- Email: [email protected]
[4] Kinetics of thiol reactions
[41
KINETICS OF THIOL REACTIONS
45
tion against oxidative damage has been reviewed. 76 Evidence for the view that ascorbate may be about as important a radical sink as oxygen with respect to radical reaction cascades that involve thiyl radicals has been obtained from observations that the ascorbyl radical concentration in skin increases when carbon-centered radicals are induced by ultraviolet photolys i s . 77 This could arise if the radicals are "repaired" by Eq. (1) followed by Eq. (20).
Acknowledgments This work is supported by the Cancer Research Campaignand the Max-Planck-Gesellschaft. 76 D, J. Reed, in "Vitamin E in Health and Disease" (L. Packer and J. Fuchs, eds.), p. 269. Dekker, New York, 1993. 77 g. A. Jurkiewicz and G. R. Buettner, Photochem. Photobiol. 59, 1 (1994).
[4]
Kinetics of Thiol Reactions By
CHRISTIAN SCHONEICH
Introduction Thiols play a role in many biochemical processes such as the detoxification of reactive oxygen species, xenobiotics, or heavy metals, the binding of transition metals in redox-active complexes, and the stabilization of protein structure through the formation of disulfide bonds. In these processes the thiol group can react either as a one-electron reductant or as a nucleophile. Depending on the actual nature of both the process and the substrate the respective rate constants can differ by several orders of magnitude, including diffusion-controlled processes (k ~ 101° M i sec-1 for the reaction of the hydroxyl radical with thiols 1) as well as comparably slow processes (k = 2.3 M 1 sec-1 for the uncatalyzed addition of glutathione to 1-chloro-2,4-dinitrobenzene2). Thiols and their oxidation products, the corresponding disulfides, are related to each other in a redox system that involves the intermediary formation of thiyl free radicals, RS. [reactions (1) and (2)]: 1 G. V. Buxton, C. L. Greenstock, W. P. Hellman, and A. B. Ross, J. Phys. Chem. Ref. Data
17, 513 (1988). 2 S. W. Huskey, W. P. Huskey, and A. Y. H. Lu, J. Am. Chem. Soc. 113, 2283 (1991).
METHODS IN ENZYMOLOGY,VOL. 251
Copyright© 1995by AcademicPress, Inc. All rightsof reproductionin any fOITnreserved.
46
THIYLRADICALS
[41
RSH ~ H ÷ + e- + RS. 2RS. ~ RSSR
(1) (2)
For many kinetic measurements it will be sufficient to follow the reaction of a thiol by monitoring the final formation of a stable disulfide, which can be conveniently done by means of high-performance liquid chromatography (HPLC). However, in some instances it will be necessary to describe accurately the one-electron oxidation of a thiol to its corresponding thiyl radical [reaction (1)], for example, by a free radical. Such measurements require fast techniques, such as pulse radiolysis or laser-flash photolysis, owing to the short half-life for both the formation and subsequent reactions of thiyl free radicals (generally t~/2 < 1 msec). The present chapter discusses some selected examples of (1) the reactions of thiols with reactive oxygen species and free radicals, (2) the nucleophilic addition of thiolate to some electrophiles, and (3) thiol-disulfide exchange reactions, in particular those involved in the formation and cleavage of protein disulfide bonds. The examples permit the discussion of various employed methods and techniques for the kinetic measurements, exhibiting a broad range of time resolution, and the concepts may easily be applied to any kinetic problem of interest. Reactions of Thiols with Reactive Oxygen Species and Free Radicals A prominent function of thiols relies on their ability to transfer hydrogen atoms to carbon-centered radicals [forward reaction (3)]Y a - C ' + RSH ~
- C - H + RS"
(3,-3)
k-3
In radiation biology this reaction is generally referred to as a "repair" reaction, as it serves the chemical repair of carbon-centered free radicals located at the carbohydrate moieties of D N A strands.
Pulse Radiolysis The radiation chemical technique of pulse radiolysis is a convenient means for the investigation of fast radical reactions 4-6 exhibiting a half-life 3 C. von Sonntag and H.-P. Schuchmann, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 409. Plenum, New York, 1990. 3a Reverse reaction is denoted by ( - 3 ) . 4 K.-D. Asmus, this series, Vol. 105, p. 167. 5 L. G. Forni and R. L. Willson, this series, Vol. 105, p. 179. 6 L. K. Patterson, in "Radiation Chemistry" (Farhataziz and M. A. J. Rodgers, eds.), p. 65. VHC, New York, 1987.
[4]
KINETICS OF THIOL REACTIONS
47
on the order of t1/2 -< 1 msec, and has been extensively used to study the fast reactions of carbon-centered radicals from carbohydrates and model alcohols and ethers with various thiols. A detailed description of the technique including experimental setup, dosimetry, and analysis of the results has been reported. 4-6 Pulse radiolysis allows the formation of a carboncentered radical of interest according to the general radiation chemical processes (4)-(7). H 2 O " ~ eaq-, U ", HO -, H2, H202 eaq- + N20 --> N2 + HO- + HO. H. + -C-H--+ H2 + - C . HO. + - C - H -~ H20 + -C"
(4) (5) (6) (7)
At sufficiently high concentration of a hydrocarbon substrate the formation of carbon-centered radicals will be completed within the duration of the electron pulse (ca. 20-200 nsec). The subsequent hydrogen transfer from a thiol to the carbon-centered radical can then be monitored by timeresolved ultraviolet (UV) spectroscopy (see below). For a quantitative formation of carbon-centered radicals, -C., prior to reaction with thiols it must be ensured that k6 [hydrocarbon] > k8 [thiol] and k 7 ]hydrocarbon] >> k9 [thiol]. Rate constants for the reactions (6)-(9) have been derived by pulse radiolysisl: for example, for - C - H (deoxyribose): k6 = 2.9 × 107 M -1 sec -1 and kv = 2.5 × 109 M -1 secM; for RSH (cysteine): k8 = 1.0 × 109 M -1 sec -1 and k9 = 3.4 × 10l° M -1 sec 1. H. + R S H - ~ H2 + RS.
H O . + RSH --->H 2 0 q- R S .
(8) (9)
The hydrogen transfer processes according to the forward reaction (3) occur with rate constants on the order of 106-108 M -1 sec -1, depending on the structure of the carbon-centered radical. 7-1° It should be pointed out, however, that not all carbon-centered radicals do react with thiols. This applies particularly to highly stabilized radicals such as the cyclohexadienyl n and pentadienyl radicals, a2 with the respective rate constants for the forward reaction (3) being k3 < 105 M -a sec -1. In fact, in the latter systems the reverse reaction ( - 3 ) becomes far more important, with rate constants 7 G. E. Adams and R. L. Willson, J. Chem. Soc. Faraday Trans. 1, 719 (1973). 8 M. S. Akhlaq, S. A1-Baghdadi, and C. yon Sonntag, Carbohydr. Res. 164, 71 (1987). 9 M. S. Akhlaq, H.-P. Schuchmann, and C. von Sonntag, Int. J. Radiat. BioL 51, 91 (1987), 10 C. von Sonntag, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 359. Plenum, New York, 1990. 11 X.-M. Pan, E. Bastian, and C. von Sonntag, Z. Naturforsch. B: Chem. Sci. 43B, 1201 (1988). 12 C. Sch6neich, U. Dillinger, F. von Bruchhausen, and K.-D. Asmus, Arch. Biochem. Biophys. 292, 456 (1992).
