Kinetics and Mechanisms of the Reaction of Hypothiocyanous Acid

Oct 12, 2009 - 5-thio-2-nitrobenzoic acid and with reduced glutathione by stopped-flow ... bell-shaped pH profile established that the reaction with ...
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Chem. Res. Toxicol. 2009, 22, 1833–1840

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Kinetics and Mechanisms of the Reaction of Hypothiocyanous Acid with 5-Thio-2-nitrobenzoic Acid and Reduced Glutathione Pe´ter Nagy,*,† Guy N. L. Jameson,‡ and Christine C. Winterbourn† Department of Pathology, UniVersity of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand, and Department of Chemistry, UniVersity of Otago, P.O. Box 56, Dunedin 9054, New Zealand ReceiVed July 22, 2009

Hypothiocyanite is a major oxidant generated by mammalian peroxidases. Although reported to react specifically with thiol groups in biological molecules, a detailed mechanistic study of this reaction has not been conducted. We have investigated the reaction of hypothiocyanous acid/hypothiocyanite with 5-thio-2-nitrobenzoic acid and with reduced glutathione by stopped-flow spectroscopy. The observed bell-shaped pH profile established that the reaction with 5-thio-2-nitrobenzoic acid proceeds via the thiolate and hypothiocyanous acid in the 2.5 < pH < 8 region. The obtained second-order rate constant of the reaction is (1.26 ( 0.02) × 108 M-1 s-1, and the effective rate constant at pH 7.4 is (4.37 ( 0.03) × 105 M-1 s-1. Analysis of the kinetic data, using a value of 4.38 ( 0.01 for the pKa of 5-thio-2-nitrobenzoic acid thiol (measured independently by spectroscopic analysis), gave a pKa of 4.85 ( 0.01 for hypothiocyanous acid at physiological salt concentration (I ) 120 mM; NaCl and phosphate buffer) and 25 °C. A second-order rate constant of (8.0 ( 0.5) × 104 M-1 s-1 for the reaction of hypothiocyanous acid/hypothiocyanite with reduced glutathione at pH 7.4 was determined. The glutathione data are also consistent with the reaction proceeding via the thiolate and hypothiocyanous acid. Our results demonstrate that hypothiocyanous acid/hypothiocyanite has very high reactivity with thiols and will be short-lived in the presence of physiological concentrations of glutathione and thiol proteins. As the reaction occurs strictly with the thiolate, this oxidant should selectively target proteins containing low pKa thiols. Introduction - 1

Hypothiocyanous acid/hypothiocyanite (HOSCN/OSCN ) is an antimicrobial oxidant that is produced by peroxidasecatalyzed oxidation of thiocyanate (SCN-) by H2O2 (1). H2O2 reacts with the ferric form of the enzyme to generate compound I, which then reacts with SCN- to give HOSCN/OSCN-. Most peroxidases are also capable of oxidizing some (or all) of the halides, but they exhibit the largest reactivity toward SCN- (2). At the relative physiological concentrations of the (pseudo)halides, lactoperoxidase (LPO), salivary peroxidase, and eosinophil peroxidase (EPO) use SCN- as their primary substrate (1, 3-5), whereas myeloperoxidase (MPO) produces comparable amounts of hypochlorous acid (HOCl) and HOSCN/OSCN- under physiological conditions (6). HOSCN/OSCN- is also formed in the very rapid reactions of HOCl and HOBr with SCN- (7, 8). It plays a major role in fighting invading pathogens (9-11) and is receiving increasing attention for its role in inflammation and oxidative stress-related diseases, such as atherosclerosis, cystic fibrosis, or asthma (11-14,). The physiological importance of secondary reactive species that are derived from HOSCN/ OSCN- such as cyanate or thiocarbamate S-oxide was recently demonstrated by their ability to cause carbamylation of protein amino groups and cysteine oxidation, respectively (15, 16). * To whom correspondence should be addressed. E-mail: peter.nagy@ otago.ac.nz. † University of Otago Christchurch. ‡ University of Otago. 1 Abbreviations: HOSCN, hypothiocyanous acid; LPO, lactoperoxidase; EPO, eosinophil peroxidase; MPO, myeloperoxidase; TNB, 5-thio-2nitrobenzoic acid; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); GSH, reduced glutathione; GSSG, oxidized glutathione; SVD, singular value decomposition; HOX, hypohalous acid.

