Biochem. J. (2009) 422, 111–117 (Printed in Great Britain)
111
doi:10.1042/BJ20090276
Hypothiocyanous acid reactivity with low-molecular-mass and protein thiols: absolute rate constants and assessment of biological relevance Ojia SKAFF*, David I. PATTISON* and Michael J. DAVIES*†1 *The Heart Research Institute, 7 Eliza Street, Newtown, Sydney, NSW 2042, Australia, and †Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia
MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate by H2 O2 to HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid, also know as cyanosulfenic acid) respectively. Specificity constants indicate that thiocyanate, SCN− , is a major substrate for MPO. HOSCN is also a major oxidant generated by other peroxidases including salivary, gastric and eosinophil peroxidases. Whereas HOCl and HOBr are powerful oxidizing agents, HOSCN appears to be a less reactive, but more thiol-specific oxidant. Although it is established that HOSCN selectively targets thiols, absolute kinetic data for the reactions of thiols with HOSCN are absent from the literature. This study shows for the first time that the reactions of HOSCN with lowmolecular-mass thiol residues occur with rate constants in the range from 7.3 × 103 M−1 · s−1 (for N-acetyl-cysteine at pH 7.4)
to 7.7 × 106 M−1 · s−1 (for 5-thio-2-nitrobenzoic acid at pH 6.0). An inverse relationship between the rate of reaction and the pK a of the thiol group was observed. The rates of reaction of HOSCN with thiol-containing proteins were also investigated for four proteins (creatine kinase, BSA, β-lactoglobulin and β-L-crystallins). The values obtained for cysteine residues on these proteins are in the range 1 × 104 – 7 × 104 M−1 · s−1 . These second-order rate constants indicate that HOSCN is a major mediator of thiol oxidation in biological systems exposed to peroxidase/H2 O2 systems at (patho)physiological concentrations of halide and SCN− ions, and that HOSCN may play an important role in inflammation-induced oxidative damage.
INTRODUCTION
[10]. Previous studies have also shown that both HOCl and HOBr readily oxidize SCN− non-enzymatically resulting in an enhanced yield of HOSCN [11,12]. HOSCN is therefore a major oxidant generated both directly by multiple members of the peroxidase family at sites of inflammation, and as a result of secondary reactions. HOCl and HOBr are powerful oxidising agents that are important components of the human immune response against invading pathogens [13]. However, there is a considerable body of evidence to support the hypothesis that excessive or misplaced production of these oxidants can lead to host tissue damage; this has been implicated in a wide range of human inflammatory diseases, including cardiovascular disease, rheumatoid arthritis, asthma, cystic fibrosis, some cancers, certain neurodegenerative conditions, kidney disease and inflammatory bowel disease (reviewed in [1,2,14]). HOCl and HOBr react rapidly with a variety of cellular and extracellular targets (reviewed in [15]), with protein components and sulfur-containing compounds being especially important targets [16]. The resulting amino acid modifications can result in alterations to protein structure and function, disruption of extracellular matrix and cell lysis ([17–20]; reviewed [1]). In contrast, HOSCN appears to be more selective, and possibly less reactive, than HOCl and HOBr, reacting predominantly with thiol (RSH) groups [21], athough tryptophan residues may also be a target under conditions when thiol residues are absent or depleted [22]. Despite these differences, recent studies have shown that HOSCN can induce a greater extent of apoptosis than HOCl and HOBr for some cell types, and it is postulated that this arises from specific targeting of critical thiol residues [23].
Myeloperoxidase (MPO) is a highly basic haem enzyme that is released extracellularly by activated neutrophils, monocytes and some macrophages. MPO catalyses the oxidation of halides Cl− and Br− and the pseudohalide thiocyanate (SCN− ) by H2 O2 to their respective HOX (hypohalous acid), HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid, also known as cyanosulfenic acid) (reviewed in [1,2]). These species exist as a mixture of the acid and the anion forms at physiological pH values (pK a values of 7.6, 8.7 and 5.3 respectively [3–5]); HOCl, HOBr and HOSCN are used from here on to indicate the physiological mixtures of these species. Of the precursor anions with which MPO reacts, Cl− is present at the highest concentration in plasma (100–140 mM), with the concentrations of Br− (20–100 μM) and SCN− (20–250 μM) considerably lower [6,7]. However, MPO has a much greater specificity constant for SCN− than for Cl− (approx. 730-fold greater [7]) or Br− , indicating that approximately half of the H2 O2 that reacts with MPO is likely to be utilized to oxidize SCN− at pH 7.4. Thus HOSCN is likely to be one of the major products generated by MPO in blood plasma [7]. Related peroxidases, including salivary, gastric and eosinophil peroxidase, also have high specificity constants for SCN− , resulting in the formation of significant yields of HOSCN at sites where these peroxidases are present (reviewed in [1]). This is of particular importance in biological fluids, such as saliva and stomach lining fluid, where SCN− is present at high concentrations [8,9]. These reactions may be of considerable significance in smokers who have markedly elevated levels of SCN− in plasma and other biological fluids
Key words: hypothiocyanous acid (HOSCN), lactoperoxidase, myeloperoxidase, thiols.