48
THIYLRADICALS
[41
k-3 in the range of 7 × 106-7 × 10 7 M -a sec-1.12-14 For comparison, the rate constants for the hydrogen abstraction by thiyl radicals from simple organic ethers and alcohols are on the order of 103-104 M -~ sec-1. 9,14'15 Thiyl radicals, RS., are characterized by absorption spectra exhibiting Ama x ~ 330 nm with relatively weak extinction coefficients between e330 = 5 8 0 M -1 s e c -1 (Ref. 16) (glutathione thiyl radical, GS .) and e330 = 1200 M -I cm -~ (Refs. 17 and 18) (penicillamine thiyl radical, PenS .). A quantitative detection of thiyl radicals requires that they be formed at low concentrations, as they suffer rapid bimolecular combination [reaction (2); 2k2 ca. 2 × 109 M -~ s e c -1 (Ref. 15)]. This, together with the low absorptivity, imposes an experimental limit on the direct detection of thiyl free radicals by pulse radiolysis coupled to time-resolved UV spectroscopy. There are several indirect methods, however, that can be used for their quantification. Thiyl radicals form complexes with thiolate anions according to equilibrium Eq. (10)19: RS. + -SR ,
" [RS.'.SR]
(10)
These complexes strongly absorb near/in the visible region with Area×being located between 390 and 500 nm, exhibiting extinction coefficients between 2990 and 9200 M -t cm-1,1°'19 depending on the actual structure of the thiol. The presence of sufficient amounts of deprotonated thiol can thus be utilized for a more sensitive, although indirect, detection of generated thiyl radicals. It should be noted, however, that many carbon-centered radicals do not react or react only slowly with thiolate anions. Therefore, the derivation of rate constants for thiyl radical formation at conditions close to the pKa of the mercapto group requires the calculation of the actual fraction of protonated thiol in the solution. A word of caution also with respect to molecular oxygen: both thiyl free radicals 2°,2t as well as their complexes with thiolate anions 22 react with molecular oxygen [reactions (11) and (12); for glutathione Kll = 3.2 × 103 M -1 (Ref. 20); for the lipoic acid radical 13 C. Sch0neich, K.-D. Asrnus, U. Dillinger, and F. yon Bruchhausen, Biochem. Biophys. Res. Commun. 161, 113 (1989). 14 C. SchOneich, M. Bonifacic, U. Dillinger, and K.-D. Asrnus, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 367. Plenum, New York, 1990. 15 C. SchOneich, M. BonifaN6, and K.-D. Asmus, Free Radical Res. Commun. 6, 393 (1989). 16 M. Z. Hoffrnan and E. Hayon, J. Phys. Chem. 77, 990 (1973). 17 j. W. Purdie, H. A. Gillis, and N. V. Klassen, J. Chem. Soc., Chem. Commun., 1163 (1971). 18j. W. Purdie, H. A. Gillis, and N. V. Klassen, Can. J. Chem. 51, 3132 (1973). 19 M. G6bl, M. Bonifacic, and K.-D. Asrnus, J. Am, Chem. Soe. 106, 5984 (1984). 20 M. Tarnba, G. Simone, and M. Quintiliani, Int. J. Radiat. Biol. 50, 595 (1986). 21 j. M0nig, K.-D. Asrnus, L. G. Forni, and R. L. Willson, Int. J. Radiat. Biol. 52, 589 (1987). 22 R. L. Willson, J. Chem. Soc., Chem. Commun., 1425 (1970).
[4]
KINETICS OF THIOL REACTIONS
49
a n i o n k12 = 9 × 108 M -1 sec -I (Ref. 22)]. Thus the contamination of solutions with molecular oxygen could eventually lead to an underestimation of the actual yields of thiyl radicals detected directly or indirectly.
RS. + O2 ~,~ R S O O . [RS.'.SR] + O2 ~ RSSR + 02-
(11) (12)
Pulse radiolysis in combination with time-resolved UV spectroscopy has also been used to measure the rate constants for the reaction of various other free radicals with thiol and/or thiolate groups, such as tert-butoxyl radicals (tert-BuO .),23 monomeric (>S +. ) and dimeric ([>S.'.S<] +) sulfur radical cations, 24 and phenoxyl radicals (PhO .).25
Stopped-Flow Measurements Nitric oxide, NO-, is an important cellular messenger that often acts via nitrosylation of protein thiol g r o u p s . 26 Superoxide anion, 02 ~, rapidly reacts with NO. to form peroxynitrite [reaction (13)], 26 which exists in equilibrium [Eq. (14)] with its protonated form, peroxynitrous acid (pKa o f 6.827).
NO" + 027 ~ O z N - O - O O=N-O-O + H + ~-- O - N - O - O - H
(13) (14)
Both species, peroxynitrite and peroxynitrous acid, are strong oxidants that have been shown to oxidize methionine, 28 lipids, 29 and sulfhydryl groups) ° The oxidation of free glutathione and protein sulfhydryls may contribute to the biologically harmful role of peroxynitrite because it affects the cellular antioxidant status as well as promotes protein cross-linking or inactivation. There is, therefore, current interest in the determination of absolute rate constants for and the characterization of the mechanism of the reaction of peroxynitrite/peroxynitrous acid with biothiols. The synthesis of peroxynitrite involves the addition of an ice-cooled acidic hydrogen peroxide solution (0.7 M H202 in 0.6 M HC1) to an icecooled 0.6 M sodium nitrite (NaNO2) solution followed by rapid quenching 23 M. Erben-Russ, C. Michel, W. Bors, and M. Saran, J. Phys. Chem. 91, 2362 (1987). 24 M. Bonifa~i6 and K.-D. Asmus, Int. J. Radiat. Biol. 46, 35 (1984). as M. J. Davies, L. G. Forni, and R. L. Willson, Biochem. J. 255, 513 (1988). 26 j. S. Stamler, D. J. Singel, and J. Loscalzo, Science 258, 1898 (1992). 27 W. H. Koppenol, J. J. Moreno, W. A. Pryor, H. Ischiropoulos, and J. S. Beckman, Chem. Res. Toxicol. 5, 834 (1992). z8 j. j. Moreno and W. A. Pryor, Chem. Res. Toxicol. 5, 425 (1992). 29 R. Radi, J. S. Beckman, K. M. Bush, and B. A, Freeman, Arch. Biochem. Biophys. 288, 481 (1991). 3o R. Radi, J. S. Beckman, K. M. Bush, and B. A. Freeman, J. Biol. Chem. 266, 4244 (1991).
