Morton 2. Hoffman and E. Hayon
990
ed value (Figure 3), of approximately 2.5 X at this concentration. In the absence of base Q . - and Q . + will undergo recombination and, as observed, no esr signal would be detected. This latter process could account for the extremely slow bleaching of the quinone observed in nonbasic media.
Acknowledgment. We are grateful to Anita VanLaeken and Roger Miller for the synthesis of I and 11, and to Mark Bailey for assistance in esr measurements. (11) J. G. Calvert and J. N. Pitts, “Photochemistry,” Wiley, New York. N. Y., 1966, p627. (12) Value estimated from Stern-Volmer quenching data.
Pulse Radialysiis Study of Sulfhydryl Compounds in Aqueous Solution Morton 2. Hoffman’ and E. Hayon* Pioneering Research Laboratory, U. S. Army Natick Laboratories, Natick, Massachusetts (Received October IO, 1972)
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Pubii’cation costs assisted by Natick Laboratories
The interactions of hydrated electrons and hydroxyl radicals with a number of sulfhydryl compounds have been studied using the technique of pulse radiolysis. Thioglycolic acid, P-mercaptopropionic acid, cysteamine, N-acetylcysteamine, cysteine, N-acetylcysteine, 5’-methylcysteine, penicillamine, glutathione, benzyl mercaptan, and thiodiacetic and thiodipropionic acids are among the compounds examined. The reaction rate constants of eaq- with these compounds were found to be strongly dependent on the acid-base properties of the sulfhydryl compounds, with the rate decreasing upon deprotonation of both the amino and the mercapto groups. The eaq- were shown to react with RSH to produce quantitativeiy the Re radical, eaq- RSH R- HS-. The reaction rate constants of OH radicals were found to be -1-3 X 1010 M - 1 sec-l and essentially independent of the ionization constants of the sulfhydryl compounds. The OH radicals form the thiyl radicals, OH RSH RS- HzO. The absorption spectra, extinction coefficients, and decay kinetics of these thiyl radicals have been determined for a number of RSII compounds. The rate Constants of the reactions of RS. radicals with RSH and RS- have been measured, and the equilibrium constants for RS. + RS- F! RSSR- have been calculated for H few systems. The high electrophilicity of the thiyl radical is indicated.
+
-
+
-
+
+
+
Introduction RSOH RS. C OH(3) The role of sulfhydryl (RSH) compounds, thiyl radicals RS. + R S - = RSSR(4) (RS-), and disulfides (RSSR) in radiation protection is Most of these studies have focussed on the formation well known.2~3The protective effect of RSH compounds (e.g., by reaction with OH radicals) and the reaction of (e.g., cysteamine, HSCHzCH2NH2) on irradiation of DNA, enzymes, and! bacteriophage was suggested4 many scientist from the Chemistry Department, Boston Univeryears ago to ,)iGL a free-radical mechanism. conse- (1) V.isiting sity, Boston, Mass., 02215. quently, the of the mechanism of the reac(2) 2. M. Bacq, “Chemical Protection against Ionizing Radiation,” C. C. Thomas Publisher, Springfield, 111., 1965. tion of free radicals and ions with sulfur compounds be(3) A. Hollaender and D. G. Doherty, “Radiation Damage and Sulfhydryl came of considerable importancein radiation and photoCompounds,” International Atomic Energy Agency, Vienna, 1969, p biochemistry. 1. (4) P. Howard-Flanders, Nature (London), 186, 485 (1960); F. HutchinThe steady-state radiation chemistry of cysteine and reson, Radiat. Res., 14, 721 (1961); K. G. Zlrnmer and A. Muller, lated compounds has been studied (see, e.g., ref 5-9 and Curr. Top. Radiat. Res. 1 (1965). references cited therein), and a number of the products (5) V. G. Wilkening, M. Lal, M. Arends, and D. A. Arrnstrong, Can. J. Chem., 45, 1209 (1967). formed in the presence and absence of oxygen were deter(6) A. AI-Thannon, R. M. Peterson, and C. N. Trurnbore, J. Phys. mined. The formation and reactivity of some of the free Chem., 72,2395 (1968). radicals produced have been studied by pulse radiolyois (7) J. A. Packer and R. V. Winchester, Can. J. Chern,, 48,417 (1970). (8) G. G. Jayson, D. A. Stirling, and A. J. Swallow, int. J. Radiat. Biol., (see ref LO -13 and references cited therein). The formation 19,143 (1971). of H characteristic transient optical absorption with a (9) J. W. Purdie, Can. J. Chern., 49, 725 (1971). maximum at -410 nm due to the RSSR- radical anion (IO) W. Karrnann and A. Henglein. Ber. Bunsenges. Phys. Chern., 71, 421 (1967); W. Karmann, A. Granzow, G. Meissner, and A. tienwas shownl0Jl to occur via reactions 1 to 4. Esr studies of glein, Int. J. Radiat. Phys. Chem., 1, 395 (1969). these radicals have also been carried out in aqueous solu(11) (a) G. E. Adams, G. S. McNaughton, and 9. D. Michael in “Chemistry of ionization and Excitation,” G.’R. A. Johnson and G. tion~1~-17 and in the stolid state.18 Scholes, Ed., Taylor and Francis, London, 1967, p 281; (b) Trans. RSH I- C)H RS. + HZO (1) Faraday Soc., 64,902 (1968).