kinetics,
Abbreviations used: CK, creatine kinase; DTNB, 5,5 -dithiobis-(2-nitrobenzoic acid); GSSG, glutathione disulfide; HOBr, hypobromous acid; HOCl, hypochlorous acid; HOSCN, hypothiocyanous acid; HOX, hypohalous acid; LPO, lactoperoxidase; MPO, myeloperoxidase; NEM, N -ethylmaleimide; TNB, 5-thio-2-nitrobenzoic acid. 1 To whom correspondence should be addressed (email
[email protected]). c The Authors Journal compilation c 2009 Biochemical Society
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The potential role of HOSCN in human disease is poorly understood, but recent studies have provided evidence for a role for SCN− -derived materials in both models of atherosclerosis and humans with cardiovascular disease [10,24,25]. These data are of particular relevance to smokers who typically have elevated levels of SCN− and an increased risk of many of the above-mentioned pathologies. In contrast to the above indicators that HOSCN formation may be damaging, other workers have postulated that oxidation of SCN− may modulate the damaging effects of HOCl and HOBr and thereby minimize plasma oxidation and cellular dysfunction [11,12]. An assessment of the role of HOSCN formation from SCN− in biological damage, relative to other MPO-derived species such as HOCl and HOBr, requires knowledge of the rate constants for reaction of these various oxidant species with biological targets. Such results for HOCl and HOBr have been reported (reviewed in [1,15]), but there are no specific kinetic results for HOSCN. If the reactions of HOSCN with thiols, the major reported target for this oxidant [21], are slow then the formation of this species by MPO is likely to diminish the overall extent of biological damage. If however these reactions are rapid, then the highly selective targeting of thiols by this oxidant may play a major role in inflammation-induced oxidative damage. This study reports such kinetic data for HOSCN. It is shown that these reactions are rapid with both low-molecular-mass and protein thiols with rate constants in the range 103 –106 M−1 · s−1 . These values implicate HOSCN as a major damaging agent at sites of inflammation, and particularly at locations where elevated levels of SCN− are present.
EXPERIMENTAL Materials
CK (creatine kinase) was obtained from Roche; BSA, from bovine serum ( 99 %), β-lactoglobulin (bovine milk) and βL-crystallins were from Sigma–Aldrich. LPO (lactoperoxidase, from bovine milk) was from Calbiochem and its concentration was determined from its optical absorption at 412 nm using ε 112 000 M−1 · cm−1 [26]. All other chemicals were from Sigma– Aldrich, with the exception of H2 O2 (30 %, v/v, Merck) and were used without further purification. The concentration of H2 O2 was determined from its optical absorption at 240 nm using ε 39.4 M−1 · cm−1 [27]. All kinetic studies were performed in 0.1 M sodium phosphate buffer (pH 7.4), except for the pH effect studies on the rates of reaction of TNB (5-thio-2-nitrobenzoic acid) and GSH with HOSCN, which were also performed in 0.1 M sodium phosphate buffer at stated pH values. Buffers were prepared using MilliQ water and treated with Chelex resin (Bio-Rad) to remove contaminating metal ions.