50
THIYLRADICALS
[41
of the reaction mixture with ice-cooled 1.5 M NaOH. 31 When synthesized in a quenched flow reactor peroxynitrite has been obtained in yields of up to 82%. 92In contrast, the synthesis by mixing of the reactants in conventional glassware afforded yields between 45 and 50%. 3l Residual hydrogen peroxide can be removed by passage of the peroxynitrite solution through an MnO~ column, although it has been pointed out that this process may lead to the contamination of the solution by metals. 27 The alkaline peroxynitrite solution further contains nitrate (a decomposition product of peroxynitrite) and small residual amounts of nitrite. Peroxynitrite solutions can be stored at alkaline p H in the cold. The direct measurement of rate constants for the reactions of peroxynitrite/peroxynitrous acid with biological thiols at physiological p H is complicated by the fact that peroxynitrous acid in particular is unstable, suffering unimolecular decomposition with a rate constant of 4.5 sec -1 at 37 ° (corresponding to a half-life of 0.15 sec). 27 Therefore, such measurements afford the stopped-flow technique, which is generally used for the investigation of reactions exhibiting a half-life between 1 msec and a few seconds (for a more recent description of the technique see Ref. 33). For recording the reaction of peroxynitrite/peroxynitrous acid with thiols the disappearance of the peroxynitrite anion as a function of thiol concentration can be monitored at 302 nm [e302 -- 1670 _+ 50 M -1 cm -~ (Ref. 31)]. The respective rate constants for the oxidation of cysteine and the bovine serum albumin (BSA) sulfhydryl group by peroxynitrite anion were determined to be k15 = 5.9 × 103 M -1 sec -1 and k16 = (2.6-2.8) × 103 M -1 sec -1, respectively. 3° O~N-O-O+ CysSH --+ CysSSCys + products O=N-O-O+ B S A - S H ~ products (no disulfides)
(15) (16)
Product studies indicate that cystine is the major oxidation product from cysteine (>90% yield) whereas intermolecular disulfide formation was not observed for B S A 9 BSA oxidation also did not yield considerable yields of sulfenic acid (RSOH). 3° Thus, either the mechanism or the extent of BSA oxidation differs considerably from the peroxynitrite-mediated oxidation of the single amino acid cysteine. As a result of its fast reaction with peroxynitrite cysteine was found to inhibit the peroxynitrite-induced oxidation of luminol. 34 An example for thiols being involved in the redox cycling of redoxsensitive drugs is provided by the reaction of glutathione with aminopyrine 31M. N. Hughes and H. G. Nicklin, J. Chem. Soc. A, 450 (1968). 32j. W. Reed, H. H. Ho, and W. L. Jolly, J. A m . Chem. Soc. 96, 1248 (1974). 33H. Strehlow, "Rapid Reactions in Solution." VCH, New York, 1992. 34R. Radi, T. P. Cosgrove, J. S. Beckman, and B. A. Freeman, Biochem. J. 290, 51 (1993).
[4]
KINETICS OF THIOL REACTIONS
51
radical cations. The absolute rate constant for this reaction (17) has been measured with the stopped-flow techniqueY Aminopyrine, A P [4-(dimethylamino)-l,2-dihydro-l,5-dimethyl-2-phenyl-3H-pyrazol-3-one], is a common drug that undergoes enzymatic oxidation (by horseradish peroxidase 36 and prostaglandin H synthase 37) to the respective radical cation, A P +. . The UV/Vis spectrum of the latter shows two distinct maxima at 325 nm [e325 -- 5350 M -1 cm -1 (Ref. 38)] and 570 nm [e570 = 1760 M -~ cm -1 (Ref. 35) to 1820 M -1 cm -1 (Ref. 38)] and the enzymatic oxidation process can be conveniently monitored by conventional UV/Vis spectroscopy. In the absence of thiols the A P +. radical cation decomposes with 2k = 52 M -1 sec -1 corresponding to a first half-life of ca. 11 rain at [AP +. ]o = 30 ~M. 3s For the investigation of the reaction of A P +. with thiols the A P +. radical cation was produced by steady state 3' radiolysis and subsequently mixed with deoxygenated solutions of glutathione in a stopped-flow spectrophotometer. A plot of the bleaching of the 570 nm absorption of AP +' vs. glutathione concentration yielded a rate constant of k17 = (2-3) × 104 M 1 sec -1 (Ref. 35) (hi2 = 120 msec for [GSH] = 0.2 m M ) . On the other hand, pulse radiolytic investigations have shown that glutathione thiyl radicals, GS., oxidize A P with k_17 = (2.5-3.0) × 10 a M -1 s e c 1,35,38 that is, with a rate constant being four orders of magnitude larger. k17 G S H + A P +. . " GS. + A P + H + (17, - 1 7 ) k 17
Thermodynamically the equilibrium (17, - 1 7 ) is, therefore, expected to be located well on the left-hand side. The overall disappearance of the AP + radical cation in the presence of G S H has thus to be ascribed to the fact that a continuous consumption of GS. radicals via different routes (most probably via 2GS. ~ GSSG; 2k ~ 2.0 × 109 M -1 sec -1) occurs much faster than a removal of A P +. (via biomolecular reaction; 2k = 52 M < sec-1), resulting in a shift of the equilibrium (17, - 1 7 ) onto the right-hand side.
Electron Spin Resonance The reactions of thiols with various free radicals and reactive oxygen species have been followed by electron spin resonance (ESR).39,4° In partic3s I. Wilson, P. Wardman, G. M. Cohen, and M. D'Arcy Doherty, Biochem. PharmacoL 35,
21 (1986). 36 R. P. Mason, Free Radicals Biol. 5, 161 (1982). 37 T. E. Eling, R. P. Mason, and K. Sivarajah, J. Biol. Chem. 260, 1601 (1985). 38 L. G. Forni, V. O. Mora-Arellano, J. E. Packer, and R. L. Willson, J. Chem. Soc., Perkin Trans. 2, 1579 (1988). 39 B. C. Gilbert, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 135. Plenum, New York, 1990. 4o M. D. Sevilla, D. Becket, and M. Yan, Int. J. Radiat. BioL 57, 65 (1990).
52
THIYLaaDICALS
[41
ular the formation of sulfinyl radicals (RSO .), sulfonyl radicals (RSO2.), and thiyl peroxyl radicals (RSOO .) have been subject to extensive investigation whereas ESR spectra of the thiyl radical itself are generally hardly observable or difficult to analyze. It should be noted, however, that ESR spectroscopy is used predominantly for identification rather than for kinetic experiments involving thiols. Nevertheless, the reactions of nucleic acid peroxyl radicals with cysteamine, glutathione, and dithiothreitol have been studied by time-resolved ESR spectroscopy. 41 The respective peroxyl radicals were generated by in situ photolysis of hydrogen peroxide, yielding hydroxyl radicals, in the presence of nucleic acid and molecular oxygen [reactions (18)-(20)]. hv
H202 .> 2HO. H O . + (nucleic acid)C-H ----+ H20 + (nucleic acid)C. (Nucleic acid)C. + 02 ----+ (nucleic a c i d ) C - O - O .
(18) (19) (20)
The lifetimes of the ESR signals of the respective nucleic acid peroxyl radicals (tl/2 = 0.2-3.3 sec in the absence of thiols) were monitored as a function of thiol concentration, yielding bimolecular rate constants on the order of 0.8 × 104-1.3 x 105 M -1 sec -1 for the three thiols, respectively. There is, however, some uncertainty as to whether this reaction involves hydrogen transfer or the formation of sulfinyl radicals according to reactions (21) and (22), respectively. (Nucleic a c i d ) C - O - O . + RSH--+ (nucleic a c i d ) C - O - O - H + RS. (Nucleic a c i d ) C - O - O . + R S H - + (nucleic acid)C-OH + RSO.
(21) (22)
Thiol as Nucleophile C o n v e n t i o n a l Ultraviolet S p e c t r o s c o p y
In the deprotonated form thiols are strong nucleophiles that react with a variety of electrophiles such as, for example, nitroaromatics or quinones. Most of these reactions can be studied under conditions in which the halflife is on the order of minutes. Therefore, they can be conveniently monitored by conventional UV/Vis spectroscopy provided that the electrophiles or their reaction products are chromophores. In an interesting study Huskey et aL 2 measured the solvent kinetic isotope effects for the reaction of glutathione with 1-chloro-2,4-dinitrobenzene in the absence and presence of two isoforms of glutathione transferase. Solvent kinetic isotope effects close to 41D. Schulte-Frohlinde, G. Behrens, and A. Onal, Int. J. Radiat. B(oL S0, 103 (1986).
