ( 0.01 0.39 ( 0.02 0.39 ( 0.02 0.35 ( 0.03 0.35 ( 0.02 0.48 ( 0.03 0.39 ( 0.03

a Incubations and data analysis were performed as described in Materials and Methods and Results. The values correspond to the mean ( SD of three independent experiments. b Values estimated from the fact that equilibrium established at least three times faster in the presence of Zn(His)2 than in the absence of Zn(His)2, that is, faster than the scanning of the first 13C NMR spectrum. c Values estimated from the fact that equilibrium established at least 1.5 times faster in the presence of BSA-cysSNEM or POPC liposomes than in the absence of BSA-cysSNEM or POPC liposomes.

Figure 3. Time-dependent increase of the relative area of the 13C NMR peak of peroxymonocarbonate in the absence (9) and presence of thiolblocked BSA (BSA-cysSNEM) (0.8 mM) (0), or POPC liposomes (0.5 mM) (∆) at 37 °C. The incubations contained 13C-labeled bicarbonate (0.2 M) and hydrogen peroxide (2 M) in a phosphate buffer (100 mM) adjusted to pH 7.2 ( 0.3 at 37 °C. The inset shows the first-order plot employed to determine kobs for peroxymonocarbonate formation in the same incubation mixtures; in the case of the incubations containing BSA-cysSNEM or POPC liposomes, kobs was estimated because equilibrium was attained before 6 min. The peroxymonocarbonate areas at equilibrium were taken as those at t∞. The shown HCO4-/HCO3ratios specify the concentration ratio at each equilibrium condition.

peroxymonocarbonate formation (k1) should be at least 1.5 times faster in the presence of BSA than that in the absence of BSA because equilibrium was attained 1.5 times faster (Figure 3, Table 1). Of relevance is the fact that zweiteronic lipids such as POPC liposomes (0.5 mM) decreased bubble formation in incubations of hydrogen peroxide with bicarbonate and affected their equilibrium in manner similar to that with BSA-cysSNEM, albeit to a lesser extent at both 25 and 37 °C (Figure 3; Table 1). These results further suggest that the equilibrium among gaseous carbon dioxide/dissolved carbon dioxide/bicarbonate is important for peroxymonocarbonate formation and is affected by proteins and lipids. The effects of a carbonic anhydrase mimetic, Zn(His)2 (1 mM) (45), on peroxymonocarbonate formation were also examined. Even at 25 °C, the equilibrium was attained before the first NMR spectrum was scanned (∼2 min) (data not shown). Although kobs could not be determined, peroxymonocarbonate formation should be at least three times faster in the presence of the Zn(II)-histidine complex than that in the absence of the Zn(II)-histidine complex because in the latter case, equilibrium was attained at around 6 min (Figure 2; Table 1). Thus, Zn(II) complexes, other Lewis acids, and eventually carbonic anhydrase (45) are likely to increase the rate of peroxymonocarbonate formation. Taken together, the above results confirmed that peroxymonocarbonate is produced and equilibrates in aqueous solutions of bicarbonate and hydrogen peroxide at neutral pH (eq 1) (Figures 1-3) (24-27). Peroxymonocarbonate production is an

Figure 4. Kinetics of BSA-cysSH (0. 8 mM) oxidation by hydrogen peroxide (1 mM) in the absence (0) and presence of bicarbonate (25 mM) (9) in a phosphate buffer (0.1 M) at pH 7.4, containing DTPA (0.1 mM) at 37 °C. The inset compares the experimental (O) and simulated kinetics (line) in the presence of bicarbonate up to 3 min as described in Materials and Methods and Results.