14 150 M−1 · cm−1 [28,29]. The oxidant was kept at 4 ◦C, pH 7.4, at concentrations of 200 μM to minimize decomposition. Fresh solutions of HOSCN were prepared for each experiment and used within 1–2 h. Quantification and blocking of thiols
The thiol concentration on proteins was measured by reduction of DTNB to yield TNB. After 30 min incubation at room temperature in the dark, the increase in absorbance at 412 nm due to TNB release was quantified using ε412 13 600 M−1 · cm−1 [28,29]. The thiol group on BSA was blocked by treatment of BSA (50 mg ml−1 ), with a 10-fold excess (over protein thiol concentration) of NEM (N-ethylmaleimide) for 30 min at room temperature. The extent of thiol blocking was assessed by quantifying residual thiols using DTNB, as described above. Under the conditions used > 99 % of the thiol groups were blocked. Excess NEM was removed from the BSA samples by passage of the treated protein through a PD-10 column (Pharmacia) using 0.1 M sodium phosphate buffer (pH 7.4) as the eluent. The concentrations of the resulting protein fractions were assessed by BCA (bicinchoninic acid) assay at 562 nm. Stopped-flow studies
An Applied Photophysics SX.18MV stopped-flow system was used in the kinetic studies as described previously in [16,30,31]. Second-order rate constants for the reaction of TNB (10–50 μM) with HOSCN (2.5 μM) were measured directly by stoppedflow kinetics at 412 nm. Rate constants for the low-molecularmass and protein thiols were determined by competition with TNB, with absorbance changes monitored at 412 nm. For the competition kinetic studies with TNB, varying concentrations of the thiol or other substrate (10–500 μM) were premixed with TNB (10 μM) then allowed to react with equal volumes of HOSCN (2.5 μM). HOSCN was typically kept as the limiting reagent with at least a 4-fold excess of the potential substrate to facilitate data analysis. The temperature of the sample chamber was maintained at 22 ◦C by circulating water from a water bath. The data were processed by single wavelength analysis at 412 nm using Pro-Data viewer 4.0 (Applied Photophysics) and OriginPro 7.0 (OriginLab) software. All second-order rate constants (with errors specified as 95 % confidence limits) were derived from at least two independent experiments each employing 4–8 different substrate concentrations (i.e. 8–16 independent measurements of the observed rate constant, kobs ), with 10 kinetic traces averaged at each concentration. The data points represent means + − S.D. RESULTS
HOSCN preparation
Determination of the rate constant for reaction of HOSCN with TNB
HOSCN was prepared enzymatically using LPO as described previously in [22,23]. In summary, LPO (1.5–2.0 μM) was incubated with NaSCN (7.5 mM) and H2 O2 (3.75 mM) in potassium phosphate buffer (pH 6.6, 10 mM) for 15 min at room temperature (22 ◦C). Catalase (1 mg per ml; from bovine liver) was added to remove any unreacted H2 O2 , followed by filtration by centrifugation at 11 300 g and 5 ◦C for 5 min to remove catalase and LPO. TNB (5–45 μM), prepared from DTNB [5,5 dithiobis-(2-nitrobenzoic acid), approx. 1.5 mM] by incubation with NaOH (50 mM) for 5 min, followed by dilution with 0.1 M phosphate buffer (pH 7.4), was used to quantify HOSCN concentrations, after reaction for 15 min, by absorbance using ε412
The rate of reaction of TNB with HOSCN was measured directly by stopped-flow methods; this value was used as a reference rate constant for subsequent competition kinetic experiments. As TNB exhibits a strong absorbance at 412 nm this wavelength was used to monitor the loss of TNB absorbance at low reactant concentrations (2.5 μM HOSCN, 10–50 μM TNB) over a period of 0.5–5 s at pH 7.4 (Figure 1a). Fitting the data obtained at 412 nm to a single exponential function yielded an observed rate constant (kobs ), which was plotted against the substrate concentration (Figure 1b). The gradient of the resulting straight line yielded the second-order rate constant given in Table 1. Kinetic studies were also carried out at lower pH values (6.0 and 6.7)
c The Authors Journal compilation c 2009 Biochemical Society
Reactivity of hypothiocyanous acid with biological thiols
113
Figure 1 Kinetic analysis of the reaction of HOSCN with TNB by direct stopped-flow studies Kinetic data obtained for the reaction of TNB (30 μM) with HOSCN (2.5 μM) at 22 ◦C and pH 7.4. (a) Kinetic trace showing depletion of TNB at 412 nm. (b) Plot of the observed rate constants (k obs ) versus TNB concentrations, with k obs determined by fitting the absorbance changes at 412 nm to a single exponential decay. Results are presented as mean + − S.D. (some error bars are smaller than the size of the symbol).