[4]
53
KINETICS OF THIOL REACTIONS
unity or of an inverse form were rationalized in terms of hydrogen bond changes about the thiolate anion involved in the reaction. Thiol-Disulfide Exchange The thiol-disulfide exchange reaction (23) has been extensively used for the investigation of the kinetics of protein folding involving the formation of disulfide bonds (for an overview see Ref. 42). I
I
R - S S - R + HS-protein-SH ~ 2 R - S H + S-protein-S
(23)
Oxidative protein folding is initiated by reacting a protein, present in the reduced state, with low molecular weight disulfides such as glutathione disulfide or oxidized dithiothreitol. The reverse reaction, reductive unfolding, requires the interaction of the disulfide-containing protein with a reduced small molecular weight thiol. Protein folding is generally studied at alkaline pH ~ 8.7, at which a significant fraction of the involved reduced thiols exists in the deprotonated state: first, the exchange kinetics are pH dependent, with the exchange reactions occurring faster at elevated pH. Second, it has been argued that an unfolded protein carrying deprotonated, negatively charged thiolate groups would tend to expose these toward the aqueous solvent whereas protonated sulfhydryls are somewhat more hydrophobic and may be partially buried. Therefore, the initiation of folding with deprotonated sulfhydryl groups would ensure their accessibility from the bulk solution by the oxidizing disulfide. The major strategy for determining thiol/disulfide exchange kinetics involves the start of the reaction at alkaline pH and the subsequent quenching of the reaction at various times by the addition of (1) acid or (2) a reagent that rapidly couples with unreacted thiolate g r o u p s . 42 The quenching of reactions by acid has been criticized because such a process is reversible and thiol/disulfide exchange might continue during an analytical procedure employed for the quantification of both thiols and disulfides. Reagents for the irreversible removal of unreacted thiolate groups include electrophiles such as iodoacetate or iodoacetamide, which rapidly add thiolate by displacement of iodide [reaction (24)]. RS- +
I - C H z C O 2 - ---> RS-CH2CO2-
+ I-
(24)
The method of thiol alkylation for determining folding kinetics and the characterization of various possible disulfide-containing intermediates 42 T. E. Creighton, in "Protein Folding" (T. E. Creighton, ed.), p. 301. Freeman, New York, 1992.
54
THIYLRADICALS
[41
should, however, also be used with care. It has been shown for bovine pancreatic trypsin inhibitor (BPTI) that the population of various disulfide intermediates was dependent on the concentration of employed trapping agent. 43 This observation has been rationalized by intramolecular disulfide rearrangements within the protein occurring on the same scale as the trapping process. High-Performance Liquid Chromatography and Capillary Electrophoresis
The high-resolution analytical techniques high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are the tools of choice for the characterization and quantification of protein-folding intermediates as well as the reduced and oxidized forms of the low molecular weight thiols/disulfides employed for the initiation of the folding/unfolding process. ~2-46The separation of all the components of the folding/unfolding experiments will most likely be successful by employing reversed-phase chromatography with gradient elution [mobile phase A, 0.1% trifluoroacetic acid in H20; mobile phase B, 0.1% trifluoroacetic acid in acetonitrile-H20 (90:10, v/v)]. A linear gradient should be started with low contents of mobile phase B to allow the separation of reduced and oxidized glutathione or dithiothreitol, respectively, whereas the elution of the larger folding intermediates should generally require higher volumes of mobile phase B. The use of trifluoroacetic acid as ion pairing agent has the advantage of (1) low pH to suppress oxidative protein folding on the column during the analysis, and of (2) volatility to ensure removal during lyophilization of a pooled HPLC fraction. If necessary, other ion-pairing agents such as HC1 (forms less hydrophobic) or perfluorobutyric acid (forms more hydrophobic peptide-ion-pairing agent complexes) may be used for the separation of the peptide/protein-folding intermediates. If the reversed-phase chromatography does not lead to satisfactory resolution of peptide/protein-folding intermediates other techniques such as ion-exchange chromatography 42 or capillary electrophoresis may be employed (for a comprehensive overview of methods for peptide/protein separations see Ref. 47). To assist in the optimization of the separation conditions for polypeptides, computer programs have been developed for the prediction of peptide retention volum43j. S. Weissman and P. S. Kim, Science 253, 1386 (1991). 44D. M. Rothwarf and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 89, 7944 (1992). 45p. L. Yeo and D. L. Rabenstein, Anal Chem. 65, 306l (1993). 46M. Wunderlich, A. Otto, R. Seekler, and R. Glockshuber,Biochemistry 32, 12251 (1993). 47M. T. W. Hearn (ed.), "HPLC of Proteins, Peptides, and Polynucleotides."VCH, New York, 1991.
[5]
PERTHIOLS AS ANTIOXIDANTS
55
ina. 4s It is also always useful to control the purity of a pooled fraction either by rechromatography under different HPLC conditions or by employing a second analytical technique such as, for example, CE (for an overview see Ref. 49). The respective analytes can be conveniently monitored by UV detection. 42-46 In general, reduced low molecular weight thiols and peptides (chromophore is the peptide bond) can be detected at h -< 214 rim, if other chromophores within the peptide, such as tryptophan or tyrosine, do not permit detection at higher wavelengths. Disulfide bonds show relatively broad absorption bands between 230 and 260 nm, with the actual Amax being dependent on the substituents in the a-position to the sulfur atoms as well as on the conformation of the disulfide bridge (dihedral angle of the C - S - S - C unit). 5° Oxidized dithiothreitol shows an exceptionally redshifted maximum at 285 nm. Other on-line methods that have been used to quantify sulfhydryls and disulfides include electrochemical detection 51-54 and FAB (fast atom bombardment) mass spectrometry. 5s
48 C. T. Mant, T. W. L. Burke, N. E. Zhou, J. M. R. Parker, and R. S. Hodges, J. Chromatogr. 485, 365 (1990). 49 S. R. Rabel and J. F. Stobaugh, Pharm. Res. 10, 171 (1993). 5o N. A. Rosenthal and G. Oster, J. Am. Chem. Soc. 83, 4445 (1961). 51 M. Ozcimder, A. J, H. Louter, H. Lingeman, W. H. Voogt, R. W. Frei, and M. Bloemendal, J. Chromatogr. 570, 19 (1991). 52 p. LllO, F. Zhang, and R. P. Baldwin, Anal, Chem. 63, 1702 (1991). 53 T. J. O'Shea and S. M. Lunte, Anal. Chem. 65, 247 (1993). 54 L. Dou and I. S. Krull, Anal. Chem. 62, 2599 (1990). 55 y. Sun, P. C. Andrews, and D. L. Smith, J. Protein Chem. 9, 151 (1990).
[51 P e r t h i o l s a s A n t i o x i d a n t s : R a d i c a l - S c a v e n g i n g Prooxidative Mechanisms
and
B y STEVEN A . EVERETT a n d PETER W A R D M A N
Introduction Thiols (RSH) are recognized for their radical-scavenging role in protection against cellular oxidative stress 1,2 and in the repair of radical-induced 1 T. M. Buttke, Immunol. Today 15, 1 (1994). 2 C. C. Winterbourn, Free Radical Biol. Med. 14, 85 (1993).
