Anal. Chem. 2003, 75, 3712-3719
Characterization of the Acid/Base and Redox Chemistry of Phytochelatin Analogue Peptides Stephen M. Spain and Dallas L. Rabenstein*
Department of Chemistry, University of California, Riverside, California 92521
The phytochelatins are a family of polydisperse, thiol-rich peptides that are synthesized by plants in response to exposure to heavy metals. The amino acid sequence of the phytochelatin peptides is (γ-glutamyl-cysteinyl)n-glycine, where n typically ranges from 2 to 5. In the first phase of a program to characterize the coordination chemistry of the phytochelatins with heavy metals, the phytochelatin analogue peptides acetyl(γ-glutamyl-cysteinyl)n-glycine amide (Ac-(γ-Glu-Cys)n-NH2, n ) 2-6) have been synthesized by solid-phase peptide synthesis methods and characterized by 1H NMR spectroscopy. The 1H NMR spectra of the analogue peptides were completely assigned by using band-selective homonuclear-decoupled (BASHD) two-dimensional NMR experiments to achieve spectral resolution. The acid dissociation constant of each cysteine residue in each peptide was determined from chemical shift-pH titration data for the CrH protons of the cysteine residues. The resonances for the CrH protons were resolved in BASHD-total correlation spectroscopy spectra that were measured as a function of pH. The pKA values for a given thiol group depend on the position of the cysteine residue in the sequence, with the thiol group of the cysteine residue attached to the C-terminal glycine being the most acidic. The pKA values also depend on the size of the peptide, increasing as the size, and thus the negative charge, of the peptide increases. The redox potential for oxidation of the two thiol groups of Ac(γ-GluCys)2-NH2 to form an intramolecular disulfide bond was also determined by measuring the equilibrium constant for its thiol/disulfide exchange reaction with glutathione. Phytochelatins (PCs) are polydisperse peptides produced by plants, algae, and certain fungi to detoxify heavy metal ions.1-11 * To whom correspondence should be addressed. Phone: 909-787-3585. Fax: 909-787-2435. E-mail:
[email protected]. (1) Grill, E.; Winnacker, E.-L.; Zenk, M. H. Science 1985, 230, 674-676. (2) Kondo, N.; Imai, K.; Isobe, M.; Goto, T.; Murasugi, A.; Wada-Nakagawa, C.; Hayashi, Y. Tetrahedron Lett. 1984, 25, 3869-3872. (3) Zenk, M. H. Gene 1996, 179, 21-30. (4) Leopold, I.; Gu ¨ nther, D.; Schmidt, J.; Neumann, D. Phytochemistry 1999, 50, 1323-1328. (5) Rauser, W. E. Cell Biochem. Biophys. 1999, 31, 19-48. (6) Corbett, C. S. Curr. Opin. Plant Biol. 2000, 3, 211-216. (7) Corbett, C. S. Plant Physiol. 2000, 123, 825-832. (8) Gekeler, W.; Grill, E.; Winnacker, E. L.; Zenk, M. H. Z. Naturforsh. 1989, 44c, 361-369. (9) Kneer, R.; Zenk, M. Phytochemistry 1992, 31, 2663-2667. (10) Rauser, W. E. Plant Physiol. 1995, 109, 1141-1149. (11) Cobbett, C.; Goldsbrough, P. Annu. Rev. Plant Biol. 2002, 53, 159-182.