apparently slow process (k ∼10-2 M-1 s-1) (Table 1), although the rate constant values determined here were more than 1 order of magnitude higher than that previously reported (26). Also, the results demonstrate that peroxymonocarbonate formation is modulated by proteins, lipids, and metal complexes. Peroxidation of BSA and GSH in the Presence of Bicarbonate/Carbon Dioxide. Our previous proposal that BSAcysSH peroxidation was accelerated by the bicarbonate/carbon dioxide pair because of peroxymonocarbonate formation was based on preliminary evidence obtained from studies of the peroxidase activity of Cu,Zn-SOD (12). Here, we systematically examined the peroxidation of BSA-cysSH (0.8-1.2 mM) and GSH (1-1.2 mM) with hydrogen peroxide (1 mM) in the absence and presence of bicarbonate (2-25 mM) in a phosphate buffer (100 mM) containing DTPA (0.1 mM) at pH 7.4 and 37 °C. The results showed that the peroxidation of both thiols was accelerated by bicarbonate in a concentration-dependent manner (Figures 4-6). To examine whether bicarbonate-accelerating effects were due to peroxymonocarbonate formation (12), the initial rates of thiol oxidation were assumed to be essentially dependent on the equilibrium concentrations of peroxymonocarbonate, and kobs values were determined from pseudo-first-order plots (Figure 6) (eqs 3, 8). The equilibrium concentrations of peroxymonocarbonate under the employed experimental conditions (hydrogen peroxide 1 mM and bicarbonate 2-25 mM) were calculated from the Keq values (eq 3) obtained at 37 °C in the absence (0.35 M-1) and presence of BSA (0.48 M-1) (Table 1). From the obtained kobs values, the second-order rate constant of the

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Figure 5. Kinetics of GSH (0.87 mM) oxidation by hydrogen peroxide (1 mM) in the absence (0) and presence of bicarbonate (25 mM) (9) in a phosphate buffer (0.1 M) at pH 7.4, containing DTPA (0.1 mM) at 37 °C. The inset compares the experimental (O) and simulated kinetics (line) in the presence of bicarbonate up to 3 min as described in Materials and Methods and Results.

Figure 6. Determination of kobs of the oxidation of BSA-cysSH (9) and GSH (∆) by peroxymonocarbonate in equilibrium with hydrogen peroxide (1 mM) and different concentrations of bicarbonate (2-25 mM) in a phosphate buffer (0.1 M) at pH 7.4, containing DTPA (0.1 mM) at 37 °C. The equilibrium concentrations of peroxycarbonate were calculated from the determined Keq values in the absence and presence of BSAcysNEM (Table 1).

reaction of peroxymonocarbonate with BSA-cysSH and GSH (k) (eq 9) was determined (Table 2).

-d[RSH] ) k[RSH][HCO4-] dt

(8)

-d[RSH] ) kobs[HCO4-] dt

(9)

In parallel, the second-order rate constant of the reaction of hydrogen with BSA-cys-SH and GSH was also determined (Table 2), and the obtained values were in reasonable agreement with previously reported values (36, 46-48). Next, the reactions shown in Table 2 and their corresponding rate constants were employed to simulate the kinetics of BSA-cysSH (Figure 4 inset) and GSH (Figure 5 inset) peroxidation in the presence of 25 mM bicarbonate. A good agreement between the experimental and simulated kinetics in the early peroxidation phase was obtained by assuming a pre-equilibrium formation of peroxymonocarbonate, whose concentration was maintained at steadystate (Materials and Methods). At longer times, experimental and simulated kinetics deviate particularly in the case of GSH, probably because of further reactions of the sulfenic acid derivative (RcysSOH) with the remaining thiol (RcysSH) (eq 11) and/or peroxide (46, 48, 49). These reactions were not included in the simulations because their second-order rate

constants are not known for either BSA-SH or GSH. In addition, thiol oxidation to produce the sulfenic acid derivative is expected to be the rate-limiting step, as has been shown for the amino acid cysteine, whose reaction with hydrogen peroxide (k ) 15.2 M-1 s-1, pH 6.0) is much slower than the reaction of cysSOH with cysSH (k ) 720 M-1 s-1) (49). This reaction (eq 11) is expected to be much faster in the case of low than high molecular weight thiol because the sulfenic acid derivatives of cysteine and GSH have never been isolated (46, 48, 49), whereas BSA-cysSOH has been (36) (also, see below). In agreement, the fit between experimental and simulated kinetics was better with BSA-cysSH (Figure 4, inset) than with GSH (Figure 5 inset) even at early oxidation stages. Taken together, these results indicate that under our experimental conditions, BSAcysSH and GSH are being oxidized mainly by the peroxymonocarbonate in equilibrium with the employed concentrations of hydrogen peroxide and bicarbonate (Figures 4-6).