to mimic those reported for phagosomes [32,33]. Direct stoppedflow studies of the reaction of HOSCN (2.5 μM) with TNB (10– 50 μM) over 0.05–0.1 s at pH 6 and 0.2–0.5 s at pH 6.7 resulted in more rapid depletion of the TNB at these lower pH values, and yielded the second-order rate constants given in Table 1. Reactions of HOSCN with low-molecular-mass thiols
Kinetic data for the reactions of HOSCN with low-molecularmass thiols, the reported major targets for this oxidant [21], were obtained from competition kinetic experiments using the rate constant determined above for TNB as a reference (as the thiols of interest do not have significant UV/visible absorbance bands, unlike TNB). The kinetics of the reaction of HOSCN (2.5 μM) with increasing concentrations of L-cysteine (10–250 μM) were investigated by competition with TNB (10 μM) at 22 ◦C over a period of 0.1–2 s. Absorbance changes arising from the reaction of TNB with HOSCN were monitored at 412 nm, as this allowed the decay of TNB to be measured, without the potentially confounding influence of other absorbing products. The change in TNB absorbance obtained in the absence of any competing substrate (yieldmax ), and in the presence of a range of different competing thiol concentrations (yieldquench ), were quantified at fixed time points for each thiol after the cessation of absorbance changes detected in time-resolved experiments (Figure 2). In the absence of added competing thiol, the extent of TNB loss was found to depend on the concentration of HOSCN, and the final absorbance values of the residual TNB were stable over the time frames used in the experiment. These data indicate that secondary reactions and/or the formation of product materials do not interfere in a significant manner in these measurements. Control
Figure 2 Kinetic analysis of the reaction of HOSCN with L-cysteine determined by competition kinetics with TNB Kinetic data were obtained for the reaction of HOSCN (2.5 μM) with TNB (10 μM) in the presence of increasing concentrations of L-cysteine (0–250 μM) at 22 ◦C and pH 7.4. (a) Kinetic trace showing consumption of TNB at 412 nm in the absence of added L-cysteine. (b) Kinetic traces showing the decreased loss of TNB absorbance with increasing concentrations of the competing thiol L-cysteine at 412 nm. (c) Plot of the linear analysis for the competitive kinetic data for L-cysteine with HOSCN at 412 nm. Results are presented as mean + − S.D. (some error bars are smaller than the size of the symbol).
experiments, in which the product of TNB oxidation, DTNB, was reacted with HOSCN, showed no significant absorbance changes over the time scales employed, indicating that secondary oxidation of the disulfide by HOSCN is not a major alternative route to consumption of the oxidant under these conditions. In the presence of competing thiol, the change in TNB absorbance was found to decrease with increasing concentrations of the added material. Typical kinetic traces at 412 nm, showing the change in absorbance due to TNB oxidation, and the decrease in the extent of absorbance loss with increasing concentrations of L-cysteine are shown in Figures 2(a) and 2(b). These data yielded a linear competition plot, in which the gradient of the line is equal to kthiol /kTNB and the y-intercept is given by the TNB concentration (Figure 2c). Using the rate constant obtained above for TNB (Table 1), the second-order rate constant for reaction of HOSCN with L-cysteine could be determined (Table 1). c The Authors Journal compilation c 2009 Biochemical Society
114 Table 1
O. Skaff and others Second-order rate constants in 0.1 M phosphate buffer at 22 ◦C for the reactions of HOSCN with low-molecular-mass and protein thiols