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PCs are atypical, nontranslationally synthesized polypeptides that have a primary structure consisting of repeating sequences of the γ-glutamyl-cysteinyl dipeptide and a carboxy-terminal glycine, (γGlu-Cys)n-Gly. The n can range from 2 to 11 (PC2 to PC11), but it is generally in the range 2-5.1,3,8 The phytochelatins are class III members of the metallothionein family of cysteine-rich polypeptides.10,11 Biosynthesis of PCs takes place by enzyme-catalyzed transpeptidation of a γ-Glu-Cys dipeptide unit from the tripeptide glutathione (GSH, γ-Glu-Cys-Gly) onto an acceptor GSH molecule to form PC2 or onto a growing PCn peptide to form a PCn+1 oligomer.3,5,10,12 Biosynthesis is catalyzed by phytochelatin synthase (PC synthase),13 a constitutive enzyme.11 Originally, biosynthesis of PCs was thought to be activated by binding of heavy metal ions by PC synthase and terminated by sequestration of the heavy metal ions by the product PCs.3,14 More recently, it has been demonstrated that direct metal binding by PC synthase is not the primary mode of metal-induced PC biosynthesis.15 Rather, heavy metal-glutathione or heavy metal-PC complexes serve as cosubstrates for catalysis via a substituted enzyme mechanism.15 PC biosynthesis is induced by a wide range of metals, including both essential and nonessential metals, and by several anionic species.1,11 For example, Cd2+, Pb2+, Zn2+, Sb3+, Ag+, Ni2+, Hg2+, AsO4,5- Cu2+, Sn2+, SeO32-, Au+, and Bi3+ induce PC formation in Rauvolfia serpentia cell suspension cultures.16 Although it is well established that PCs are widely distributed in the plant kingdom and that they play a role in detoxification and homeostasis of heavy metal ions in plants,11 relatively little is know about the complexes they form with heavy metal ions. As with glutathione (PC1) and other members of the metallothionein family of peptides, complexation takes place through the cysteine thiolate groups.3,10,17-19 However, there is relatively little published information about the stoichiometries and the kinetic and thermodynamic stabilities of the complexes formed among the various (12) Cobbett, C. Plant Physiol. 2000, 123, 825-833. (13) Grill, E.; Loffler, S.; Winnacker, E.-L.; Zenk, M. H. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6838-6842. (14) Loeffler, S.; Hochberger, A.; Grill, E.; Winnacker, E.-L.; Zenk, M. H. FEBS Lett. 1989, 258, 42-46. (15) Vatamaniuk, O. K.; Mari, S.; Lu, Y.-P.; Rea, P. A. J. Biol. Chem. 2000, 275, 31451-31459. (16) Grill, E.; Winnacker, E.-L.; Zenk, M. H. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 439-443. (17) Strasdiet, H.; Duhme, A.-K.; Kneer, R.; Zenk, M. H.; Hermes, C.; Nolting, H.-F. J. Chem. Soc., Chem. Commun. 1991, 1129-1130. (18) Rabenstein, D. L.; Guevremont, R.; Evans, C. A. Metal Ions in Biological Systems; Sigel, H., Ed.; Marcel Dekker: New York, 1979; pp 103-142. (19) Rabenstein, D. L. In Coenzymes and Cofactors; Dolphin, D., Poulson, R., Avramovic, O., Eds.; John Wiley and Sons: New York, 1989; pp 147-186. 10.1021/ac0207426 CCC: $25.00
© 2003 American Chemical Society Published on Web 06/24/2003
PC oligomers and heavy metal ions. This information is of interest because of potential applications of PCs as metal sequestering agents, for example, as agents for the treatment of heavy metal poisoning. As the first step in a program to characterize the coordination chemistry of PCs with heavy metal ions, we have synthesized and characterized analogues of PCs with the N-terminal amino group acetylated and the C-terminal carboxyl group amidated, Ac-(γGlu-Cys)n-Gly-NH2. These analogues were chosen for study in this first phase so as to focus on the coordination chemistry of the cysteine thiol groups. We report here the synthesis and characterization of PC analogues with n ) 2-6, the complete assignment of their 1H NMR spectra, and the determination of specific acid dissociation constants for each of their thiol groups. We also report the oxidation potential for the thiol groups of Ac-(γ-Glu-Cys)2-GlyNH2, as determined indirectly from the equilibrium constant for the thiol/disulfide exchange reaction of Ac-(γ-Glu-Cys)2-Gly-NH2 with GSH.20 Complete assignment of the 1H NMR spectra of the PC analogue peptides is complicated by the considerable overlap of resonances for the repeating γ-Glu-Cys dipeptide sequences in one-dimensional and regular two-dimensional 1H NMR spectra. However, with the superior resolution of band-selective homonuclear-decoupled (BASHD) two-dimensional 1H NMR experiments,21,22 it was possible to completely assign the 1H NMR spectra of the Ac-(γ-Glu-Cys)n-Gly-NH2, n ) 2-6, peptides. With the complete assignments, the acid dissociation constant of each cysteine thiol group in each analogue peptide was determined from chemical shift-pH titration data measured by two-dimensional 1H NMR.23 EXPERIMENTAL SECTION Peptide Synthesis. The peptides Ac-(γ-Glu-Cys)n-Gly-NH2, n ) 2-6, were synthesized using solid-phase peptide synthesis methodology on a