RcysSOH + RcysSH f RcysS-ScysR + H2O

(10)

Because the 13C NMR experiments showed that thiol-blocked BSA influences peroxymonocarbonate equilibrium (Figure 2, Table 1), it was interesting to examine whether it also affected the rate of biothiol peroxidation. Thus, the initial rate of BSAcysSH (0.4 mM) and GSH (1.1 mM) oxidation by hydrogen peroxide (1 mM) in the presence of bicarbonate (25 mM) was determined in the presence of increasing concentrations of thiolblocked BSA (up to ∼1.1 mM) (Figure 7). Of relevance is the fact that BSA-cysSNEM increased the peroxidation rate of both thiols in a concentration-dependent manner. In the case of GSH, the rate increase with 0.8 mM BSA-cysSNEM (1.2 times) was similar to the expected increase of the peroxymonocarbonate concentration in the presence of protein (1.4 times) because of the higher Keq (Table 1). These results further support the hypothesis that peroxymonocarbonate is the main thiol-oxidizing agent under the tested experimental conditions. BSA and GSH Oxidation Products. The major products obtained from biothiol oxidation by hydrogen peroxide in the presence of bicarbonate were analyzed and shown to be the same as those obtained in the presence of hydrogen peroxide alone. In the case of BSA, the major product was the sulfenic acid derivative (BSA-cysSOH) as shown by the representative light absorption spectra of adducts resulting from NBD-Cl trapping at zero time and after 20 min of incubation of BSA-cysSH (0.9 mM) with hydrogen peroxide (1 mM) and bicarbonate (25 mM) (Figure 8) (12, 35, 36). Under these experimental conditions, it can be estimated that 80% of BSA-cysSH was converted to BSA-cysSOH by assuming that BSA-cysSNDB (peak at 408 nm) and BSAcysS(O)NDB (peak at 378 nm) have the same extinction coefficient (35, 36). In the case of GSH (1 mM), an almost quantitative production of GSSG was demonstrated by HPLC-EC analysis (Figure 8, inset). The data shown in Figure 8 confirm the higher stability of BSA-cysSOH compared to that of GSOH, which reacts with the remaining GSH to produce GSSG. The production of BSAcysSOH and GSSG from peroxycarbonate-mediated oxidations also confirmed that peroxymonocarbonate is a two-electron oxidant (26, 27). Indeed, parallel EPR spin-trapping experiments showed no significant production of DMPO radical adducts in the presence and absence of bicarbonate up to 30 min incubations (data not shown) (also see Materials and Methods). Also, the addition of DMPO (10-50 mM) to the incubation mixtures did not affect the rate of biothiol oxidation (data not shown).

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Table 2. Reactions Employed to Simulate the Kinetics of BSA-cysSH and GSH Peroxidation in the Presence of Bicarbonate at 37 °C reaction

constantsa

H2O2 + HCO3- {\} H2O + HCO4H2O2 + BSA-cysSH f BSA-cysSOH + H2O HCO4- + BSA-cysSH f BSA-cysSOH + HCO3H2O2 + HCO3- a H2O + HCO4H2O2 + GSH f GSOH + H2O HCO4- + GSH f GSOH + HCO3-

k1 ) 3.8 × 10-3 M-1 s-1; k-1 ) 8.0 × 10-3 s1 k ) 1.18 M-1 s-1 k ) 2.3 × 102 M-1 s-1 k1 ) 2.4 × 10-3 M-1 s-1; k-1 ) 6.9 × 10-3 s-1 k ) 1.85 M-1 s-1 k ) 1.6 × 102 M-1 s-1

BSAb

a Rate constants determined in this work as described in Results. The values correspond to the mean of three independent experiments. b Peroxymonocarbonate equilibrium in the presence of BSA.