Second-order rate constants (k 2 ) with 95 % confidence limits were determined for TNB by stopped-flow methods using single wavelength analysis at 412 nm. The k 2 for the other thiols was determined by competition kinetics relative to TNB at 412 nm using k (HOSCN + TNB) = 1.62 × 106 M−1 · s−1 at pH 6.7 for GSH at pH 6.7, k (HOSCN + TNB) = 7.7 × 106 M−1 · s−1 at pH 6.0 for GSH at pH 6.0 and k (HOSCN + TNB) = 3.8 × 105 M−1 · s−1 at pH 7.4 for all other thiols. pH
k 2 (HOSCN) (M−1 · s−1 )
TNB
7.4 6.7 6.0
5 (3.8 + − 0.2) × 10 6 (1.62 + 0.03) × 10 − 6 (7.7 + − 0.9) × 10
L-Cysteine
7.4
4 (7.8 + − 1.4) × 10
Cysteamine
7.4
4 (7.1 + − 0.7) × 10
N -Acetyl-cysteine
7.4
3 (7.3 + − 1.6) × 10
L-Cysteine methyl ester
7.4
5 (1.6 + − 0.1) × 10
Penicillamine
7.4
5 (3.0 + − 0.7) × 10
GSH
7.4 6.7 6.0
4 (2.5 + − 0.4) × 104 (3.1 + 0.3) × 10 − 4 (4.0 + − 0.4) × 10
7.4 7.4 7.4 7.4
4 (7.6 + − 1.5) × 104 (3.1 + 0.6) × 10 − 4 (1.0 + − 0.1) × 10 + (1.4 − 0.1) × 104
Substrate
Structure
Low-molecular-mass thiols
Thiol-containing proteins BSA Creatine kinase β-Lactoglobulin β-L-Crystallins
– – – –
The effect of net charge and pK a on thiol reactivity with HOSCN (2.5 μM) was examined using the following range of other thiols: cysteamine (10–250 μM); N-acetyl-cysteine (10– 500 μM); L-cysteine methyl ester (10–200 μM); and penicillamine (10–100 μM). Reaction of N-acetyl-cysteine with HOSCN was comparatively slow, thereby requiring higher concentrations of thiol to generate a substantial modulation of the loss of TNB absorbance. In contrast, low concentrations of penicillamine gave rise to a marked decrease in the extent of TNB absorbance loss, indicative of a higher rate constant for reaction of this thiol with HOSCN. The rate constants obtained are summarized in Table 1. Analogous experiments using a range of other potential targets of HOSCN oxidation [GSSG (glutathione disulfide), methionine, tryptophan, histidine and lysine, all at 100 μM] did not result in any significant modulation of the extent of TNB loss under the conditions employed, indicating that reaction of HOSCN with these materials is at least 6-fold slower than reaction with the least reactive thiols. c The Authors Journal compilation c 2009 Biochemical Society
Reaction of HOSCN with the tripeptide GSH and its pH dependence
The cysteine-containing tripeptide GSH is the most abundant low-molecular-mass thiol in most cells [34] and a major target for HOCl [35,36]; it is therefore a potential target for HOSCN in cells. The rate constant for reaction of HOSCN with GSH was therefore determined at both pH 7.4 and lower pH values to model the reported pH values at sites of inflammation [32,33]. HOSCN (2.5 μM) was allowed to react with TNB (10 μM) in the absence and presence of increasing concentrations of GSH (10–250 μM) at 22 ◦C using stopped-flow techniques. At pH 7.4, the absorbance due to TNB at 412 nm decayed completely over a period of 0.5–1 s, and the resulting data (Figure 3) yielded the second-order rate constant given in Table 1. In experiments at pH 6.0 and 6.7, increased concentrations of GSH (0.25–5 mM GSH at pH 6.0, 0.125–2 mM GSH at pH 6.7) were required to modulate the loss of TNB (10 μM) absorbance. Analysis of the competitive kinetic data (Figure 3) provided the appropriate
Reactivity of hypothiocyanous acid with biological thiols
Figure 3 Kinetic analysis of the reaction of HOSCN with GSH determined by competition kinetics with TNB and the effect of reaction pH Plot of the linear analysis for the competitive kinetic data showing the pH-dependence of the second-order rate constant for the reaction of HOSCN (2.5 μM) with TNB (10 μM) in the presence of increasing concentrations of GSH at 22 ◦C at pH 7.4 (䊉), at pH 6.7 (䉱) and at pH 6.0 (䊏). Results are presented as means + − S.D. (some error bars are smaller than the size of the symbol).
