Species-Specific ThiolâDisulfide Equilibrium Constant - American

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The Journal of Physical Chemistry

TITLE PAGE Species-Specific Thiol-Disulfide Equilibrium Constant: a Tool to Characterize Redox Transitions of Biological Importance

AUTHORS Arash Mirzahosseini, Máté Somlyay, Béla Noszál* Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary Research Group of Drugs of Abuse and Doping Agents, Hungarian Academy of Sciences, Hungary

*to whom correspondence should be addressed: Prof. Béla Noszál Department of Pharmaceutical Chemistry, Semmelweis University H-1092 Budapest, Hőgyes Endre utca 9, Hungary Phone/Fax: +3612170891 E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Microscopic redox equilibrium constants, new, species-specific type of physico-chemical parameters were introduced and determined to quantify thiol-disulfide equilibria of biological significance. The thiol-disulfide redox equilibria of glutathione with cysteamine, cysteine, and homocysteine were approached from both sides, and the equilibrium mixtures were analyzed by quantitative NMR methods to characterize the highly composite, co-dependent acid-base and redox equilibria. The directly obtained, pH-dependent, conditional constants were then decomposed by a new evaluation method, resulting in pH-independent, microscopic redox equilibrium constants for the first time. The 80 different, microscopic redox equilibrium constant values show close correlation with the respective thiolate basicities and provide sound means for the development of potent agents against oxidative stress.

KEYWORDS Glutathione; thiol; disulfide; redox equilibrium; quantitative NMR, species-specific constants


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1. INTRODUCTION Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) is the single most important nonenzymatic intracellular antioxidant. As the prime molecule of the physiological defense system against reactive oxygen species (ROS), the agents of oxidative stress, GSH is oxidized to glutathione-disulfide (GSSG) as seen in Figure 1A. Glutathione is a key factor in the maintenance of the redox homeostasis of biological systems, due partly to its cellular concentration exceeding that of other antioxidants. The concentration of GSH ranges between 0.5 and 10 mM1, and is largely maintained in its reduced form by glutathione reductase2-3. Glutathione is also responsible for the regulation of the thiol-disulfide balance in proteins and biogenic thiols4-5. Another reason why glutathione works as ideal redox buffer is its optimal, pH-modulated redox potential, which is not yet thoroughly understood and quantified, and is, actually the subject of this and forthcoming papers. Nevertheless, the ubiquitous GSH-GSSG redox system is the most widely studied one6. It does not require enzyme catalysis to function, and can be applied in aqueous media under a wide range of conditions. These advantages make the GSH-GSSG system a “gold standard” in thiol-disulfide biochemistry; hence, every thiolcontaining antioxidant is compared to glutathione4. It is a key feature of any thiol-disulfide redox system, however, that only the deprotonated thiol species are active in the redox process, i.e. only the anionic thiolate can be oxidized directly7. The redox potential of thiolcontaining biomolecules is usually doubly pH-dependent. Primarily, the deprotonated fraction of every thiol depends on the pH of the solution, with strong dependence typically in the 6-11 pH range. Secondarily, the thiolate oxidizability in most biomolecules is multiply modulated by the adjacent basic groups, since protonation of any such group exerts an electronwithdrawing effect on the thiolate. We therefore postulate that a thiolate has 2n different oxidizabilities, if the number of the basic sites in its intramolecular environment is n. Thus,


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macroscopic physico-chemical parameters cannot characterize the thiolate moiety specifically. A thorough characterization of the thiol-disulfide equilibria can be achieved in terms of species-specific, so-called microscopic parameters, which are, in fact, well-established for acid-base systems8-9. In this work the species-specific redox equilibria of three biogenic thiols (namely, cysteamine, cysteine, homocysteine) with glutathione were studied. The constitutional formulae of the three biological thiols are depicted in Figure 1B. The important biological role of these thiols is epitomized by cysteamine as a subunit of coenzyme A10, and cysteine as a semi-essential proteinogenic amino acid with an important role as a nucleophilic agent. Homocysteine takes part in the methyl-equivalent transfer in biochemical reactions and has been shown to be a major risk in heart-disease11, due to the low solubility of its disulfide. The oxidation of these three thiols yields cystamine, cystine, and homocystine, respectively. The microscopic protonation constants of cysteamine, cysteine, homocysteine, glutathione and their respective homodisulfides are sine qua non components to determine species-specific thiol-disulfide equilibrium constants, and have been reported previously12-13. The general scheme of thiol-disulfide redox equilibria are depicted in Figure 1C, where RSH and RSSR stand for the reduced and oxidized form of the thiol, and RSSG stands for the heterodisulfide of the corresponding thiol and glutathione. The three conditional equilibrium constants are as follows:

(1) =

(2) =

[][ ] [ ][]

[][ ] [ ][ ]


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(3) = =

[] [ ] [ ] []

Since redox and acid-base reactions coexist, the observed apparent redox equilibrium constants, K1C, K2C, and K3C are pH-dependent. In order to get a clear insight into the redox equilibria, purified from the protonation effects, an improved evaluation method had to be introduced, including a set of species-specific, new type of redox equilibrium constants (k1, k2, k3). As equation (3) shows the complete redox transition includes two thiols and two disulfides. We therefore confined our efforts to the determination of the K3C macroconstants, and the related k3 microconstants, the number of which can certainly be numerous. This confinement overcomes the highly complex quantification of the heterodisulfides, which actually improves perspicuity. Keire et al.4 have already demonstrated that a) there is correlation between the thiolate basicities and redox properties and b) purely redox thiol-disulfide equilibrium constants can only be obtained if these transitions can be decoupled from the acid-base processes by an appropriate evaluation method. Determination of the redox equilibrium constants requires all the species-specific protonation constants, including those of the minor microspecies; otherwise, redox processes could only be characterized at the level of phenomenon, especially for acidic media. Having now elaborated the proper evaluation procedure and the complete microscopic protonation constant repertoire of the studied thiols12-13, here we report the comprehensive characterization of the species-specific redox properties of thiol-disulfide systems. Our experiments were carried out under consistent, biomimetic conditions of 95 v/v% H2O and 0.15 mol/l ionic strength. This work is the first attempt to completely characterize thioldisulfide redox equilibria in terms of pH-independent, species-specific constants.


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Figure 1. A: The thiol-disulfide redox half-reaction between GSH and GSSG. The biochemical oxidation of GSH is mostly paired with glutathione peroxidase enzyme in the elimination of hydrogen peroxide. However, GSH is the most important non-enzymatic antioxidant in neutralizing reactive oxygen species. GSSG is converted back to GSH by glutathione reductase with the use of NADPH. B: The structural formulae of the three biogenic thiols. C: The scheme of thiol-disulfide equilibria at the macroscopic level. RSH denotes cysteamine, cysteine, and homocysteine; RSSR denotes their respective homodisulfides; RSSG denotes their respective heterodisulfides formed with glutathione.


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2. EXPERIMENTAL SECTION 2.1. Materials Homocysteine and homocystine were purchased from Bachem (Bubendorf, Switzerland), glutathione, glutathione disulfide, cysteamine, cystamine dihydrochloride, cysteine, cystine, and all other chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and used without further purification.

2.2 Preparation of solutions Acidic (pH = 0.85) and basic (pH = 13.15) stock solutions containing either: (GSH + cystamine, cystine, or homocystine) or (GSSG + cysteamine, cysteine, homocysteine) were prepared in an 815-PGB glove box (Plas-Labs Ic., Lansing, MI, USA) under N2 atmosphere to preclude oxidation by air. The concentrations of the reagents were optimized for quantitative NMR at around 15 mmol/L, except for the slightly soluble cystine and homocystine (ca. 7 mmol/L). Three series of solutions with 16-16 different pH were prepared by mixing the acidic with the basic stock solutions. D2O, DSS (sodium 4,4-dimethyl-4-silapentane-1sulfonate), and a pH indicator, which also served as a concentration standard, were added to the solutions. The concentration of the pH indicators/concentration standards were comparable to the analyte concentrations in the final samples, and included: dichloroacetic acid for pH 0-2, formic acid for pH 2-4, imidazole for pH 5-9, tert-butylamine for pH 9-12. The samples were protected from sunlight and kept in the glove box under N2 atmosphere for 6 weeks, until the reactions in all the samples had reached equilibrium.

2.3 NMR spectroscopy measurements


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NMR spectra were recorded on a Varian 600 MHz spectrometer at 25.0 °C. The solvent in every case was an aqueous solution with H2O:D2O, 95:5, v/v (0.15 mol/l ionic strength), using DSS as the reference compound. The sample volume was 600 µl, pH values were determined by internal indicator molecules optimized for NMR14-15. 1H NMR spectra were recorded with the WET solvent suppression sequence (number of transients = 64, number of points = 65536, acquisition time = 3.407 s, relaxation delay = 15 s).