Millipore 9050 Plus peptide synthesizer. All solutions were prepared in N,N-dimethylformamide (Fisher Scientific). The resin Fmoc-PAL-PEG-PS (Applied Biosystems) had a loading of 0.17 meq/g and was allowed to swell in DMF for 1 h before beginning the synthesis. N-R-Fmoc-glycine was obtained from Chem-Impex International, N-R-Fmoc-S-tert-butylthio-L-cysteine, N-R-Fmoc-S-trityl-L-cysteine, and N-R-Fmoc-L-glutamic acid R-tert-butyl ester were from Nova Biochem. N-R-Fmoc-S-tertbutylthio-L-cysteine was used for the synthesis of Ac-(γ-Glu-Cys)2Gly-NH2; N-R-Fmoc-S-trityl-L-cysteine was used for Ac-(γ-Glu-Cys)nGly-NH2, n ) 3-6. Before each coupling step, the N-terminal Fmoc groups were removed from the resin-bound peptide with 20% piperidine in N,N-dimethylformamide (Fisher Scientific). Coupling reactions were carried out using a 5-fold excess of Fmocprotected amino acid. The incoming Fmoc-protected amino acids were activated by reaction with 1-hydroxy-7-azabenzotriazole (HOAT, Applied Biosystems) in 0.3 M 1,3-diisopropylcarbodiimide (DIPCDI, Aldrich Chemical Co.). The activated amino acids were allowed to react with the resin for 1 h. The peptides were cleaved from the resin by reaction of 3 mL of cleavage cocktail (95% (20) Yeo, P. L.; Rabenstein, D. L. Anal. Chem. 1993, 65, 3061-3066. (21) Krishnamurthy, V. V. Magn. Reson. Chem. 1997, 35, 9-12. (22) Kaerner, A.; Rabenstein, D. L. Magn. Reson. Chem. 1998, 36, 601-607. (23) Rabenstein, D. L.; Hari, S. P.; Kaerner, A. Anal. Chem. 1997, 69, 43104316.
trifluoroacetic acid (TFA), 2.5% H2O, and 2.5% triisopropylsilane) per 100 mg of resin-bound peptide for 4 h under nitrogen. Cleavage was quenched by addition of 25 mL of H2O. The solution was lyophilized, and for Ac-(γ-Glu-Cys)2-Gly-NH2, the lyophilized powder was dissolved in a minimum of 0.1 M NH4HCO3 that contained a 20-fold excess of 1,4-dithiol-DL-threitol (DTT, Fluka). The solution was allowed to react under N2 for 1 h to remove the S-tert-butylthio protecting groups. For the other peptides, dissolution in NH4HCO3/DTT solution was not necessary, because the trityl protecting group had been removed by TFA in the cleavage cocktail. After lyophilization, the peptides were purified by high performance size-exclusion chromatography using a Biopharmacia “Superdex Peptide” size-exclusion column on a Biorad HPLC. A mobile phase of 0.1% TFA in H2O was used. The product fraction was collected, lyophilized, and verified by either ESI-MS or MALDI-TOFMS. NMR Measurements. NMR spectra were measured on a Varian Unity Inova 500 MHz spectrometer equipped with waveform generators, a Performa X, Y, Z gradient module, and a 1H{13C, 15N} triple-resonance, X, Y, Z triple-axis pulsed-field gradient probe. One- and two-dimensional NMR spectra were measured with suppression of the water resonance by presaturation. Twodimensional TOCSY and ROESY spectra were measured with standard pulse sequences. The spectral window was set to 5500 Hz in both dimensions. Typically, 2D data sets were acquired with 128 increments in the t1 dimension and 8 K points in the t2 dimension at a temperature of 5 or 25 °C. BASHD-TOCSY and BASHD-ROESY 1H NMR spectra were measured using literature pulse sequences.21,22 In the two-dimensional BASHD experiments, resolution is increased by collapsing multiplets from 1H-1H spinspin coupling to singlets in the F1 dimension. This is accomplished by use of a double-pulsed field gradient spin-echo (DPFGSE) technique with band selective pulses interspersed in the t1 time period to bring all components of each multiplet within the bandselected region (in this case, either the amide NH resonances or the CRH resonances) back in phase at the end of the t1 evolution period. The band-selective pulses used in the BASHD-TOCSY and BASHD-ROESY experiments were Gaussian cascade Q3 pulses24 phase-modulated to shift the center of excitation 1740 Hz to the amide NH region or -385.5 Hz to the CRH region of the 1H NMR spectra. F1 spectral windows of 540 and 525 Hz were used for band selection of the amide NH and CRH resonances, respectively. A total of 64 × 8 K complex points were acquired in the t1 and t2 dimensions. Mixing times of 0.12 and 0.2 ms were used in the BASHD-TOCSY and BASHD-ROESY experiments. Pulsed field gradients were applied in the Z direction. Data were processed with linear prediction to 256 points in the t1 dimension and zerofilled to 512 points. A shifted sine bell apodization function was applied to the F1 dimension, and a Gaussian function to the F2 dimension. The difference between the transmitter offset and the center of the excitation window was applied to the t1 interferograms prior to Fourier transformation for the BASHD-TOCSY and BASHD-ROESY data. Peptides were dissolved in 90% H2O/10% D2O at concentrations of 5 mM, and the pH was adjusted using 0.1 M NaOH. Samples were contained in Shigimi NMR tubes, and sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP) was added as the chemical shift reference. (24) Emsley, L.; Bodenhausen, G. J. Magn. Reson. 1992, 97, 135-148.