METHODS IN ENZYMOLOGY, VOL. 251
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
KINETICS OF THIOL REACTIONS
45
tion against oxidative damage has been reviewed. 76 Evidence for the view that ascorbate may be about as important a radical sink as oxygen with respect to radical reaction cascades that involve thiyl radicals has been obtained from observations that the ascorbyl radical concentration in skin increases when carbon-centered radicals are induced by ultraviolet photolys i s . 77 This could arise if the radicals are "repaired" by Eq. (1) followed by Eq. (20).
Acknowledgments This work is supported by the Cancer Research Campaignand the Max-Planck-Gesellschaft. 76 D, J. Reed, in "Vitamin E in Health and Disease" (L. Packer and J. Fuchs, eds.), p. 269. Dekker, New York, 1993. 77 g. A. Jurkiewicz and G. R. Buettner, Photochem. Photobiol. 59, 1 (1994).
[4]
Kinetics of Thiol Reactions By
CHRISTIAN SCHONEICH
Introduction Thiols play a role in many biochemical processes such as the detoxification of reactive oxygen species, xenobiotics, or heavy metals, the binding of transition metals in redox-active complexes, and the stabilization of protein structure through the formation of disulfide bonds. In these processes the thiol group can react either as a one-electron reductant or as a nucleophile. Depending on the actual nature of both the process and the substrate the respective rate constants can differ by several orders of magnitude, including diffusion-controlled processes (k ~ 101° M i sec-1 for the reaction of the hydroxyl radical with thiols 1) as well as comparably slow processes (k = 2.3 M 1 sec-1 for the uncatalyzed addition of glutathione to 1-chloro-2,4-dinitrobenzene2). Thiols and their oxidation products, the corresponding disulfides, are related to each other in a redox system that involves the intermediary formation of thiyl free radicals, RS. [reactions (1) and (2)]: 1 G. V. Buxton, C. L. Greenstock, W. P. Hellman, and A. B. Ross, J. Phys. Chem. Ref. Data
17, 513 (1988). 2 S. W. Huskey, W. P. Huskey, and A. Y. H. Lu, J. Am. Chem. Soc. 113, 2283 (1991).
METHODS IN ENZYMOLOGY,VOL. 251
Copyright© 1995by AcademicPress, Inc. All rightsof reproductionin any fOITnreserved.
46
THIYLRADICALS
[41
RSH ~ H ÷ + e- + RS. 2RS. ~ RSSR
(1) (2)
For many kinetic measurements it will be sufficient to follow the reaction of a thiol by monitoring the final formation of a stable disulfide, which can be conveniently done by means of high-performance liquid chromatography (HPLC). However, in some instances it will be necessary to describe accurately the one-electron oxidation of a thiol to its corresponding thiyl radical [reaction (1)], for example, by a free radical. Such measurements require fast techniques, such as pulse radiolysis or laser-flash photolysis, owing to the short half-life for both the formation and subsequent reactions of thiyl free radicals (generally t~/2 < 1 msec). The present chapter discusses some selected examples of (1) the reactions of thiols with reactive oxygen species and free radicals, (2) the nucleophilic addition of thiolate to some electrophiles, and (3) thiol-disulfide exchange reactions, in particular those involved in the formation and cleavage of protein disulfide bonds. The examples permit the discussion of various employed methods and techniques for the kinetic measurements, exhibiting a broad range of time resolution, and the concepts may easily be applied to any kinetic problem of interest. Reactions of Thiols with Reactive Oxygen Species and Free Radicals A prominent function of thiols relies on their ability to transfer hydrogen atoms to carbon-centered radicals [forward reaction (3)]Y a - C ' + RSH ~
- C - H + RS"
(3,-3)
k-3
In radiation biology this reaction is generally referred to as a "repair" reaction, as it serves the chemical repair of carbon-centered free radicals located at the carbohydrate moieties of D N A strands.
Pulse Radiolysis The radiation chemical technique of pulse radiolysis is a convenient means for the investigation of fast radical reactions 4-6 exhibiting a half-life 3 C. von Sonntag and H.-P. Schuchmann, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 409. Plenum, New York, 1990. 3a Reverse reaction is denoted by ( - 3 ) . 4 K.-D. Asmus, this series, Vol. 105, p. 167. 5 L. G. Forni and R. L. Willson, this series, Vol. 105, p. 179. 6 L. K. Patterson, in "Radiation Chemistry" (Farhataziz and M. A. J. Rodgers, eds.), p. 65. VHC, New York, 1987.
[4]
KINETICS OF THIOL REACTIONS
47
on the order of t1/2 -< 1 msec, and has been extensively used to study the fast reactions of carbon-centered radicals from carbohydrates and model alcohols and ethers with various thiols. A detailed description of the technique including experimental setup, dosimetry, and analysis of the results has been reported. 4-6 Pulse radiolysis allows the formation of a carboncentered radical of interest according to the general radiation chemical processes (4)-(7). H 2 O " ~ eaq-, U ", HO -, H2, H202 eaq- + N20 --> N2 + HO- + HO. H. + -C-H--+ H2 + - C . HO. + - C - H -~ H20 + -C"
(4) (5) (6) (7)
At sufficiently high concentration of a hydrocarbon substrate the formation of carbon-centered radicals will be completed within the duration of the electron pulse (ca. 20-200 nsec). The subsequent hydrogen transfer from a thiol to the carbon-centered radical can then be monitored by timeresolved ultraviolet (UV) spectroscopy (see below). For a quantitative formation of carbon-centered radicals, -C., prior to reaction with thiols it must be ensured that k6 [hydrocarbon] > k8 [thiol] and k 7 ]hydrocarbon] >> k9 [thiol]. Rate constants for the reactions (6)-(9) have been derived by pulse radiolysisl: for example, for - C - H (deoxyribose): k6 = 2.9 × 107 M -1 sec -1 and kv = 2.5 × 109 M -1 secM; for RSH (cysteine): k8 = 1.0 × 109 M -1 sec -1 and k9 = 3.4 × 10l° M -1 sec 1. H. + R S H - ~ H2 + RS.
H O . + RSH --->H 2 0 q- R S .
(8) (9)
The hydrogen transfer processes according to the forward reaction (3) occur with rate constants on the order of 106-108 M -1 sec -1, depending on the structure of the carbon-centered radical. 7-1° It should be pointed out, however, that not all carbon-centered radicals do react with thiols. This applies particularly to highly stabilized radicals such as the cyclohexadienyl n and pentadienyl radicals, a2 with the respective rate constants for the forward reaction (3) being k3 < 105 M -a sec -1. In fact, in the latter systems the reverse reaction ( - 3 ) becomes far more important, with rate constants 7 G. E. Adams and R. L. Willson, J. Chem. Soc. Faraday Trans. 1, 719 (1973). 8 M. S. Akhlaq, S. A1-Baghdadi, and C. yon Sonntag, Carbohydr. Res. 164, 71 (1987). 9 M. S. Akhlaq, H.-P. Schuchmann, and C. von Sonntag, Int. J. Radiat. BioL 51, 91 (1987), 10 C. von Sonntag, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 359. Plenum, New York, 1990. 11 X.-M. Pan, E. Bastian, and C. von Sonntag, Z. Naturforsch. B: Chem. Sci. 43B, 1201 (1988). 12 C. Sch6neich, U. Dillinger, F. von Bruchhausen, and K.-D. Asmus, Arch. Biochem. Biophys. 292, 456 (1992).