second-order rate constants by use of the values for TNB at the same pH (Table 1). The second-order rate constants obtained exhibit a pH dependency, with the rate constant for reaction of GSH with HOSCN increasing at lower pH values. Reactions of HOSCN with proteins
As the results in Table 1 indicate variation in the rate constant for reactions of HOSCN with thiols with varying substrate structure, experiments were also carried out with four thiolcontaining proteins, CK, BSA, β-lactoglobulin and β-Lcrystallins. Experiments with other proteins were precluded by the amount of protein required for these analyses. The rate constant for reaction of HOSCN (2.5 μM) with the thiol residues (of which there are four in the rabbit muscle isoenzyme) of CK (10–100 μM protein thiol, based on concentrations determined by DTNB assay) was investigated by including TNB (10 μM) in the reaction mixture and monitoring the extent of absorbance loss at 412 nm with increasing protein thiol concentration as outlined above. The addition of increasing concentrations of CK resulted in a decrease in the extent of loss of the TNB absorbance at 412 nm, and the resulting linear competition plots allowed a second-order rate constant to be determined (Table 1); this value is likely to be a composite value for the rate constant for reaction with each of four cysteine residues present in CK. Similar studies carried out with BSA (Figure 4; which has a single free cysteine residue at position 34), β-lactoglobulin (which has a single free cysteine) and β-Lcrystallins (which have multiple free cysteine residues) yielded the second-order rate constants shown in Table 1. Blocking of the thiol residue on BSA with NEM resulted in a marked, and highly significant, decrease in the extent of TNB absorbance loss (> 53 % with four preparations, results not shown) when compared with that induced by the same concentration of the native protein. This observation indicates that reaction of HOSCN with the protein is occurring predominantly at the single Cys34 residue in BSA. DISCUSSION
Considerable evidence supports a role for peroxidase-derived oxidants in human pathologies [1,14]. SCN− oxidation by MPO accounts for 50 % of the H2 O2 consumed by peroxidases [7], indicating that HOSCN is a major oxidant at inflammatory sites.
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Figure 4 Kinetic analysis of the reaction of HOSCN with the single free cysteine residue on BSA determined by competition kinetics with TNB Plot of the linear analysis for the competitive kinetic data for the reaction of HOSCN (2.5 μM) with TNB (10 μM) in the presence of increasing BSA thiol concentrations (5–72.25 μM) determined by stopped-flow methods. Results are presented as mean + − S.D. (some error bars are smaller than the size of the symbol).
However, quantitative results on the importance of HOSCN are lacking, and no ‘biomarker’ for this oxidant has been reported. Elevated levels of carbamylated proteins have been detected in human and murine atherosclerotic lesions [10], with these ascribed to reactions of OCN− generated from HOSCN, or as a result of uraemia [37]. Serum SCN− levels correlate with early markers of atherosclerosis including the deposition of oxidized lipoproteins and numbers of macrophage foam cells [24,25]. In contrast, SCN− oxidation by HOCl and HOBr has been postulated as a protective process [11,12]. It has been shown here that HOSCN reacts rapidly with low-molecular-mass peptide (GSH) and protein thiols. The observed rate constants vary, for the low-molecular-mass thiols, over the range 7.3 × 103 M−1 · s−1 (N-acetyl-cysteine, pH 7.4) to 7.7 × 106 M−1 · s−1 (TNB, pH 6.0). Rate constants for the reaction of HOSCN with protein cysteine residues lie within the range 1 × 104 – 7.6 × 104 M−1 · s−1 . No detectable reaction (k < 103 M−1 · s−1 ) has been observed with methionine, histidine, tryptophan, lysine or GSSG, in contrast with the behaviour of HOCl and HOBr [1,16]. Experiments with NEM-blocked BSA confirm that reaction occurs predominantly at the free cysteine residue. In the absence of such residues, or after their depletion, oxidation of tryptophan residues occurs on proteins [22]. The rate constants show a dependence on pH, with higher values observed as the reaction pH approaches the pK a of HOSCN/− OSCN (5.3 [5]). This increase is more marked for TNB than with GSH (a 20-fold compared with a 2-fold increase). This increase is consistent with HOSCN, rather than − OSCN, being the major reactive species. The rate constants vary with structure of the thiol, with this being ascribed, at least in part, to the thiol pK a values. Neutral thiols (RSH) are weaker nucleophiles than the corresponding anions (RS− ), so compounds with a low pK a thiol would be expected to be more reactive, other factors being equal. Thus TNB, which has the lowest thiol pK a (5.1 [38]) is the most reactive. A plot of thiol pK a against rate constant (Figure 5), indicates a significant inverse correlation between these parameters, with the exception of penicillamine, which has the most sterically-hindered thiol. This analysis mirrors that reported for the reaction of thiols with taurine chloramine [38]. In this previous study cysteamine did not fit the inverse relationship, with this being ascribed to its higher net charge [38]; this variance was not observed in the current study. Whether this correlation also holds for protein cysteine residues cannot be determined from the current data, as the rate constants c The Authors Journal compilation c 2009 Biochemical Society
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arises from HOSCN, making this species a major mediator of thiol oxidation in vivo. Furthermore, reaction of HOCl and HOBr with SCN− to give HOSCN is unlikely to be protective [11,12] given the specificity and high reactivity of HOSCN with thiols. Experiments to test these hypotheses are underway. HOSCN-mediated oxidation of low-molecular-mass and protein thiols is also likely to be of considerable significance in other situations where significant peroxidase activity is present and the SCN− concentration is higher than that in plasma, including in saliva [9,46], lung bronchioalveolar fluid [47,48] and the stomach [8]. AUTHOR CONTRIBUTION Figure 5 Graph showing the relationship between thiol pK a and the secondorder rate constants for the reaction with HOSCN at pH 7.4 Cys MeE, L-cysteine methyl ester; Pen, penicillamine; NAC, N -acetyl-cysteine. Thiol pK a values are taken from [38].