N- S RS-
+
H20
The Journal of Physical Chemistry, Voi. 77, No. 8, 1973
(2)
+
(12) J. P. Barton and J. E. Packer, Int. J. Radiat. Phys. Chem., 2, 159 (1970).
991
Pulse Radiolysis Study of Sulfhydryl Compounds
thiyl radicals, RS-. The predominant mechanisms of protection have been suggested to be due either (a) to hydrogen transfer from sulfhydryl compounds to organic free radicals FP.(known as the “repair” mechanism)
R1*+ RSH and/or (b) to a competition pounds and organic raolecules cals iR’H -*e
-
R’H + RS. (5) between sulfhydryl comfor the oxidizing OH radi-
(6) II,Q i.e., competitioii among reactions 1, 3, and 6. The rates of reaction 5 are not high,11b 5108 M-1 sec-1. A third possibility which has apparently not been seriously considered hitherto is that sulfhydryl compounds may compete for hydrated electrons and/or be a good acceptors19 in electron transfer processes, kfRlH-- + RSH R1H + R. HS-) 108 M-1 see-1. It is interesting to note that inactivation (in dilute solutions) of enzymes containing -SH groups were found20 to be considerably less than for en-t
-
-
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R1.
Verg little kinetic information is available on the reactivity of eaq- and of OH radicals with sulfhydryl compounds. The effects of substituents and ionic forms on these reaction rates constants have not been studied. This work deals with a s,ystematic examination of these rate constants, the intermediates produced, and the dependence of the rates of ,*eactions4 and 7 on the nature ofthe sulfhydryl compounds. A similar study with disulfide compounds, RSSR, has recently been carried out.21 ~ x p e ~ ~ n i ~ n t ~ ~ Experimental details of the pulse radiolysis set-up used have been described elsewhere.Z2 All solutions were prepared just prior to use and buffered, and the p1-E was) adjusted in the absence of oxygen to minimize the oxidation of the sulfhydryl compounds. In order to avoid photolysis of these solutions by the monitoring light from a. boosted 450-W xenon lamp, a synchronized shutter (open for -1-4 msec) was used. Perchloric acid, phosphate (1--3 mM), tetraborate (1-2 mM), and potassium hydroxide were used as buffers. A fresh solution was used for each pulse. Quartz cells with 2-em optical paths were used. Dosimetry was carried out using a KCNS solution, and the extinction coefficients were derived based on G(eaq-1 =: G(OH) = 2.8anLX G(H) =: 0.55. The chemica.ls used were obtained from Calbiochem (cysteamine, cysteine, penicillamine, glutathione), Sigma (thioglycollate, Na+ salt), Cyclochemicals (N-acetylcystamine, cysteine methyl ester, S-methylcysteine, N-acetylcysteine), Eastman (p-thiopropionic acid was redistilled, methionine), A!drich (thiodiacetic acid was recrystallized, P-thiodipropionic acid, methyl thioglycollate, thiolactic acid),,and Evans (benzyl mercaptan). ~~~~~~~
Reactions with eiiq--. The reaction rate constants of with sulfhydryl compounds were determined (in presence of -0.1 Ad tert-butyl alcohol in order to scavenge the OH radicals) from the pseudo-first-order decay of the hydrated elect,ron a t 700 nm. From the dependence of these rates upon the substrate concentration, the values of .k(eacl- i- S) viere calculated. These results are shown in Table I. eaq -