All peroxidase oxidants show preferential reactivity with thiols (17-19). However, HOCl and HOBr also react with methionine and most other amino acid residues, whereas HOSCN/OSCN- almost exclusively targets sulfhydryl groups (9, 20). HOSCN/OSCN- has been reported to oxidize protein thiols more efficiently than HOCl or HOBr in murine macrophages and was observed to be a more potent inducer of apoptosis (21). The role of HOSCN/OSCN- in cellular signaling is also increasingly recognized (22, 23), and its selective reactivity toward thiol groups should also have a pivotal role in signal transduction. There is limited information on the kinetics and mechanism of thiol oxidation by HOSCN/OSCN-. Skaff et al. (24) have recently reported rate constants for 5-thio-2-nitrobenzoic acid (TNB) that they measured directly, then used them in competition kinetics to obtain rate constants for a range of other thiols. In the present work, we have used stopped-flow spectroscopy to carry out a comprehensive kinetic analysis of the reaction of HOSCN/OSCN- with TNB and to obtain a direct measurement of the rate constant for reduced glutathione (GSH). The study establishes the rates of the reactions via the different protonated and deprotonated forms of the reactants as well as the pKa of HOSCN. Our rate constant for TNB agrees with that of Skaff et al., but our value for GSH is 3-fold higher than was estimated from the competition studies. This information allows the estimation of rates of HOSCN with thiol proteins having different pKa values and will be valuable for investigating the fate of HOSCN/OSCN- in vivo.

Experimental Procedures Reagents. All chemicals were ACS certified grade or better. Water was purified by running through a Milli-Q system (Millipore)

10.1021/tx900249d CCC: $40.75  2009 American Chemical Society Published on Web 10/12/2009

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so that its resistivity was greater than 18 MΩ cm. All reagents and enzymes were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Standard HCl solutions were prepared by dilution of exactly 0.1 mol of analytical grade HCl, packaged in an ampule (obtained from Fixanal, Seelzee, Germany). The buffer solutions were prepared from NaH2PO4·2H2O, Na2HPO4, and CH3COONa·3H2O, the ionic strength was adjusted with NaCl, and the pH was adjusted with ∼6 M HCl or freshly prepared ∼6 M NaOH (mostly free of CO32- contamination). Unless stated otherwise, reactions were carried out at 25 °C and a constant ionic strength of 120 mM, with diethylenetriamine-penta-acetic acid (DTPA; 50 µM) present to chelate contaminating trace metal ions. Preparation of HOSCN/OSCN- Stock Solutions. HOSCN/ OSCN- was generated by the LPO-catalyzed reaction of SCN- with H2O2. The concentration of H2O2 stock solutions was determined using ε(H2O2)240nm ) 43.6 M-1 cm-1. LPO concentrations were determined spectrophotometrically using ε(LPO)412nm ) 112 mM-1 cm-1. The reaction was initiated by adding a small aliquot of H2O2 (0.8 mM after mixing) to a mixture of 1.2 µM LPO and 7.5 mM SCN- in 10 mM phosphate buffer (pH ) 7.4) at 4 °C. Two additional aliquots of H2O2 were added in 1 min intervals (2.4 mM final). After 10 min at 4 °C, 20 µg (300 activity unit)/mL catalase was added to destroy residual H2O2. The enzymes were removed by centrifugation at 3000g and 4 °C using a 10 kDa cutoff Amicon Ultra centrifugal filter device. Concentrations of the stock solutions were measured by detecting the loss of absorbance at 412 nm after adding an aliquot to a ∼4-5-fold excess of TNB solution under vigorous vortexing conditions. This method typically gave ∼1.2 mM HOSCN/OSCN-. Preparation of TNB Stock Solutions. TNB solutions were prepared by alkaline hydrolysis of a 10 mM 5,5′-dithiobis(2nitrobenzoic acid) (DTNB) solution made up to pH > 12 by adding NaOH. After 5-10 min, the solution was titrated back to pH 7 with HCl, and 50 µM DTPA was added. The TNB concentration was measured spectrophotometrically using ε(TNB-S2-)412nm ) 14.1 mM-1 cm-1 (25). This gave exactly 3/2 mol equiv of TNB-S2- as compared to the original DTNB solution. On the basis that alkaline hydrolysis of DTNB results in a 3/2:1/2 mixture of TNB-S2- and TNB-SO2- (26), this gives us confidence that TNB-SO2- has negligible absorbance at 412 nm, which is important when measuring the pKa of TNB at this wavelength. This observation is in agreement with a previous report, which obtained similar extinction coefficients for TNB at 412 nm, where TNB was generated either by reduction using different thiols or by the alkaline hydrolysis method (27). This same study presents corroborating evidence for the two different methods resulting in essentially similar compounds (i.e., TNB-S2-) by precipitation, recovery, and elemental analysis. Our polychromatic data suggest that the resultant TNB-SO2- is inert toward HOSCN under our conditions. The stock solutions of HOSCN and TNB were stored on ice and protected from light. pH Measurements. A Metler Toledo S40-K pH meter with an Ag/AgCl combination pH electrode, calibrated using potassiumborate/carbonate (pH 10.00), potassium-phosphate (pH 7.00), and potassium-hydrogen-phthalate (pH 4.00) buffers. To obtain the [H+] or [OH-] of the buffered solutions from the measured pH values, all pH measurements were corrected for the “Irving factor” (28) that was measured by using standard solutions of HCl containing 120 mM NaCl in the 2 > pH > 2.5 range at 25 °C. The pH of the reaction mixtures was measured after each stopped-flow run by taking aliquots from the reservoirs of the sample handling unit and mixing them in a 1:1 ratio. UV/Vis Spectroscopy. Electronic spectra were measured using an Agilent 8453 diode array spectrophotometer using quartz cells with calibrated 1 cm path lengths or the PDA detector of the Applied Photophysics SX20 stopped-flow instrument equipped with a 150W Xe lamp. Stopped-Flow Studies. Kinetic measurements were completed with an Applied Photophysics SX20 stopped-flow spectrophotometer using a Xe arc lamp. Monochromatic kinetic traces were collected using a photomultiplier detector and polychromatic data