Figure 7. Effects of increasing concentrations of thiol-blocked BSA (BSA-cysSNEM) on the initial rates of biothiol oxidation. (A) BSAcysSH (0.4 mM) oxidation by hydrogen peroxide (1 mM) and bicarbonate (25 mM) in a phosphate buffer (0.1 M) at pH 7.4, containing DTPA (0.1 mM) at 37 °C. (B) GSH (1.1 mM) oxidation by hydrogen peroxide (1 mM) in the presence of bicarbonate (25 mM) in a phosphate buffer (0.1 M) at pH 7.4, containing DTPA (0.1 mM) at 37 °C.

Figure 8. Representative UV-visible spectra of the adducts trapped with NDB-Cl during BSA-cysSH (0.9 mM) oxidation by hydrogen peroxide (1.0 mM) and bicarbonate (25 mM) at zero time (---) and after 20 min of incubation (-). The inset shows a representative ECHPLC chromatogram of a 20 min incubation of GSH (1.1 mM) with hydrogen peroxide (1 mM) and bicarbonate (25 mM). Incubations and analysis were performed as described in Materials and Methods.

Discussion Our results confirmed and extended previous studies (2429) in demonstrating that peroxymonocarbonate is produced

from mixtures of hydrogen peroxide with bicarbonate in aqueous solution at neutral pH and attains relatively stable concentrations (Figures 1-3). By monitoring the time-dependent peroxymonocarbonate formation by 13C NMR, it was possible to obtain Keq and to estimate the rate constants for peroxymonocarbonate formation (k1) and decomposition (k-1) under different conditions (Table 1). Also, peroxymonocarbonate equilibrium was shown to be affected by BSA-cysSNEM, liposomes, and a carbonic anhydrase mimetic, probably because they alter the equilibrium among gaseous carbon dioxide/dissolved carbon dioxide/bicarbonate (eq 5) (Table 1) (also see Results). Moreover, the rate constant for peroxymonocarbonate production in aqueous buffer at pH 7.2 ( 0.3 estimated here (k1 ∼ 10-2 M-1 s-1) is likely to represent the lowest possible value because of the inherently slow 13C NMR measurements (Figures 1-3). Overall, the 13C NMR experiments indicate that peroxymonocarbonate formation from hydrogen peroxide may occur at considerable rates in the protein-, lipid- and bicarbonate/carbon dioxide-rich physiological media. The 13C NMR experiments also provided data to examine the participation of the oxidant in the accelerating effects of bicarbonate on BSA-cysSH (12) and GSH peroxidation (Figures 4-6; Table 2). Kinetic data of BSA-cysSH and GSH oxidation by equimolar hydrogen peroxide (1-1.2 mM) and various concentrations of bicarbonate (2-25 mM) were analyzed by assuming that the peroxidation rate was dependent on the equilibrium concentrations of peroxymonocarbonate (eqs 3, 9, 10) (Figure 6, Table 2). The assumption was proved to be essentially correct by the good fit obtained between the experimental kinetics and their simulation with the determined rate constant values (inset of Figures 4 and 5). In addition, BSAcysSNEM was shown to increase both peroxymonocarbonate concentration at equilibrium (Table 1) and the biothiol peroxidation rate (Figure 7). Taken together, these results indicate that peroxymonocarbonate is the main species responsible for biothiol peroxidation in the presence of physiological concentrations of the bicarbonate/carbon dioxide pair. In fact, the determined second-order rate constant of the reaction of peroxymonocarbonate with BSA-cysSH and GSH was 2 orders of magnitude higher than the corresponding rate constant of the hydrogen peroxide reaction (Table 2). A similarly increased reactivity of peroxymonocarbonate over hydrogen peroxide toward aryl sulfides (26) and methionine (27) has been previously reported by Richardson and co-workers. The higher reactivity of peroxymonocarbonate compared to that of hydrogen peroxide is attributed to carbonate being a better leaving group than hydroxide (26, 27, 50). In agreement, a plot of the log of the second-order rate constants of BSA-cysSH and GSH oxidation by peroxynitrite (46, 51), peroxymonocarbonate, and hydrogen peroxide (Table 2) against the pKa of the conjugated acid of the corresponding leaving group (52-54) showed the expected linear correlation (Figure 9). Peroxymonocarbonate is negatively charged in aqueous solution at neutral pH, and it is likely to react with the neutral thiol. In contrast, recent reports