2.4 Data analysis For the analysis of quantitative NMR measurements, the peak fitting algorithm (without apodization) of the ACD/NMR Processor Academic Edition v12.01 software package (Advanced Chemistry Development, Toronto, ON, Canada) was used. For the regression analyses, the software Origin Pro 8 (OriginLab Corp., Northampton, MA, USA) was used. The standard deviations of the peak areas obtained by fitting Lorentzian peak shapes, the error of pH determination, and the standard errors of the microscopic protonation constants12-13 were used to calculate the Gaussian propagation of uncertainty to the redox equilibrium constants derived in the Results chapter. In practice, the measured 1H NMR spectra were fitted to Lorentzian curves in the frequency domain using the curve fitting algorithm of ACD 1D NMR Processor. The Fourier number was always set to maximum, automatic baseline and phase correction, and no apodization was used. Using the chemical shift-pH profiles determined previously12-13 the chemical shifts of the various nuclei in the coexisting molecules (e.g. CysSH, GSH, CysSSCys, GSSG) at a given pH could be predicted (sample spectra are depicted in Figure 2). For the sake of simplicity, whenever possible the following characteristic peaks were observed: α-glutamyl-, α-cysteinyl-, glycinyl proton of GSH and GSSG; α-proton of cysteine, homocysteine, and their homodisulfides; the methylene protons of cysteamine and cystamine. In order to calculate the concentration of the species, the


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integral of the fitted peaks were related to the integral of that of the pH indicator, which also served as concentration standard. The indicators were chosen so that their peaks were always discernable. When no any peak was discernable for one of the species due to overlapping signals, its concentration was calculated using the concentration of the stock solutions and the law of conservation of mass.

Figure 2. The expanded 1H NMR spectra of samples containing cysteamine-glutathione mixtures (A) and cysteine-glutathione mixtures (B). Cα, Gα, and Eα, denote the cysteinyl, glycinyl, glutamyl α protons of glutathionyl residues.

3. RESULTS


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Figures 3 and 4 represent the species-specific protonation scheme of cysteine, cystine, glutathione, and glutathione disulfide. Macroequilibria (top lines) indicate the stoichiometry of the successively protonated ligand and the stepwise macroscopic protonation constants. In the microspeciation schemes, the different microspecies with their one-letter symbols (a, b, c …), and the microscopic protonation constants are depicted (kN, kNS …). The superscript at k for any microconstant indicates the protonating group while the subscript (if any) shows the site(s) already protonated. S, N, O, G and E symbolize the thiolate, amino, carboxylate (in cysteine and cystine), glycinyl carboxylate and glutamyl carboxylate (in GSH and GSSG after their one-letter symbol) sites, respectively. The acid-base macro- and microequilibria are inevitable constituents in the evaluation of the species-specific, pH-independent redox equilibrium constants, as shown in equations (9) and (10). Some protonation constant examples for cysteine are shown below:

(4) =

[ ] [][ ]

(5) = =

(6) =

[ ] [ ][ ]

[] [][]

where K1, K2, K3, are successive macroconstants, β3 is one of the cumulative macroconstants,

kN is the microconstant representing the amino protonation in cysteine, when its carboxylate and thiolate sites are unprotonated. The concentrations of the various macrospecies comprise the sum of the concentration of those microspecies that contain the same number of protons, for example in GSH:


10

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(7) [H L ] = [F] [G] [H] [I] [J] [K]

Figure 3. The protonation macro- and microequilibrium schemes of CysSH (A) and CysSSCys (B) in terms of stepwise macroscopic protonation constants (K1, K2, K3 …), where L2-, HL-, etc. are the successively protonating ligands (top lines). Below are the species-


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specific protonation schemes in terms of microspecies (a, b, c …) and microscopic protonation constants (kN, kNS …). The components of the major pathways are in bold.


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Figure 4. The protonation macro- and microequilibrium schemes of GSH (A) and GSSG (B) in terms of stepwise macroscopic protonation constants (K1, K2, K3 …), where L3-, HL2-, etc. are the successively protonating ligands (top lines). Below are the species-specific protonation schemes in terms of microspecies (A, B, C …) and microscopic protonation constants (kN, kNS …). For GSSG, only non-identical microspecies and microconstants are shown, and only a few of the microconstants are depicted. The components of the major pathways are in bold.