Nagy et al. with a photo diode array spectrophotometer attached to the observation cell. All kinetic data were collected at 120 mM ionic strength (buffer and NaCl) and at 25 °C. The temperature was maintained at 25 °C in the observation cell with a Haake model DC10-K10 Refrigerated Circulator thermostat during the kinetic runs. A small aliquot of the stock solutions (T ) 4 °C) of the reagents was added to the prepared buffers (T ) 25 °C) and immediately subjected to stopped-flow analysis. In the stoppedflow experiments, error bars represent the standard deviation of the average of at least six kinetic measurements on the same solution. Spectrophotometric Titration of TNB. A 50 mL aliquot of 47 µM TNB solution in a mixture of 5 mM phosphate and 5 mM acetate buffer at I ) 120 mM (NaCl + phosphate buffer + acetate buffer) was titrated using a ∼6 M HCl or a ∼6 M NaOH solution. The titration was performed at 25.0 °C. Spectra were recorded immediately after the pH values were determined. The acid dissociation constants were calculated using KaleidaGraph 3.5 (Synergy Software). The concentration of the TNB solution was checked at the end of the titration by returning the pH to pH 7. No significant decomposition was observed. Reaction of HOSCN with TNB and GSH. The pH, [HOSCN], and [TNB] or [GSH] dependencies of the reaction rates were investigated under pseudo first-order conditions with at least a 5-fold excess of one of the reactants over the other (10-fold for the pH dependency). Reactions were carried out in 10 mM phosphate buffer at 6.3 < pH < 7.8 and 2.5 < pH < 3.8 and 10 mM acetate buffer at 3.8 < pH < 6.0 with I ) 120 mM. Kinetic Data Analysis. The monochromatic kinetic traces were fitted with SX20 Pro-Data Control Software (Applied Photophysics, United Kingdom). Polychromatic data were analyzed using PC Pro-K Global Analysis and Data Simulation Software (Applied Photophysics, United Kingdom). The concentration dependencies of the pseudo first-order rate constants were fitted with KaleidaGraph 3.5 (Synergy Software).

Results and Discussion Decomposition of HOSCN/OSCN- at pH 7.4. It was first necessary to confirm the stoichiometry of the reaction between HOSCN/OSCN- and TNB. This was investigated by following decomposition of HOSCN/OSCN-, both by monitoring its characteristic band at 376 nm (29) and by measuring the amount of HOSCN/OSCN- remaining at different time points by adding excess TNB and measuring loss of TNB and formation of TNB disulfide (DTNB) (Figure 1). The two methods resulted in similar kinetic traces that fitted a single exponential equation and gave similar rate constants: (1.13 ( 0.13) × 10-4 and (1.10 ( 0.13) × 10-4 s-1, respectively, establishing a 1:2 stoichiometry. It also shows that the decomposition products of HOSCN exhibit no reactivity toward TNB within ∼1 min and need not be considered in the following stopped-flow experiments. Measurement of the Acid Dissociation Constant of the Sulfhydryl Group of TNB. To analyze the mechanism of TNB oxidation by HOSCN/OSCN-, the acid dissociation constants of TNB were required. These were measured by spectrophotometric titration at 25 °C and 120 mM ionic strength (NaCl and buffer) (Figure 2). Two inflection points were observed at 325 nm in accordance with the two functional groups containing a labile proton (carboxylate and sulfhydryl). The inflection point at ∼pH 4.4 was assigned to pKaTNB(SH) (macroscopic pKa of the sulfhydryl group) and the one at ∼1.6 to pKaTNB(COOH) (macroscopic pKa of the carboxylate group). As the two pKa values are well-separated, we assumed that the concentration of the TNB derivative with protonated carboxylic acid and deprotonated thiolate groups is negligible at all pH values and therefore did not take into account the possibility of microscopic acid dissociation constants.