48
THIYLRADICALS
[41
k-3 in the range of 7 × 106-7 × 10 7 M -a sec-1.12-14 For comparison, the rate constants for the hydrogen abstraction by thiyl radicals from simple organic ethers and alcohols are on the order of 103-104 M -~ sec-1. 9,14'15 Thiyl radicals, RS., are characterized by absorption spectra exhibiting Ama x ~ 330 nm with relatively weak extinction coefficients between e330 = 5 8 0 M -1 s e c -1 (Ref. 16) (glutathione thiyl radical, GS .) and e330 = 1200 M -I cm -~ (Refs. 17 and 18) (penicillamine thiyl radical, PenS .). A quantitative detection of thiyl radicals requires that they be formed at low concentrations, as they suffer rapid bimolecular combination [reaction (2); 2k2 ca. 2 × 109 M -~ s e c -1 (Ref. 15)]. This, together with the low absorptivity, imposes an experimental limit on the direct detection of thiyl free radicals by pulse radiolysis coupled to time-resolved UV spectroscopy. There are several indirect methods, however, that can be used for their quantification. Thiyl radicals form complexes with thiolate anions according to equilibrium Eq. (10)19: RS. + -SR ,
" [RS.'.SR]
(10)
These complexes strongly absorb near/in the visible region with Area×being located between 390 and 500 nm, exhibiting extinction coefficients between 2990 and 9200 M -t cm-1,1°'19 depending on the actual structure of the thiol. The presence of sufficient amounts of deprotonated thiol can thus be utilized for a more sensitive, although indirect, detection of generated thiyl radicals. It should be noted, however, that many carbon-centered radicals do not react or react only slowly with thiolate anions. Therefore, the derivation of rate constants for thiyl radical formation at conditions close to the pKa of the mercapto group requires the calculation of the actual fraction of protonated thiol in the solution. A word of caution also with respect to molecular oxygen: both thiyl free radicals 2°,2t as well as their complexes with thiolate anions 22 react with molecular oxygen [reactions (11) and (12); for glutathione Kll = 3.2 × 103 M -1 (Ref. 20); for the lipoic acid radical 13 C. Sch0neich, K.-D. Asrnus, U. Dillinger, and F. yon Bruchhausen, Biochem. Biophys. Res. Commun. 161, 113 (1989). 14 C. SchOneich, M. Bonifacic, U. Dillinger, and K.-D. Asrnus, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 367. Plenum, New York, 1990. 15 C. SchOneich, M. BonifaN6, and K.-D. Asmus, Free Radical Res. Commun. 6, 393 (1989). 16 M. Z. Hoffrnan and E. Hayon, J. Phys. Chem. 77, 990 (1973). 17 j. W. Purdie, H. A. Gillis, and N. V. Klassen, J. Chem. Soc., Chem. Commun., 1163 (1971). 18j. W. Purdie, H. A. Gillis, and N. V. Klassen, Can. J. Chem. 51, 3132 (1973). 19 M. G6bl, M. Bonifacic, and K.-D. Asrnus, J. Am, Chem. Soe. 106, 5984 (1984). 20 M. Tarnba, G. Simone, and M. Quintiliani, Int. J. Radiat. Biol. 50, 595 (1986). 21 j. M0nig, K.-D. Asrnus, L. G. Forni, and R. L. Willson, Int. J. Radiat. Biol. 52, 589 (1987). 22 R. L. Willson, J. Chem. Soc., Chem. Commun., 1425 (1970).
[4]
KINETICS OF THIOL REACTIONS
49
a n i o n k12 = 9 × 108 M -1 sec -I (Ref. 22)]. Thus the contamination of solutions with molecular oxygen could eventually lead to an underestimation of the actual yields of thiyl radicals detected directly or indirectly.
RS. + O2 ~,~ R S O O . [RS.'.SR] + O2 ~ RSSR + 02-
(11) (12)
Pulse radiolysis in combination with time-resolved UV spectroscopy has also been used to measure the rate constants for the reaction of various other free radicals with thiol and/or thiolate groups, such as tert-butoxyl radicals (tert-BuO .),23 monomeric (>S +. ) and dimeric ([>S.'.S<] +) sulfur radical cations, 24 and phenoxyl radicals (PhO .).25
Stopped-Flow Measurements Nitric oxide, NO-, is an important cellular messenger that often acts via nitrosylation of protein thiol g r o u p s . 26 Superoxide anion, 02 ~, rapidly reacts with NO. to form peroxynitrite [reaction (13)], 26 which exists in equilibrium [Eq. (14)] with its protonated form, peroxynitrous acid (pKa o f 6.827).
NO" + 027 ~ O z N - O - O O=N-O-O + H + ~-- O - N - O - O - H
(13) (14)
Both species, peroxynitrite and peroxynitrous acid, are strong oxidants that have been shown to oxidize methionine, 28 lipids, 29 and sulfhydryl groups) ° The oxidation of free glutathione and protein sulfhydryls may contribute to the biologically harmful role of peroxynitrite because it affects the cellular antioxidant status as well as promotes protein cross-linking or inactivation. There is, therefore, current interest in the determination of absolute rate constants for and the characterization of the mechanism of the reaction of peroxynitrite/peroxynitrous acid with biothiols. The synthesis of peroxynitrite involves the addition of an ice-cooled acidic hydrogen peroxide solution (0.7 M H202 in 0.6 M HC1) to an icecooled 0.6 M sodium nitrite (NaNO2) solution followed by rapid quenching 23 M. Erben-Russ, C. Michel, W. Bors, and M. Saran, J. Phys. Chem. 91, 2362 (1987). 24 M. Bonifa~i6 and K.-D. Asmus, Int. J. Radiat. Biol. 46, 35 (1984). as M. J. Davies, L. G. Forni, and R. L. Willson, Biochem. J. 255, 513 (1988). 26 j. S. Stamler, D. J. Singel, and J. Loscalzo, Science 258, 1898 (1992). 27 W. H. Koppenol, J. J. Moreno, W. A. Pryor, H. Ischiropoulos, and J. S. Beckman, Chem. Res. Toxicol. 5, 834 (1992). z8 j. j. Moreno and W. A. Pryor, Chem. Res. Toxicol. 5, 425 (1992). 29 R. Radi, J. S. Beckman, K. M. Bush, and B. A, Freeman, Arch. Biochem. Biophys. 288, 481 (1991). 3o R. Radi, J. S. Beckman, K. M. Bush, and B. A. Freeman, J. Biol. Chem. 266, 4244 (1991).