obtained for both CK and the β-L-crystallins are likely to reflect reaction at multiple cysteine sites. For CK, reaction may occur with any of the four cysteine residues, and these are known to have markedly different pK a s [39]. The active site Cys282 of CK with a pK a of ∼ 5.4 [39] might be expected to react more rapidly than Cys34 of BSA, which has a pK a of 8.5 [40]. However, the higher value determined for BSA suggests that other factors play a role. Neighbouring residues may affect the local pH and access to the cysteine residues for steric and electronic (i.e. charge repulsion and electrostatic attractions) reasons. The Cys34 residue in BSA is contained within a cleft [41], whereas the cysteine residues of CK are in a variety of environments, with none highly exposed and Cys282 partially buried in a cleft [42]. Comparison of the rate constants for protein cysteine residues with that for GSH (the most abundant cellular thiol with concentrations 10 mM [34]), suggests that GSH is a major intracellular target, but that protein thiols will also be oxidized, which is consistent with published results [23]. For extracellular oxidant generation (e.g. released MPO or eosinophil peroxidase at sites of inflammation), where low-molecular-mass thiols are present at < 10 μM (compared with the concentrations of hundreds of μM for protein thiols [6]), oxidation of protein cysteine will predominate and be highly specific. The rate constants for reaction of HOCl and HOBr with cysteine residues are approx. 3 × 107 M−1 · s−1 ([16,43,44]; reviewed in [15]). These values are 2–3 orders of magnitude higher than for HOSCN. However, unlike HOCl and HOBr, HOSCN does not appear to undergo rapid alternative reactions (e.g. with other protein residues, lipids, DNA and antioxidants), which will consume some HOCl and HOBr. For HOCl, other reactive residues are present at higher concentrations than cysteine (concentrations of 7.2 mM methionine, 9 mM cystine, 14 mM histidine, 46 mM lysine, and 4 mM tryptophan for plasma proteins, compared to 520 μM cysteine residues; [45]). A similar argument applies to HOBr, where the difference in rate constants is even smaller than for HOCl [15,16,43]. Computational modelling of the reactions of HOCl with plasma indicates that 30–35 % of the HOCl reacts with cysteine residues with the remainder consumed by other targets [45]. Furthermore, the percentage of HOCl consumed by cysteine decreases as the HOCl concentration increases. As up to 50 % of the H2 O2 consumed by MPO is used to oxidize SCN− (with concentrations of 100 mM Cl− and 100 μM SCN− [7]), and only 35 % of the HOCl gives rise to thiol loss, the majority of thiol oxidation observed in plasma on exposure to MPO/H2 O2 systems (under the above conditions) probably c The Authors Journal compilation c 2009 Biochemical Society
Ojia Skaff performed the research, analysed the data and contributed to the manuscript preparation. David Pattison assisted with the data analysis and experimental design, and contributed to the manuscript preparation. Michael Davies designed the research and experimental plan, and contributed to the manuscript preparation.
ACKNOWLEDGEMENTS We thank Professor Peter Lay and Dr Aviva Levina (University of Sydney) for the use of the Applied Photophysics SX.18MV stopped-flow system, and Dr Clare Hawkins and Dr Mitchell Lloyd (The Heart Research Institute) for technical assistance and helpful discussions.
FUNDING This work was supported by grants from the Australian Research Council under the ARC Centres of Excellence and Discovery programs [grant numbers CEO561607, DP0663967, DP0988311]; and by the National Health and Medical Research Council [grant number 570829].
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Received 13 February 2009/7 May 2009; accepted 3 June 2009 Published as BJ Immediate Publication 3 June 2009, doi:10.1042/BJ20090276
c The Authors Journal compilation c 2009 Biochemical Society