Thiol Oxidation by Hypothiocyanous Acid

Figure 1. Kinetic traces for the decomposition of HOSCN/OSCN- and the corresponding fits to a single exponential equation. Circles represent the loss of absorbance at 376 nm (a characteristic band for OSCNwith ε376nm ) 26.5 M-1 cm-1) using a cuvette with 5 cm optical path length. Squares represent 0.5 mol equiv of the TNB losses measured at 412 nm [using ε(TNB-S2-)412nm ) 14.1 mM-1 cm-1] upon mixing a 50-100-fold diluted reaction mixture with 63 µM TNB. Conditions: [HOSCN]tot ) 1 mM, [SCN-] ) 6.4 mM, and T ) 37 °C in Hanks’ buffer: NaCl ) 140 mM, phosphate buffer ) 10 mM, CaCl2 ) 1 mM, MgCl2 ) 0.5 mM, glucose ) 5 mg/mL, and pH ) 7.4. Error bars represent the range of duplicate experiments using different solutions.

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information about its extinction coefficient at 325 nm. Therefore, the measured pKaTNB(COOH) is less reliable. However, this pKa will not play a role in the kinetics of the reaction of TNB with HOSCN/OSCN- over the pH range we used and is only presented to show that the two pKa values of TNB are wellseparated. A previous study measured pKaTNB(SH) to be 4.75 by spectrophotometric titration at 25 °C (27). The difference from our value is possibly due to a difference in ionic strength, which was not reported in the previous study (27). Consistent with this, we observed that decreasing the ionic strength of the pH 4.5 buffer used in Figure 2 by omission of NaCl decreased slightly the extent of TNB ionization. Kinetics of the Reaction of HOSCN/OSCN- with TNB. Polychromatic Kinetic Analysis at pH 7.37. Time-resolved spectra collected using stopped flow under second-order conditions (Figure 3a) show the consumption of TNB (λmax ) 412 nm) and the formation of DTNB (λmax ) 325 nm). The observed isosbestic point indicates the lack of a detectable non-steadystate intermediate, and analysis of the polychromatic data using singular value decomposition (SVD) indicates the presence of only two species with measurable absorbance in the studied region during the course of the reaction (TNB and DTNB). All kinetic traces fit well to a second-order equation derived from the following simple mechanism, where the different protonated states of the species are not taken into account and the compounds represent all acid/base derivatives: k1

TNB + [HOSCN]tot 98 TNB-S-SCN + H2O k2

TNB-S-SCN + TNB 98 DTNB + SCN-+H+ 2TNB + [HOSCN]tot f DTNB + SCN- + H+ + H2O Figure 2. Measurement of the acid dissociation constants of TNB by spectrophotometric titration. Circles and squares represent the change in absorbance at 412 and 325 nm in the spectrum of TNB, respectively. The solid line represents nonlinear least-squares fit to an equation using one pKa [pKaTNB(SH)] and the dashed line to an equation with two pKa values [pKaTNB(SH) and pKaTNB(COOH)]. Conditions are given in the Experimental Procedures. Data are from one experiment. Results from a duplicate experiment under different concentration conditions using a different electrode and spectrophotometer were almost identical. All solutions were made up freshly (including buffers) on the day of the experiments in both cases. The calculated pKa values are as follows: pKaTNB(SH) ) 4.38 ( 0.01 and pKaTNB(COOH) ) 1.68 ( 0.04. These represent the mean values of the two independent experiments with the calculated ranges.

Only one inflection point was observed at 412 nm, indicating the lack of absorbance of the protonated forms of TNB at this wavelength. The pKa values were obtained by fitting the absorbance vs pH curves by the nonlinear least-squares method (Figure 2). At pH > 2.5, an equation was derived for the Abs412nm data assuming one acid dissociation constant. This resulted in pKaTNB(SH) ) 4.38 ( 0.01 (for details, see the Experimental Procedures). The curve at 325 nm was fitted to an equation including two acid dissociation constants. The fits of two independent experiments gave means and ranges of pKaTNB(SH) ) 4.38 ( 0.01 and pKaTNB(COOH) ) 1.68 ( 0.04. It was also possible to fit the two pKa values separately with similar results (not shown). It should be noted that the TNB-sulfinate contamination in the TNB stock solutions did not contribute to the absorbance at 412 nm (see the Experimental Procedures), but we have no