For the thiol-disulfide redox equilibria, only the apparent or conditional equilibrium constants (K3C) are directly available, by determining the equilibrium concentrations of RSH, GSSG, RSSR, and GSH in the reaction mixtures. In other words: these conditional equilibrium constants are pH-dependent parameters in terms of total concentrations of the thiol and disulfide species that are present, since the integrals of the observed NMR signals correspond to the total concentration of a thiol reactant that is actually composed of the concentration of the variously protonated microspecies. The concentration of the macrospecies can be written as the sum of the microspecies of the same number of bound protons, as shown in equation (7) for H2L- of GSH. The pH-dependent, apparent constants can be decomposed into pHindependent, species-specific equilibrium constants, the number of which is large, but definite. For example, the thiolate moiety in cysteine is adjacent to two other basic sites; the amino and the carboxylate groups. Therefore, the thiolate-bearing microspecies can exist in 4 different protonation states. Concurrently, the thiolates in the 4 different microspecies will be in different electrostatic environments, thus having 4 different oxidizabilities. Considering the opposite direction of the redox equilibrium, GSH with an amino and two carboxylate sites can have 8 different thiolate-bearing microspecies with 8 different electron densities on its sulfur. This leads to 4x8=32 different microscopic redox equilibria between cysteine and glutathione. To epitomize the determination of microscopic redox equilibrium constants (k3) from the


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conditional equilibrium constants (K3C), the calculation of the k3bB for the reaction involving the b microspecies of cysteine, the corresponding f’ cystine microspecies, the B glutathione microspecies, and the corresponding E’ glutathione disulfide microspecies will be demonstrated. Note that b and B define that the disulfide microspecies are f’ and E’, since these are the only ones with identical protonation states in the side-chain. Superscripts b and B therefore unambiguously identify all the 4 microspecies in the microequilibrium in question. This example of the pH-independent, microscopic thiol-disulfide equilibrium constants is expressed by equation (8):

[ ] [!" ]

(8) = " [] [# ]

Quantification of redox equilibria in terms of such pH-independent, species-specific constants has long been a conceptual endeavor. The obvious reason why it has not so far been achieved is the fact that the microspecies concentrations cannot be measured. As shown below, the species-specific constants can be, however, obtained as the product of total species concentration and the relative abundance of the respective microspecies. The relative abundance of the microspecies in turn, is a function of pH and the microscopic protonation constants. For the b cysteine microspecies, the concentration can be written as follows:

(9) [b] = [CysSH] × * = [CysSH]

+ , [ ] 01234 01234 01234 -./ [ ]-. [ ] -. [ ]

where [CysSH] is the total solution concentration of cysteine, χb is the relative abundance of microspecies b (i.e. χb = [b]/[CysSH]), kN is the microscopic protonation constant of cysteine involved in the formation of microspecies b. Substituting analogous equations as equation (9)


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for the concentrations of the microspecies in k3bB, one can write the equation of the microscopic equilibrium constant as follows:

[ ] [!" ]

[] 56 [7878]5 "

56 5"

9 (10) = " = = 9 [] [# ] [78] 5: []5;" 5: 5;"

Thus, if K3C, the apparent equilibrium constant and the 4 χ values (as functions of the protonation constants and pH) have previously been determined, the k3bB value and all the analogous, pH-independent, species-specific redox microconstants can be calculated. The conditional equilibrium constants were calculated from the concentrations in the equilibrium reaction mixtures according to equation (3). The conditional redox equilibrium constant values are listed in Table 1 and represented as a function of pH in Figure 5A.

Table 1. Conditional redox equilibrium constants Cysteamine pH logK3C 0.99 -0.155 1.31 -0.097 1.59 -0.071 1.83 -0.097 2.62 -0.222 2.93 -0.252 3.17 -0.260 3.95 -0.328 5.43 -0.319 7.87 -0.495 7.92 -0.509 9.62 -1.347 10.20 -0.523 10.60 0.021 11.44 0.701

Cysteine pH logK3C 0.86 0.028 1.25 0.078 1.54 0.113 1.81 0.176 2.61 0.197 2.89 0.146 3.20 0.122 3.40 0.116 5.60 -0.273 6.42 -0.230 7.67 -0.204 8.83 0.105 9.67 0.601 9.98 0.779 10.44 1.244 11.86 1.668

Homocysteine pH logK3C 0.91 0.099 1.22 0.137 1.54 0.203 1.81 0.263 2.61 0.335 2.89 0.323 3.20 0.288 3.40 0.174 5.62 -0.051 6.37 -0.056 7.60 -0.092 8.83 -0.048 9.67 0.280 9.98 0.693 10.44 1.003 11.86 1.443


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The conditional redox equilibrium constants of cysteamine-, cysteine-, homocysteineglutathione thiol-disulfide systems. The uncertainties in the pH and logK3C values are 0.010.02 and 0.002-0.004, respectively.