50
THIYLRADICALS
[41
of the reaction mixture with ice-cooled 1.5 M NaOH. 31 When synthesized in a quenched flow reactor peroxynitrite has been obtained in yields of up to 82%. 92In contrast, the synthesis by mixing of the reactants in conventional glassware afforded yields between 45 and 50%. 3l Residual hydrogen peroxide can be removed by passage of the peroxynitrite solution through an MnO~ column, although it has been pointed out that this process may lead to the contamination of the solution by metals. 27 The alkaline peroxynitrite solution further contains nitrate (a decomposition product of peroxynitrite) and small residual amounts of nitrite. Peroxynitrite solutions can be stored at alkaline p H in the cold. The direct measurement of rate constants for the reactions of peroxynitrite/peroxynitrous acid with biological thiols at physiological p H is complicated by the fact that peroxynitrous acid in particular is unstable, suffering unimolecular decomposition with a rate constant of 4.5 sec -1 at 37 ° (corresponding to a half-life of 0.15 sec). 27 Therefore, such measurements afford the stopped-flow technique, which is generally used for the investigation of reactions exhibiting a half-life between 1 msec and a few seconds (for a more recent description of the technique see Ref. 33). For recording the reaction of peroxynitrite/peroxynitrous acid with thiols the disappearance of the peroxynitrite anion as a function of thiol concentration can be monitored at 302 nm [e302 -- 1670 _+ 50 M -1 cm -~ (Ref. 31)]. The respective rate constants for the oxidation of cysteine and the bovine serum albumin (BSA) sulfhydryl group by peroxynitrite anion were determined to be k15 = 5.9 × 103 M -1 sec -1 and k16 = (2.6-2.8) × 103 M -1 sec -1, respectively. 3° O~N-O-O+ CysSH --+ CysSSCys + products O=N-O-O+ B S A - S H ~ products (no disulfides)
(15) (16)
Product studies indicate that cystine is the major oxidation product from cysteine (>90% yield) whereas intermolecular disulfide formation was not observed for B S A 9 BSA oxidation also did not yield considerable yields of sulfenic acid (RSOH). 3° Thus, either the mechanism or the extent of BSA oxidation differs considerably from the peroxynitrite-mediated oxidation of the single amino acid cysteine. As a result of its fast reaction with peroxynitrite cysteine was found to inhibit the peroxynitrite-induced oxidation of luminol. 34 An example for thiols being involved in the redox cycling of redoxsensitive drugs is provided by the reaction of glutathione with aminopyrine 31M. N. Hughes and H. G. Nicklin, J. Chem. Soc. A, 450 (1968). 32j. W. Reed, H. H. Ho, and W. L. Jolly, J. A m . Chem. Soc. 96, 1248 (1974). 33H. Strehlow, "Rapid Reactions in Solution." VCH, New York, 1992. 34R. Radi, T. P. Cosgrove, J. S. Beckman, and B. A. Freeman, Biochem. J. 290, 51 (1993).
[4]
KINETICS OF THIOL REACTIONS
51
radical cations. The absolute rate constant for this reaction (17) has been measured with the stopped-flow techniqueY Aminopyrine, A P [4-(dimethylamino)-l,2-dihydro-l,5-dimethyl-2-phenyl-3H-pyrazol-3-one], is a common drug that undergoes enzymatic oxidation (by horseradish peroxidase 36 and prostaglandin H synthase 37) to the respective radical cation, A P +. . The UV/Vis spectrum of the latter shows two distinct maxima at 325 nm [e325 -- 5350 M -1 cm -1 (Ref. 38)] and 570 nm [e570 = 1760 M -~ cm -1 (Ref. 35) to 1820 M -1 cm -1 (Ref. 38)] and the enzymatic oxidation process can be conveniently monitored by conventional UV/Vis spectroscopy. In the absence of thiols the A P +. radical cation decomposes with 2k = 52 M -1 sec -1 corresponding to a first half-life of ca. 11 rain at [AP +. ]o = 30 ~M. 3s For the investigation of the reaction of A P +. with thiols the A P +. radical cation was produced by steady state 3' radiolysis and subsequently mixed with deoxygenated solutions of glutathione in a stopped-flow spectrophotometer. A plot of the bleaching of the 570 nm absorption of AP +' vs. glutathione concentration yielded a rate constant of k17 = (2-3) × 104 M 1 sec -1 (Ref. 35) (hi2 = 120 msec for [GSH] = 0.2 m M ) . On the other hand, pulse radiolytic investigations have shown that glutathione thiyl radicals, GS., oxidize A P with k_17 = (2.5-3.0) × 10 a M -1 s e c 1,35,38 that is, with a rate constant being four orders of magnitude larger. k17 G S H + A P +. . " GS. + A P + H + (17, - 1 7 ) k 17
Thermodynamically the equilibrium (17, - 1 7 ) is, therefore, expected to be located well on the left-hand side. The overall disappearance of the AP + radical cation in the presence of G S H has thus to be ascribed to the fact that a continuous consumption of GS. radicals via different routes (most probably via 2GS. ~ GSSG; 2k ~ 2.0 × 109 M -1 sec -1) occurs much faster than a removal of A P +. (via biomolecular reaction; 2k = 52 M < sec-1), resulting in a shift of the equilibrium (17, - 1 7 ) onto the right-hand side.
Electron Spin Resonance The reactions of thiols with various free radicals and reactive oxygen species have been followed by electron spin resonance (ESR).39,4° In partic3s I. Wilson, P. Wardman, G. M. Cohen, and M. D'Arcy Doherty, Biochem. PharmacoL 35,
21 (1986). 36 R. P. Mason, Free Radicals Biol. 5, 161 (1982). 37 T. E. Eling, R. P. Mason, and K. Sivarajah, J. Biol. Chem. 260, 1601 (1985). 38 L. G. Forni, V. O. Mora-Arellano, J. E. Packer, and R. L. Willson, J. Chem. Soc., Perkin Trans. 2, 1579 (1988). 39 B. C. Gilbert, in "Sulfur-Centered Reactive Intermediates in Chemistry and Biology" (C. Chatgilialoglu and K.-D. Asmus, eds.), p. 135. Plenum, New York, 1990. 4o M. D. Sevilla, D. Becket, and M. Yan, Int. J. Radiat. BioL 57, 65 (1990).
52
THIYLaaDICALS
[41
ular the formation of sulfinyl radicals (RSO .), sulfonyl radicals (RSO2.), and thiyl peroxyl radicals (RSOO .) have been subject to extensive investigation whereas ESR spectra of the thiyl radical itself are generally hardly observable or difficult to analyze. It should be noted, however, that ESR spectroscopy is used predominantly for identification rather than for kinetic experiments involving thiols. Nevertheless, the reactions of nucleic acid peroxyl radicals with cysteamine, glutathione, and dithiothreitol have been studied by time-resolved ESR spectroscopy. 41 The respective peroxyl radicals were generated by in situ photolysis of hydrogen peroxide, yielding hydroxyl radicals, in the presence of nucleic acid and molecular oxygen [reactions (18)-(20)]. hv
H202 .> 2HO. H O . + (nucleic acid)C-H ----+ H20 + (nucleic acid)C. (Nucleic acid)C. + 02 ----+ (nucleic a c i d ) C - O - O .
(18) (19) (20)
The lifetimes of the ESR signals of the respective nucleic acid peroxyl radicals (tl/2 = 0.2-3.3 sec in the absence of thiols) were monitored as a function of thiol concentration, yielding bimolecular rate constants on the order of 0.8 × 104-1.3 x 105 M -1 sec -1 for the three thiols, respectively. There is, however, some uncertainty as to whether this reaction involves hydrogen transfer or the formation of sulfinyl radicals according to reactions (21) and (22), respectively. (Nucleic a c i d ) C - O - O . + RSH--+ (nucleic a c i d ) C - O - O - H + RS. (Nucleic a c i d ) C - O - O . + R S H - + (nucleic acid)C-OH + RSO.
(21) (22)
Thiol as Nucleophile C o n v e n t i o n a l Ultraviolet S p e c t r o s c o p y
In the deprotonated form thiols are strong nucleophiles that react with a variety of electrophiles such as, for example, nitroaromatics or quinones. Most of these reactions can be studied under conditions in which the halflife is on the order of minutes. Therefore, they can be conveniently monitored by conventional UV/Vis spectroscopy provided that the electrophiles or their reaction products are chromophores. In an interesting study Huskey et aL 2 measured the solvent kinetic isotope effects for the reaction of glutathione with 1-chloro-2,4-dinitrobenzene in the absence and presence of two isoforms of glutathione transferase. Solvent kinetic isotope effects close to 41D. Schulte-Frohlinde, G. Behrens, and A. Onal, Int. J. Radiat. B(oL S0, 103 (1986).