(1)

(2) (3)

where reaction 2 is rate determining, k2 . k1, and therefore k2 is a fixed constant (Figure S1a of the Supporting Information). The obtained second-order rate constant from the polychromatic data is k1 ) 5.2 × 105 M-1 s-1. [HOSCN]tot represents the total HOSCN/OSCN- concentration regardless of ionization state. SVD analysis indicates no formation of higher oxidation states of TNB such as the sulfinic or the sulfonic acid derivatives even under second-order conditions and corroborates the 1:2 stoichiometry established from the decomposition of HOSCN/ OSCN-. Reaction 2 could occur as written or could involve a hydrolysis step of sulfenyl thiocyanate as in eqs 4 and 5.

TNB-S-SCN + H2O f TNB-S-OH + SCN- + H+ (4) TNB-S-OH + TNB f DTNB + H2O

(5)

The latter would be consistent with a large body of literature on the two-electron oxidation of nucleophiles with hypohalous acids (HOX). Margerum and others have shown that these reactions proceed mostly via transfer of X+ rather than oxygen, and in many cases, the resultant sulfenyl-halide hydrolyzes quickly in aqueous media (17, 30-32). A previous study showed that (SCN)2/HOSCN reacts with GSH and penicillamine under acidic conditions to give sulfenyl-thiocyanates (20), which are unstable at physiological pH and hydrolyze relatively quickly (33). However, in some proteins, relatively stable sulfenylthiocyanates were observed, which would suggest slow hydrolysis (9, 34).

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order dependencies for both reactants and is in agreement with the kinetics measured under second-order conditions. However, the slopes of the [HOSCN]tot vs kobs and the TNB dependency plots differ by a factor of 2 [(8.74 ( 0.08) × 105 and (4.37 ( 0.03) × 105 M-1 s-1, respectively], in accordance with the 2:1 (TNB:HOSCN) stoichiometry of the reaction, which defines the rate law as follows:

rate )

d[HOSCN]tot -1 d[TNB]tot ) ) 2 dt dt keff[HOSCN]tot[TNB]tot (6)

Under the applied conditions, the observable in the system is TNB. Therefore, on the basis of eq 6 when [HOSCN]tot is in excess, a proportionality constant of 2 has to be incorporated into the rate equation (35), and the observed pseudo first-order rate constant will be equal to 2keff[HOSCN]tot. When [TNB]tot is in excess, the exponential decay represents the loss of [HOSCN]tot, and the observed pseudo first-order rate constant will be keff[TNB]tot. This is in agreement with the second-order rate constant that was obtained from the SVD analysis of the polychromatic data using a mechanism that incorporates the 1:2 ) [HOSCN]tot:[TNB]tot stoichiometry (eqs 1 and 2). pH Dependence. At pH > 6.5, a linear [H+] dependency was observed (Figure 4a). With a pKaTNB(SH) of 4.38, TNB is present entirely as the thiolate under these conditions; therefore, the drop in the rate with increasing pH cannot be explained by the protonation of TNB. It indicates that despite the dominant species being OSCN-, HOSCN is more reactive. The zero intercept indicates that even at the highest pH (pH 7.88) the reaction proceeds via HOSCN and k8 cannot be measured. This is explained by HOSCN serving as the electrophile and agrees with previous reports that the protonated forms of HOX react typically 3-5 orders of magnitude faster with nucleophiles than the deprotonated forms (8, 17, 30, 36, 37). Therefore, the reaction of TNB with HOSCN exhibits overall third-order kinetics at pH > 6.5, as described by the following rate law: Figure 3. Spectral changes and concentration dependencies for the reaction of HOSCN/OSCN- with TNB. (a) Time-resolved spectra collected under second-order conditions. Spectra were recorded at 18.9 ms time intervals. The absorbance decrease at 412 nm and increase at 325 nm represent the loss of TNB and formation of DTNB, respectively. Conditions: [TNB]tot ) 35 µM, [HOSCN]tot ) 15 µM, [phosphate buffer] ) 10 mM, [DTPA] ) 50 µM, I ) 120 mM (NaCl + phosphate buffer), pH ) 7.37, and T ) 25 °C. (b) [TNB] and (c) [HOSCN] dependencies of the observed pseudo first-order rate constants and the corresponding linear fits. Conditions: [TNB]tot ) 8.8-80 (b) or 1.8 µM (c), [HOSCN]tot ) 1.8 (b) or 8-32 µM (c), [phosphate buffer] ) 10 mM, [DTPA] ) 50 µM, I ) 120 mM (NaCl + phosphate buffer), pH ) 7.35, and T ) 25 °C. After correcting for the appropriate proportionality constants (see the text), identical second-order rate constants were obtained when an excess of [TNB]tot or an excess of [HOSCN]tot were used (i.e., plot from panels b and c, respectively).