Biothiol Peroxidation by Bicarbonate/CO2

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1481

targets by two-electron mechanisms or, in the presence of metal ions and metal centers, acts as a precursor of the carbonate radical. Finally, it should be emphasized that free radicals and oxidants are currently considered to mediate responses that range from signaling circuits involved in physiology and pathology to cellular and tissue injury. The elucidation of these many interrelated processes requires a better understanding of cellular oxidative mechanisms, many of which are likely to be modulated by the ubiquitous bicarbonate/carbon dioxide pair as evidenced here and in many other studies (1-21, 27-29, 60-62). Hence, it is appropriate and timely to recognize that the main physiological buffer is active in redox processes.

Figure 9. Schematic and graphic representation of the nucleophilic displacement on peroxymonocarbonate by biothiols. In the plot, log of second-order rate constants of BSA-cysSH (9) and GSH (∆) oxidation by peroxynitrite, peroxymonocarbonate, and hydrogen peroxide plotted against the pKa of the conjugated acid of the corresponding leaving group (XOH) (nitrous acid, carbonate, and water, respectively). Secondorder rate constant (51; this work) and pKa (52-54) values were taken from the specified references.

have presented kinetic evidence that cysteine oxidation by peroxynitrite (55) and hydrogen peroxide (49) occurs by a twostep nucleophilic reaction mechanism involving rate-determining nucleophilic attack of the thiolate anion on the un-ionized oxidant to generate the sulfenic acid intermediate. Hence, mechanistic details of thiol oxidation by peroxymonocarbonate, peroxynitrite, and hydrogen peroxide may differ but are yet to be explored. A role for peroxymonocarbonate in the acceleration of peroxidations by the bicarbonate/carbon dioxide pair has been overlooked because of many reasons, including the low reported second-order rate constant of formation (26). In showing that this rate constant is more than 1 order of magnitude higher and affected by components of the physiological milieu, our results indicate that peroxymonocarbonate is a feasible biological oxidant. Certainly, the second-order rate constant estimated here for peroxymonocarbonate formation (k ∼10-2 M-1 s-1) is several orders of magnitude lower than those of hydrogen peroxide decomposition by catalases and peroxidases (k ∼107 M-1 s-1) (56). Still, peroxymonocarbonate formation may occur in specific microenvironments (Figures 2 and 3; Table 1) (22, 23). Bicarbonate concentration is high under physiological conditions (∼25 mM), and for a fixed hydrogen peroxide concentration, the rate of peroxymonocarbonate formation (k × 25 mΜ ∼2.5 × 10-4 s-1) is likely to be similar to that expected for hydroxyl radical formation from redox-active iron (II) (k × 5 µM ∼5 × 10-4 s-1) (57, 58). Although the physiological concentration and ligands of redox active iron (II) remain debatable (59), the fact that Fenton chemistry has been widely used to explain oxidative damage in ViVo is noteworthy. Our data confirmed that peroxymonocarbonate is a twoelectron oxidant (26, 27). In the presence of metal ions and metalloenzymes, however, peroxymonocarbonate is likely to be reduced to the carbonate radical (eq 2) as has been evidenced in recent studies (14-16, 28). Thus, peroxymonocarbonate is a potentially important biological oxidant that directly oxidizes

Acknowledgment. This work was supported by grants from the Fundac¸ a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (Projeto Mileˆnio: Redoxoma). We thank M. G. Bonini and R. Cunha for some preliminary experiments, M. Uemi for technical assistance in the NMR experiments, and I. Cuccovia, B. Alvarez, and G. Ferrer-Sueta for helpful discussions.

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