[4]
53
KINETICS OF THIOL REACTIONS
unity or of an inverse form were rationalized in terms of hydrogen bond changes about the thiolate anion involved in the reaction. Thiol-Disulfide Exchange The thiol-disulfide exchange reaction (23) has been extensively used for the investigation of the kinetics of protein folding involving the formation of disulfide bonds (for an overview see Ref. 42). I
I
R - S S - R + HS-protein-SH ~ 2 R - S H + S-protein-S
(23)
Oxidative protein folding is initiated by reacting a protein, present in the reduced state, with low molecular weight disulfides such as glutathione disulfide or oxidized dithiothreitol. The reverse reaction, reductive unfolding, requires the interaction of the disulfide-containing protein with a reduced small molecular weight thiol. Protein folding is generally studied at alkaline pH ~ 8.7, at which a significant fraction of the involved reduced thiols exists in the deprotonated state: first, the exchange kinetics are pH dependent, with the exchange reactions occurring faster at elevated pH. Second, it has been argued that an unfolded protein carrying deprotonated, negatively charged thiolate groups would tend to expose these toward the aqueous solvent whereas protonated sulfhydryls are somewhat more hydrophobic and may be partially buried. Therefore, the initiation of folding with deprotonated sulfhydryl groups would ensure their accessibility from the bulk solution by the oxidizing disulfide. The major strategy for determining thiol/disulfide exchange kinetics involves the start of the reaction at alkaline pH and the subsequent quenching of the reaction at various times by the addition of (1) acid or (2) a reagent that rapidly couples with unreacted thiolate g r o u p s . 42 The quenching of reactions by acid has been criticized because such a process is reversible and thiol/disulfide exchange might continue during an analytical procedure employed for the quantification of both thiols and disulfides. Reagents for the irreversible removal of unreacted thiolate groups include electrophiles such as iodoacetate or iodoacetamide, which rapidly add thiolate by displacement of iodide [reaction (24)]. RS- +
I - C H z C O 2 - ---> RS-CH2CO2-
+ I-
(24)
The method of thiol alkylation for determining folding kinetics and the characterization of various possible disulfide-containing intermediates 42 T. E. Creighton, in "Protein Folding" (T. E. Creighton, ed.), p. 301. Freeman, New York, 1992.
54
THIYLRADICALS
[41
should, however, also be used with care. It has been shown for bovine pancreatic trypsin inhibitor (BPTI) that the population of various disulfide intermediates was dependent on the concentration of employed trapping agent. 43 This observation has been rationalized by intramolecular disulfide rearrangements within the protein occurring on the same scale as the trapping process. High-Performance Liquid Chromatography and Capillary Electrophoresis
The high-resolution analytical techniques high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are the tools of choice for the characterization and quantification of protein-folding intermediates as well as the reduced and oxidized forms of the low molecular weight thiols/disulfides employed for the initiation of the folding/unfolding process. ~2-46The separation of all the components of the folding/unfolding experiments will most likely be successful by employing reversed-phase chromatography with gradient elution [mobile phase A, 0.1% trifluoroacetic acid in H20; mobile phase B, 0.1% trifluoroacetic acid in acetonitrile-H20 (90:10, v/v)]. A linear gradient should be started with low contents of mobile phase B to allow the separation of reduced and oxidized glutathione or dithiothreitol, respectively, whereas the elution of the larger folding intermediates should generally require higher volumes of mobile phase B. The use of trifluoroacetic acid as ion pairing agent has the advantage of (1) low pH to suppress oxidative protein folding on the column during the analysis, and of (2) volatility to ensure removal during lyophilization of a pooled HPLC fraction. If necessary, other ion-pairing agents such as HC1 (forms less hydrophobic) or perfluorobutyric acid (forms more hydrophobic peptide-ion-pairing agent complexes) may be used for the separation of the peptide/protein-folding intermediates. If the reversed-phase chromatography does not lead to satisfactory resolution of peptide/protein-folding intermediates other techniques such as ion-exchange chromatography 42 or capillary electrophoresis may be employed (for a comprehensive overview of methods for peptide/protein separations see Ref. 47). To assist in the optimization of the separation conditions for polypeptides, computer programs have been developed for the prediction of peptide retention volum43j. S. Weissman and P. S. Kim, Science 253, 1386 (1991). 44D. M. Rothwarf and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 89, 7944 (1992). 45p. L. Yeo and D. L. Rabenstein, Anal Chem. 65, 306l (1993). 46M. Wunderlich, A. Otto, R. Seekler, and R. Glockshuber,Biochemistry 32, 12251 (1993). 47M. T. W. Hearn (ed.), "HPLC of Proteins, Peptides, and Polynucleotides."VCH, New York, 1991.
[5]
PERTHIOLS AS ANTIOXIDANTS
55
ina. 4s It is also always useful to control the purity of a pooled fraction either by rechromatography under different HPLC conditions or by employing a second analytical technique such as, for example, CE (for an overview see Ref. 49). The respective analytes can be conveniently monitored by UV detection. 42-46 In general, reduced low molecular weight thiols and peptides (chromophore is the peptide bond) can be detected at h -< 214 rim, if other chromophores within the peptide, such as tryptophan or tyrosine, do not permit detection at higher wavelengths. Disulfide bonds show relatively broad absorption bands between 230 and 260 nm, with the actual Amax being dependent on the substituents in the a-position to the sulfur atoms as well as on the conformation of the disulfide bridge (dihedral angle of the C - S - S - C unit). 5° Oxidized dithiothreitol shows an exceptionally redshifted maximum at 285 nm. Other on-line methods that have been used to quantify sulfhydryls and disulfides include electrochemical detection 51-54 and FAB (fast atom bombardment) mass spectrometry. 5s
48 C. T. Mant, T. W. L. Burke, N. E. Zhou, J. M. R. Parker, and R. S. Hodges, J. Chromatogr. 485, 365 (1990). 49 S. R. Rabel and J. F. Stobaugh, Pharm. Res. 10, 171 (1993). 5o N. A. Rosenthal and G. Oster, J. Am. Chem. Soc. 83, 4445 (1961). 51 M. Ozcimder, A. J, H. Louter, H. Lingeman, W. H. Voogt, R. W. Frei, and M. Bloemendal, J. Chromatogr. 570, 19 (1991). 52 p. LllO, F. Zhang, and R. P. Baldwin, Anal, Chem. 63, 1702 (1991). 53 T. J. O'Shea and S. M. Lunte, Anal. Chem. 65, 247 (1993). 54 L. Dou and I. S. Krull, Anal. Chem. 62, 2599 (1990). 55 y. Sun, P. C. Andrews, and D. L. Smith, J. Protein Chem. 9, 151 (1990).
[51 P e r t h i o l s a s A n t i o x i d a n t s : R a d i c a l - S c a v e n g i n g Prooxidative Mechanisms
and
B y STEVEN A . EVERETT a n d PETER W A R D M A N
Introduction Thiols (RSH) are recognized for their radical-scavenging role in protection against cellular oxidative stress 1,2 and in the repair of radical-induced 1 T. M. Buttke, Immunol. Today 15, 1 (1994). 2 C. C. Winterbourn, Free Radical Biol. Med. 14, 85 (1993).
METHODS IN ENZYMOLOGY, VOL. 251
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