Concentration Dependencies. To establish the rate law and to obtain more accurate second-order rate constants, the reaction was followed under pseudo first-order conditions. In all cases, the obtained kinetic traces fit well to single exponential equations (an example is shown in Figure S2 of the Supporting Information). When [TNB]tot was used in excess over [HOSCN]tot, the resultant pseudo first-order rate constants exhibit a linear dependence on [TNB]tot and the linear fit passes through the origin (Figure 3b). Similarly, a linear relationship was observed between the pseudo first-order rate constants and [HOSCN]tot using a deficit of [TNB]tot (Figure 3c). This establishes first-

rate ) k∗[HOSCN]tot[TNB]tot[H+]

(7)

The following mechanism is consistent with the observations at pH > 6.5: k8

OSCN- + TNB-S2- 98 products

(8) 1

H+ + OSCN- h HOSCN

KHOSCN a

k10

HOSCN + TNB-S2- 98 products

(9)

(10)

The pre-equilibrium approximation was used for reaction 9, and KaHOSCN was used to derive the following rate equation for this mechanism:

(

)

k10[H+] -d[OSCN-] ) k8 + HOSCN [OSCN-][TNB-S2-] dt Ka

(11)

As reasoned above, the reactivity of OSCN- is not detectable at pH < 8; therefore, k8 is negligible. Therefore, comparison of eq 11 to the observed rate law (eq 7) gives k* ) k10/KaHOSCN ) (1.07 ( 0.02) × 1013 M-2 s-1.

Thiol Oxidation by Hypothiocyanous Acid

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The pH profile observed at pH < 6.5 (Figure 4b, top) exhibits a more complex bell-shaped behavior with a maximum rate at about pH 4.6. The increase in the rate below pH 8 is explained by the reaction being due to HOSCN rather than OSCN-. The observed decrease below pH 4.6 suggests that the thiolate of TNB reacts faster than the thiol. Therefore, the following mechanism is proposed for the reaction of TNB with HOSCN at 2.5 < pH < 8:

TNB-SH- h TNB-S2- + H+

KTNB (SH) a 1

H+ + OSCN- h HOSCN

KHOSCN a

k10

HOSCN + TNB-S2- 98 TNB-S-SCN- + OHk13

HOSCN + TNB-SH- 98 TNB-S-SCN- + H2O

(12) (9)

(10)

(13)

fast

TNB-S-SCN- + TNB-S2- 98 DTNB2- + SCN-

(14)

Assuming that eqs 12 and 9 are pre-equilibria and that reaction 14 is fast, the following rate equation can be derived

-d[OSCN-] (SH) + k13[H+]) × ) (k10KTNB a dt 1 1 × TNB + (KHOSCN + [H ]) (K (SH) + [H+]) a a [HOSCN]tot[TNB]tot[H+]

(15)

Equally good fits of the data were obtained to eq 15 [using pKaTNB(SH) ) 4.38 and k10, k13, and KaHOSCN as floating parameters] and to eq 16, where the term k13[H+] was excluded based on the assumption that k10pKaTNB(SH) . k13[H+] (i.e., even at pH 2.5, the reaction will proceed mainly via the totally deprotonated form of TNB).

-d[OSCN-] 1 × (SH) HOSCN ) k10KTNB a dt (Ka + [H+]) 1 [HOSCN]tot[TNB]tot[H+] TNB + (Ka (SH) + [H ])

(16)

The solid line in the top graph of Figure 4b represents the fit of the data to eq 16, which gave k10 ) (1.26 ( 0.02) × 108 M-1 s-1 and pKaHOSCN ) 4.85 ( 0.01. Figure 4b, bottom, shows the speciation of the different charged states of TNB and HOSCN/ OSCN- based on the obtained pKaTNB(SH), pKaTNB(COOH), and pKaHOSCN. The data cannot be fitted to eq 16 when the previously reported pKaHOSCN ) 5.3 and pKaTNB(SH) ) 4.75 or 4.38 are used as constants. The value k10/KaHOSCN (equivalent to k* in eq 7) of 8.84 × 1012 M-2 s-1 obtained from the fit of the entire pH range to eq 16 is in a reasonable agreement with the value of k* ) 1.07 × 1013 M-2 s-1 calculated for the data measured at high pH. HOSCN/OSCN- is widely accepted to be largely ionized at neutral pH. There are problems with determining its pKa value accurately by spectrophotometric titration due to its instability. Hogg and Jago reported a value of 5.1 at 30 °C and low ionic strength for the antimicrobial product of LPO (subsequently identified as HOSCN) based on polarographic half wave potential and spectral measurements (38). A more extensive