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Figure 5. A: The pH dependence of the conditional redox equilibrium constant K3C. B: The correlation between species-specific redox equilibrium constants and the species-specific thiolate protonation constants. Each symbol represents the thiol-disulfide equilibria involving various thiol microspecies of cysteamine, cysteine, and homocysteine with a selected glutathione microspecies (A, B, D, E, G, H, K, N). C: The correlation between speciesspecific redox equilibrium constants (logk3xY) and the difference between species-specific thiolate protonation constants of the involved RSH and GSH microspecies (∆logk). The ∆logk values were calculated from the species-specific protonation constants previsously determined12-13.

The microscopic redox equilibrium constants were calculated from the conditional equilibrium constants using analogous equations as equation (10). Obviously, the conditional equilibrium constants were measured at different pH media, all of which theoretically afford the same value for a certain microscopic equilibrium constant. However, each microscopic redox equilibrium constant was calculated at pH values where the corresponding equation (10) is well-conditioned, i.e. the mole fractions of the involved microspecies are nearmaximal. For k3bB the optimal pH of determination are 5.60, 6.42, 7.67, 8.83 (see Table 1). The mean of the calculated microscopic redox equilibrium constants are listed in Table 2.


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Table 2. Species-specific redox equilibrium constants Site(s) of protonation and one-letter symbols of thiolate microspecies Cysteamine Glutathione

Cysteine

Homocysteine

-

amino

-

amino

carboxylate

amino and carboxylate

-

amino

carboxylate

amino and carboxylate

a

b

a

b

d

f

a

b

d

f

-

A

0,91

-1,73

1,74

-1,04

0,12

-3,16

1,42

-0,43

0,77

-1,86

amino

B

1,56

-1,40

2,33

-0,30

0,44

-2,74

2,09

0,19

1,09

-1,17

gly carboxylate

D

1,73

-1,35

2,39

-0,14

0,46

-2,68

2,15

0,18

1,18

-1,09

glu carboxylate

E

1,38

-1,48

2,23

-0,50

0,36

-2,82

1,94

-0,01

1,00

-1,25

amino and gly carboxylate

G

2,30

-0,66

3,19

0,44

1,18

-2,00

2,83

0,86

1,83

-0,41

amino and glu carboxylate

H

2,00

-0,95

2,89

0,16

0,88

-2,30

2,53

0,45

1,53

-0,71

both carboxylates

K

2,10

-0,85

3,10

0,24

0,98

-2,20

2,63

0,82

1,63

-0,61

amino and both carboxylates

N

2,88

0,03

3,64

0,93

1,86

-1,32

3,42

1,40

2,51

0,18

The species-specific redox equilibrium constants of glutathione with cysteamine, cysteine, and homocysteine in log units. Each column corresponds to a cysteamine, cysteine, or homocysteine microspecies, the one-letter symbols of which are described in Fig. 2. The microscopic protonation scheme of homocysteine is analogous to that of cysteine, however for the case of cysteamine only four microspecies exist (here microspecies ‘a’ corresponds to the completely unprotonated cysteamine microspecies, while ‘b’ corresponds to the cysteamine microspecies protonated only at the amino moiety). On the other hand, each row corresponds to a glutathione microspecies. Note, that a certain pair of RSH and GSH microspecies unambiguously determines the RSSR and GSSG microspecies that also take part in the thiol-disulfide equilibrium. For example, logk3aA of the cysteine-glutathione system has the value of 1.74, and can be found in the left uppermost corner of the cysteine box. The uncertainties in the logk3 values are 0.01-0.03.


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The correlation between the species-specific redox equilibrium constants (within each block of GSH microspecies) and the thiolate species-specific protonation constants of CysASH, CysSH, hCysSH (taken from13) is depicted in Figure 5B, while the correlation between the logk3xY and ∆logk (the difference in species-specific protonation constants of the involved RSH and GSH thiolates) are depicted in Figure 5C.