Sulfhydryl compounds and sulfur amino acids are present in various ionic forms depending upon the pH of the solution. A systematic examination of the dependence of k(easS) upon the ionization constants of these compounds was carried out, and the values are shown in Table I and Figure 1. These results reveal that the rates are markedly dependent upon the state of protonation of the various functional groups in the molecule. In all cases, the rates decrease with increase in pH. It is evident from the “titration curves” in Figure 1 that the rate constants can be directly related to the pK, values of the -SH groups and, in the case of amino acids or amines, to the -NH3+ groups. Previously,23 the lowering of the rate of reaction of eaq- with cysteine (the only sulfhydryl compound hitherto studied) in alkaline solution was attributed only to the deprotonation of the -SH group. It is clear from Figure 1, that for S-metbylcysteine (pKa = 8.75), deprotonation of the -NH3+ group reduces the eaqrate from 7.2 X 108 to 1.5 X 108 M-I sec-l a t pH 12.2. It is to be noted that the values of k(eaq- 4-penicillamine) as a function of pH are the same as those shown for cysteine, The absolute values of k(eaqS) generally follow the overall charge on the mercaptan. Cysteamine, with a + I overall charge in neutral solution, exhibits a high rate, as does similarly charged cysteine methyl ester. The neutral (overall charge 0) molecules, methyl thioglycolate, cysteine, penicillamine, and N-acetylcysteamine, show rates which are close to the diffusion-controlled region, -1 X 1010 M-1 sec-1. As the charge on the molecule becomes more negative (but with the -SH group remaining protonated) the rate constant decreases. Note the near identity of the values in neutral solution for thioglycolate, thiolactate, P-mercaptopropionate, N-acetylcysteine, and glutathione, all have a - 1 charge. The effect of deprotonation of the -SEI group to -Sleads to a marked reduction in the rate of reaction with eaq- (Table I). This can be seen most clearly with thioglycolate; thiolactate, methyl thioglycolate, P-mercaptopropionate, N-acetylcysteamine, and N-acetylcysteine, The overall charge again appears to affect the rate, e.g., k(eaq-SCH&H2NHCOCH3) = 1.9 X 109 M-1 sec-1 while k[eaq-SCH2CW(NHCOCH3)COO-I = 3.3 X 10s M - I sec-l. The effect of deprotonation of the -NH3+ group in the absence of an -S- group can be seen only in the case of S-methylcysteine, where the loss of the positive charge (and of an electron-withdrawing group -NH3+) diminishes the rate. In contrast, deprotonation of -SH in the absence of the amino group causes an extremely large reduction of the rate, often by a factor of 10 or more. When deprotona-
+
+
+
+
(13) J. W. Purdie, H. A. Giilis, and N. V , Klassen, Chern. Commun., 1163 (1971). (14) W. W. Wolf, J. C. Kertesz, and W. C. Landgraf, J. Magn. Resonance, 1,616 (1969). (15) W. A. Armstrong and W. C. Humphreys, Can. J. Chem,, 45, 2569 (1967). (16) P. Neta and R. W.Fessenden, J. Phys. Chem., 75,2277 (1971). (17) G. Nucifora, B. Smaller, R. Remko, and E. C. Avery, Radial. Res., 49, 96 (1972). (16) See, e. g.#T. Henriksen, Radiat. Res., 27, 694 (1966). (19) E. Hayonand M. Simic, J. Amer. Chem. Soc., 93,6781 (1971). (20) R. Lange. A. Pihl. and L. Eidjarn, Int. J. Radial. Biol., 1, 73 (1959). (21) M. Z. Hoffman and ,E. Hayon, J. Amer. Chem. SOC., 94, 7950 (1972). See also A. Shafferman, Isr. J . Chem., 10, 725 (1972), dealing with the kinetics of the decay of the RSSR- radicai from glutathione disulfide. (22) M. Simic, P. Neta, and E. Hayon. J. Phys. Chem., 73, 3794 (1969): .I P. Keene, E. D. Black, and E. Hayon, Rev. Sci. Instrum., 40, 1199 (1969). (23) R. Braams, Radiat. Res., 27,319 (1966). The Journal of Physical Chemistry, Vol. 77, No. 8,1973
Morton Z. Hoffman and E. Hayon
99
TABLE I: Rate Constants for the Reaction of eaq- with Various Sulfhydryl Compounds in Aqueous Solutiona k(e,,- f S), Compound,
Structure
pKab
PH
M - ' sec-'
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I -
Thioglycotic acid Methyl thiogl ycolate 'r'hiolactic acid 6-Mercaptopropionic: aeid Cysteamine
HSC H &02H HSCHzCOzCH3
7.8
HSCH (CH3)COzH
-4,
HSCHzCHzCOzH
4.3, 10.3
HSCHZCH~NH~+
8.6, 10.7
N-Acetyicysteamine Cysteine
HSCH2CH2NHCOCH3
-9.5
HSCHZCH(NH~+)CO~II