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Figure 1. 500 MHz 1H NMR spectrum of Ac-(γ-Glu-Cys)4-Gly-NH2 in 90% H2O/10% D2O at pH ) 5.0 and 5 °C.
Measurement of Acid Dissociation Constants. Acid dissociation constants were measured for each cysteine thiol group in the Ac-(γ-Glu-Cys)n-Gly-NH2, n ) 2-6, peptides from the pH dependence of the chemical shifts of their cysteine CRH resonances. Because of extensive overlap of the cysteine CRH resonances in one-dimensional and regular two-dimensional TOCSY spectra, the chemical shift-pH titration data were obtained from 2D-BASHD-TOCSY spectra.22,23 Chemical shiftpH titrations were carried out directly in the NMR tube at 25 °C using a Corning NMR micro combination pH electrode (Fisher Scientific). The procedure involved adjusting the initial pH of a 5 mM solution of each peptide to ∼5 and measurement of a BASHDTOCSY spectrum. The pH was then increased by addition of 0.1 M NaOH, and another BASHD-TOCSY spectrum was measured. This procedure was repeated until a pH of ∼12 was reached. pKA values were determined by nonlinear least-squares fits of chemical shift-pH titration data to an equation describing the dependence of chemical shift on pH and pKA.23 The nonlinear fits were performed using the Scientist (Micromath, Inc.) curve-fitting program. Measurement of Redox Potential. The redox potential of the two thiol groups of Ac-(γ-Glu-Cys)2-Gly-NH2 was determined by reaction of a solution of Ac-(γ-Glu-Cys)2-Gly-NH2 with a GSH/ GSSG redox buffer.20 All solutions were prepared under an argon atmosphere in a glovebox and allowed to react under argon for 3 h at 25 °C. Concentrated HCl was then added to lower the pH to 3, the pKA values of the other thiols are essentially identical for a given peptide. The increase in pKA correlates well with an increase in peptide size and the corresponding increase in total negative charge. At neutral pH, Ac-(γ-Glu-Cys)2-Gly-NH2 would have a charge of -2 from the carboxylate groups, and Ac-(γ-Glu-Cys)6-Gly-NH2 would have a charge of -6. This buildup of negative charge would destabilize the formation of thiolate anions. The lower pKA or greater acidity of the thiol group nearest the glycine also correlates well with structure, since this thiol group is only flanked on one side by a negative carboxylate group. Redox Potential of Ac-(γ-Glu-Cys)2-Gly-NH2. The E°′ for oxidation of the two thiol groups of Ac-(γ-Glu-Cys)2-Gly-NH2 to form an intramolecular disulfide bond was found to be -0.224 V. This value is similar to E°′ values for other peptides that form intramolecular disulfide bonds, even though the number of atoms in the disulfide-containing ring is somewhat smaller. For example, E°′ values for arginine vasopression, oxytocin, and somatostatin at pH 7.00 are -0.228, -0.216, and -0.221 V, respectively. The number of atoms in the disulfide-containing rings is 20, 20, and 38, respectively, as compared to 13 in the disulfide-containing ring of oxidized Ac-(γ-Glu-Cys)2-Gly-NH2.27,28
Received for review December 6, 2002. Accepted May 4, 2003. AC0207426
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