4. DISCUSSION The highly interwoven acid-base and redox properties of the GSH/GSSG-RSH/RSSR system can be decomposed into elementary, component equilibria if a) the species-specific protonation constants of GSH, GSSG, RSH, and RSSR are determined, and b) the conditional equilibrium constants of the pH-dependent redox equilibria are also quantified. In this work the thiol-disulfide equilibria of glutathione with cysteamine, cysteine, and homocysteine were characterized in terms of conditional and species-specific equilibrium constants. As anticipated, the dissection of conditional equilibria into elementary redox ones in biological thiol-disulfide systems reveals significant differences between reactions of apparently highly similar, covalently identical reactants, improving thus our understanding of redox homeostasis and providing new means to influence it. As Table 2 reveals, this is all the more important in light of the highly complex, parallel protonation equilibria of these multibasic biomolecules: not only the protonation of the thiolate, its protonation fraction, and the concomitant redox behavior will be very sensitive to minor pH changes, but the protonation state of neighboring basic moieties can influence the redox behavior by various inductive effects on the thiolate moiety. The correlation between thiolate basicity and redox behavior in this paper is in excellent agreement with the previously published claims that thiolate basicity and oxidizability are proportional parameters4.


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Major trends among the 16, 32, and 32 k3xY redox microconstants in the respective cysteamine, cysteine, and homocysteine boxes in Table 2 are that their values generally increase downwards and leftwards. In fact, the largest values appear in the left bottom corners, while the smallest values can be found in the right uppermost corners. More precisely, the values strictly increase as the strength and number of electron-withdrawing moieties increases on the glutathione species and vice versa on the cysteamine, cysteine, and homocysteine species. Both the rightwards and upwards decreasing values can well be interpreted by means of the gradually protonating side-chain sites and the concomitantly decreasing electron density at the thiolate site. Such effects in the side-chain protonation are strongest in cysteine (k3aY >> k3fY), since its amino and carboxylate moieties are located in the vicinity of the thiolate, and both the amino and the carboxylate sites are closer to the thiolate as compared to homocysteine and glutathione. Protonation of the cysteine amino site decreases the k3xY value by a factor of 600, while protonation of the cysteine carboxylate exerts an effect of the same direction by a factor of 40. Even though in glutathione three groups protonate in the vicinity of its thiolate, their cumulative effect is typically less than 200. Upon comparing k3aN and k3fA (103.64 and 10-3.16) in the cysteine-glutathione system, the ratio of these values is some 6.3 million, indicating that side-chain protonations can modify thiol-disulfide equilibria by several orders of magnitude, while covalent structures remain exactly the same. The strong correlation between the species-specific thiolate protonation constants and speciesspecific redox equilibrium constants (Figure 5B) confirm the assumption that the thioldisulfide redox reactions proceed via the thiolate form. As the electron density around the thiolate moiety increases, the proton binding affinity and oxidizability (inversely proportional to electron affinity) of the thiolate should increase proportionally, since both processes are governed by electron-density effects. The linear relationship between logk thiolate protonation microconstants and logk3xY redox equilibrium constants indicates that the acid-base and redox


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equilibria have been broken down to elementary reactions. The consistent slope of the correlation (mean value: 2.13 ±0.04) for the different thiol-disulfide equilibria involving various GSH microspecies also supports the hypothesis that these acid-base and redox processes are driven by the same force, and the slope values are also in excellent agreement with the previous works of Keire et al.4 (2.17 ±0.19) and Szajewski and Whitesides7 (2.42). The different offset in the regression lines in Figure 5B is due to the different reaction partner for each thiol microspecies, i.e. different GSH microspecies. When the species-specific redox equilibrium constants are depicted as a function of ∆logk this reaction partner dependence is eliminated. In Figure 5C all the data points align on the same line (slope = 2.11 ±0.03), with a very convincing linear regression going through the origin, indicating the presence of a collective fundamental process governing all thiol-disulfide transitions. In the future we intend to determine the species-specific standard redox potentials (E°) of the various GSH microspecies, which can also be done indirectly only. The calculation of the species-specific redox potentials for the redox half-reaction of each thiol microspecies will enable the independent evaluation of these processes from the redox equilibrium partner.