Cysteine methyl ester S-Methylcysteine N-Acetylcysteine Penicillamine
HSCHzCH (NH3+)COzCH3
1.8, 8.3, 10.8 6.5, 9.0
CH~SCHZCH(NH~+)CO~H
-2,
HSCHzCH (NHCOCH3)COzH
-2,9.5
HSC(CH3)zCH (NHs+)COzH
-2, 7.9, 10.4 2.1, 3.6, 8.8, 9.7 9.4 2.3, 9.2 3.3, 4.5
Glutathione
d
Benzyl mercaptan Methionine Thiodiacetic acid 6-Thiodipropionic acid
C6HsCHzSH CH~SCHZCH~CH(NH~')CO~H S(CHzC0zH)z S(CH2CHzCOzH)z
6.5 12.0 5.2 10.3 7.2 12.2 7.4 13.0
3.7, 10.3
-10.7
5.5
1.4 5.0 7.7 5.0
x x x x
?. 22.5 X
109 109 108 109
lo8
3.0 X 10'O 1.5 x 109 9.1 x 109 1.9 x 109 1.3 x 10'0 2.0 X 1Q8 1.8 x 10'0 6.9 x 109 7.2 x 108
5.1 10.1 5.4 12.2 7.1 12.5 5.3 12.0 7.2 12.7
x x 3.3 x 1.0 x 1.5 5.6
108 109 108 10'0 5.6 X l o 8 4.5 x 109 4.7 x 108
x x 8.3 x 5.8 x
7.0
8.7 4.5
7.3 10.8 10.8
-4
x 109
5.6 X 1Q8 1.4 x 10'0
5.5 12.5 7.1 12.6 5.8 12.5
8.8
-
109
107 107 107
a Measured in the presence of -0.1 M ferf-butyl alcohol as an OH scavenger. Values taken from the "Handbook of Biochemistry," 2nd ed, Chemical Rubber Publishing Co., Cleveland, Ohio, 1970.The pKa values of the acidic functional groups are in the order -COzH < -SH < -NH3+. C Estimated error (N HsC)CH2CH$ONHC(CH2SH) HCONHCH2C02H. in the values $: 10%. HCI~CCH
tion of both -SH and -NH3+ occurs (e.g., cysteine) the effect appears to be additive. The effect of the sulfur atom, in compounds that exhibit the -S- thio ether linkage, on the rate of reaction with eap- can be demonetrated for thiodiacetic acid, 6-thiodipropionic acid, methionine, and S-methylcysteine. These compounds all give relatively low rate constants. The presence of an amino group fl to the CH3S- group, in the case of S-methylcysteine, allows the rate to be considerably faster than for methionine (where the -NH3+ group is y to the -SC& group). Whether the source of this effect is inductive or steric is not known. It is interesting to note that the reactivity toward eaqof RSH and RrSR2 compounds is considerably higher than that of ROH and RllOR2 compounds. The covalent radius of the sulfur atom irj appreciably larger than that of the oxygen (or carbon) at0m,2* making the C-S bond more sterically accessible. Furthermore, and perhaps most important is the fact that the sulfur atom can use d orbitals to accommodate a negative charge, while this is impossible for the oxygen atom. It is more difficult to explain the difference between ihe rates of RSH and RlSRz compounds with eaq-. It may be due to the much faster dissociative electron capture reactions of RSH (see more below) and to the higher electron affinity of the HS. radical (-53 kca125) compared to that of the RS. radical. Intermediates Produced from the Reaction with eaq-. The transient species produced from the reaction with ea,- were observed in solutions containing -1.5 Ad t The Journal of Physical Chemistry, Vol. 77, No. 8, 1973
BuOH (to scavenge the OH radicals) and argon (1 atm), Figure 2 shows the transient absorptions obtained with thioglycolic acid at pH 4.1 and 7.4. These spectra are very similar to the spectra of the aCH2COQH and eCH2C00radicals previously obtained,26 and are formed via reactions 8 and 9. eaq- + HSCHzCOOH CH2C00H HS(8)
-
eaq-
+ HSCH,COO-
-
.CHzCOO-
+ + HS-
(9)
The extinction coefficient €320 670 M - l cm-I for the .CHzCOOH radical and €350 880 M - I em-1 for the .CH2COO- radical are in good agreement with earlier26 values. The decay rates were 2k = 3.6 X 109 M - l and 2k = 9.8 X 10s.M-1 sec-I at pH 4.1 and 7.2, respectively. The insert in Figure 2 shows that the two radicals exist in an acid-base equilibrium with PKa 4.9 f 0.1, compared to PKa 4.5.26
-
-
-*CH2COO- t- H+
-CH,COOH
(10)
pK,-49
The reaction of free radicals with the sulfur atom has been suggested27 to occur via an addition-elimination process. The interaction of eaq- with RSH compounds undergoes a rapid (7