Figure 4. Effect of pH on the rate of the reaction of HOSCN/OSCNwith TNB. (a) Change in the observed pseudo first-order rate constants as a function of [H+] at pH > 6.5 and the corresponding linear fit. (b) Top: Complete pH profile of the rate of the reaction at 2.5 < pH < 8. The solid line represents the nonlinear least-squares fit to eq 16. Conditions: [TNB]tot ) 35 µM, [HOSCN]tot ) 3.5 µM, [phosphate buffer] or [acetate buffer] ) 10 mM, [DTPA] ) 50 µM, I ) 120 mM (NaCl + phosphate buffer/acetate buffer), pH ) 2.5-8, and T ) 25 °C. The pH dependency data were collected on three different days using different solutions and buffers that were made up freshly on the day of the experiment. Bottom: Computed speciation of OSCN- (solid), HOSCN (short dashed), the fully deprotonated form of TNB (dashed), the monoprotonated form of TNB (dotted), and the fully protonated form of TNB (long dashed) using pKaHOSCN ) 4.85, pKaTNB(SH) ) 4.38, and pKaTNB(COOH) ) 1.68.

study by Thomas of the pH-dependent decomposition kinetics of HOSCN/OSCN- and solvent extraction yielded a pKa of 5.3 (25 °C, 100 mM NaCl) (39). A subsequent study using the rate of HOSCN decomposition at 37 °C was broadly consistent with a value around 5 (40), and a pKa of 5.3 is widely quoted. However, the decomposition mechanism of HOSCN/OSCNis complex and pH-dependent (41, 42), and the above-mentioned experiments were carried out in unbuffered solutions, which may have influenced the pKa values obtained. Our value of 4.85 was obtained as the best fit to well-characterized kinetic data measured in buffered solutions at constant ionic strength and temperature (I ) 120 mM and 25 °C) and is likely to be more accurate. Our results indicate, therefore, that the pKa of HOSCN is slightly lower than the commonly accepted value. Kinetics of the Reaction of HOSCN/OSCN- with GSH at Physiological pH. The reaction of HOSCN/OSCN- with GSH gives stoichiometric conversion to glutathione disulfide (GSSG) (43). It was possible to follow the kinetics of the reaction by stopped flow. Kinetic traces were collected at 230 nm, where GSH and GSSG have very small and similar absorbances, and there is a measurable change on conversion of HOSCN to SCN- (Figure 5a). Thus, under the conditions of the reaction, the absorbance change is due to depletion of

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Figure 5. Kinetics of the reaction of GSH with HOSCN/OSCN-. (a) Spectra of 100 µM GSH as compared with 80 µM GSH plus 10 µM GSSG and 10 µM HOSCN plus 54 µM SCN- as compared with 64 µM SCN-. These represent the [GSH]tot and [HOSCN]tot species present at the beginning (solid lines) and end of the reaction (dashed lines). (b) Spectrum at the end of the reaction between 10 µM HOSCN and 100 µM GSH (dashed line) as compared with the added spectra of the individual starting solutions (solid line). (c) Representative kinetic trace collected at 230 nm using 100 µM [GSH]tot and 10 µM [HOSCN]tot and the corresponding exponential fit (solid line). d) [GSH]tot dependency of the observed pseudo first-order rate constants and the corresponding linear fit. This experiment was repeated (at a couple of data points) on a different occasion using different solutions and slightly different concentration conditions, and similar rate constants were obtained. Conditions: [GSH]tot ) 50-300 µM, [HOSCN]tot ) 10 µM, [phosphate buffer] ) 10 mM, [DTPA] ) 50 µM, I ) 120 mM (NaCl + phosphate buffer), pH ) 7.07, and T ) 25 °C.