5. CONCLUSION The pH-dependent and 80 pH-independent equilibrium constants of the thiol-disulfide redox reactions between glutathione and the three important biogenic thiols were determined. The pH-independent species-specific redox equilibrium constant was implemented for the first time for thiol-disulfide systems; these values characterize the redox processes at the microspecies level. The 80 different species-specific equilibrium constant values provide sound means to predict thiolate oxidizabilities, a key parameter to understand and influence oxidative stress. The delicate task of medicinal antioxidants lies in their confinement: ROS (reactive oxygen species) surplus has to be eliminated, while physiological disulfides in


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proteins and peptides have to be kept intact. The major difficulty in designing effective antioxidants without decomposing the physiological disulfides in biomolecules is that the redox potential of the latter could not so far be determined. The correlation between the redox and acid-base properties serves now as a basis to quantify reducibility of disulfide moieties in physiological proteins and peptides, allowing thus the development of potent, selective antioxidant compounds.

ACKNOWLEDGEMENT This research was supported by TÁMOP 4.2.1.B-09/1/KMR and OTKA T 73804. The authors report no conflict of interest.

REFERENCES 1. Kosower, N. S.; Kosower, E. M. The Glutathione Status of Cells. Int. Rev. Cytol. 1978, 54, 109-160. 2. Huxtable, R. J. Biochemistry of Sulfur; Plenum Press: New York, 1986. 3. Reed, D.J. Oxidative Stress; Academic Press: New York, 1985. 4. Keire, D. A.; Strauss, E.; Guo, W.; Noszál, B.; Rabenstein, D. L. Kinetics and Equilibria of Thiol/Disulfide Interchange Reactions of Selected Biological Thiols and Related Molecules with Oxidized Glutathione. J. Org. Chem. 1992, 57, 123-127. 5. Rabenstein, D. L.; Yeo, P. L. Kinetics and Equilibria of the Formation and Reduction of the Disulfide Bonds in Arginine Vasopressin and Oxytocin by Thiol/Disulfide Interchange with Glutathione and Cysteine. J. Org. Chem. 1994, 59, 4223-4229. 6. Millis, K. K.; Weaver, K. H.; Rabenstein, D. L. Oxidation/Reduction Potential of Glutathione. J. Org. Chem. 1993, 58, 4144-4146.


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7. Szajewski, R. P.; Whitesides, G. M. Rate Constants and Equilibrium Constants for Thiol-Disulfide Interchange Reactions Involving Oxidized Glutathione. J. Am. Chem. Soc. 1980, 102, 2011-2026. 8. Szakács, Z.; Noszál, B. Protonation Microequilibrium Treatment of Polybasic Compounds with Any Possible Symmetry. J. Math. Chem. 1999, 26, 139–155. 9. Szakács, Z.; Kraszni, M.; Noszál, B. Determination of Microscopic Acid-Base Parameters from NMR-pH Titrations. Anal. Bioanal. Chem. 2004, 378, 1428-1448. 10. Miller, S. L.; Schlesinger, G. Prebiotic Syntheses of Vitamin Coenzymes: I. Cysteamine and 2-Mercaptoethanesulfonic Acid (Coenzyme M). J. Mol. Evol. 1993, 36, 302-307. 11. Ueland, P. M.; Refsum, H.; Stabler, S. P.; Malinow, M. R.; Andersson, A.; Allen, R. H. Total Homocysteine in Plasma or Serum: Methods and Clinical Applications. Clin. Chem. 1993, 39, 1764-1779. 12. Mirzahosseini, A.; Somlyay, M.; Noszál, B. The Comprehensive Acid-Base Characterization of Glutathione. Chem. Phys. Lett. 2015, 622, 50-56. 13. Mirzahosseini, A.; Noszál, B. The Species- and Site-Specific Acid-Base Properties of Biological Thiols and their Homodisulfides. J. Pharm. Biomed. Anal. 2014, 95, 184192. 14. Szakács, Z.; Hägele, G.; Tyka, R. 1H/31P NMR pH Indicator Series to Eliminate the Glass Electrode in NMR Spectroscopic pKa Determinations. Anal. Chim. Acta. 2004, 522, 247–258. 15. Orgován, G.; Noszál, B. Electrodeless, Accurate pH Determination in Highly Basic Media Using a New Set of 1H NMR pH Indicators. J. Pharm. Biomed. Anal. 2011, 54, 958-964.


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Species-Specific ThiolâDisulfide Equilibrium Constant - American

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