HOSCN (i.e., the observable is [HOSCN]tot) (Figure 5b). The kinetics of the reaction were investigated with a large excess of [GSH]tot over [HOSCN]tot; therefore, the observed pseudo first-order rate constants refer to the decay of HOSCN at constant GSH concentration. Although relatively small changes were followed, the kinetic traces were reproducible and the signal-to-noise ratio was high (Figure 5c). All kinetic traces fit well to a single exponential equation (as in Figure 5c), and the obtained pseudo first-order rate constants show a linear dependence on [GSH]tot (Figure 5d), indicating overall second-order kinetics. The second-order rate constant at pH 7.07 was calculated from the slope of Figure 5d: keff,pH7.07GSH ) (8.4 ( 0.1) × 104 M-1 s-1. At pH 7.40, we obtained keff,pH7.40GSH ) (8.0 ( 0.5) × 104 M-1 s-1 (measurement was done under pseudo first-order conditions at 100 µM [GSH]tot and 10 µM [HOSCN]tot). The rate constant for GSH is considerably smaller than keff,pH7.40TNB (4.4 × 105 M-1 s-1). This is because at this pH the sulfhydryl group of GSH is mostly protonated due to its higher pKa. The amount of GSH thiolate is the sum of GS- and GS2(where the amine group is protonated and deprotonated, respectively). Using the reported microscopic acid dissociation constants for GSH [pKa(H3N+GSH h H3N+GS-) ) 8.75, pKa(H3N+GSH h H2NGSH) ) 9.28, pKa(H3N+GS- h H2NGS-) ) 9.28, and pKa(H2NGSH h H2NGS-) ) 8.72] (44, 45) and assuming that the reaction proceeds predominantly via HOSCN and the thiolate derivatives GS- and GS2-, which have similar reactivity, we estimated the second-order rate constant of this reaction. Values of 6.2 × 108 or 6.4 × 108 M-1

s-1 were obtained from the measured keff at pH 7.4 or 7.07, respectively. A simulation of keff at 5 < pH < 11 results in a bell-shaped curve with a plateau at the maximum in the 6.2 < pH < 7.4 region. The small difference in these values corroborates our assumption that the reactivity of the thiol is negligible and indicates that the reaction rate will change little over the physiological pH range. A recent study by Skaff et al. (24) investigated the reactivity of HOSCN with GSH and other thiols. Their value for the rate constant with TNB, measured directly at pH 7.4 (3.8 × 105 M-1 s-1), compares well with the value that we obtained. The rate constants for the other thiols were investigated in competition experiments with TNB, and the value that they obtained for GSH is 3-fold lower than ours. As shown in reactions 1-5, the bimolecular elementary reaction of HOSCN with a thiol generates a sulfenyl thiocyanate (or a sulfenic acid) derivative that reacts with another thiol molecule to give a disulfide. Our data for TNB indicate that the second reaction is fast. It will consume another equivalent of thiol, which could be either TNB or the competing substrate. This means that the two reactions are not independent, thus complicating the kinetic analysis and possibly explaining their lower rate constant for GSH. We argue therefore that the direct measurement of the GSH rate constant is more accurate and that differences in reactivity of the thiols that compete for the sulfenyl thiocyanate intermediate (which are likely especially for protein thiols) will confound measurements determined by competition kinetics with TNB (in other words, the competition is not just at reaction 1 but reaction 2 as well).

Thiol Oxidation by Hypothiocyanous Acid

Conclusions Mechanistic investigation of the reaction of hypothiocyanite with the aromatic thiol TNB revealed the reaction pathway to occur solely via the protonated electrophile, HOSCN, and the deprotonated thiolate over a wide 2.5 < pH < 8 range. The reaction is fast, with a second-order rate constant of 1.26 × 108 M-1 s-1 for the TNB thiolate and 6 × 108 M-1 s-1 for the GSH thiolate. These rate constants are not very different from that for the reaction of HOCl with cysteine thiolate (1.2 × 109 M-1 s-1) (46, 17), indicating a very fast reaction. At neutral pH, the effective rate constant is less due to protonation of the thiolate and deprotonation of the hypothiocyanous or hypochlorous acid. This has a greater effect for HOSCN as it is mostly ionized under physiological conditions. Nevertheless, the effective rate constant for GSH at pH 7.4 is still 8 × 104 M-1 s-1. This is at least 100 times higher than the equivalent reaction of chloramines (47), which are other major MPO products formed from the reaction of HOCl with amino compounds. Interestingly, HOSCN/OSCN- is often considered as a weak, long-lived oxidant; yet, this high reactivity means that in the presence of physiological concentrations of GSH and thiol proteins, its lifetime would be restricted to a few milliseconds. It is also unreactive with methionine (Turner, R., et al. Unpublished results) and other biological compounds that are targeted by other oxidants such as HOCl and chloramines and is therefore highly selective for thiols. Further specificity is introduced by the thiolate being the reactive species. Protein thiols with a low pKa will be favored targets, and the modification of these proteins is likely to be important in the antibacterial activity and cellular responses to hypothiocyanite. Acknowledgment. This work was supported by the Marsden Fund and the Health Research Council of New Zealand and used equipment provided by the National Research Centre for Growth and Development. We are grateful to Dr. Stephanie Bozonet who helped with the experiment on the decomposition of HOSCN. Supporting Information Available: Figures S1 and S2. This material is available free of charge via the Internet at http:// pubs.acs.org.

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