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The interaction of hydrophilic thiols with cadmium: investigation with a simple model, 3-mercaptopropionic acid
Marine Chemistry 70 Ž2000. 181–189 www.elsevier.nlrlocatermarchem
The interaction of hydrophilic thiols with cadmium: investigation with a simple model, 3-mercaptopropionic acid q Murthy A. Vairavamurthy a,) , Wayne S. Goldenberg a , Shi Ouyang a , Syed Khalid b a
Department of EnÕironmental Sciences, BrookhaÕen National Laboratory, Upton, NY 11973, USA b National Synchotron Light Source, BrookhaÕen National Laboratory, Upton, NY 11973, USA Received 15 May 1999; received in revised form 22 September 1999; accepted 27 October 1999
Abstract Environmental contamination by cadmium is widely recognized as a serious problem. All cadmium compounds are considered toxic and are regulated by the US Environmental Protection Agency. In organisms, thiols or organic sulfhydryl compounds are primarily involved in mobilizing and detoxifying cadmium through the formation of cadmium–thiol complexes inside the cell. This complexation is also of environmental importance because a variety of thiols are generated by biotic and abiotic processes in anaerobic environments, such as estuarine wetlands and coastal sediments, which have increasingly become the targets of metal pollution. We investigated the complexation of cadmium by 3-mercaptopropionic acid ŽMPA., using it as a simple model to better understand the potential pathways of cadmium transformations by hydrophilic thiols. Our results show that cadmium forms primarily 1:1 and 1:2 complexes with MPA in aqueous solutions at near-neutral pH values. While the dithio complex was highly soluble in water, the monothio complex rapidly precipitated from solution. Based on mass spectrometric and X-ray absorption spectroscopic data, we propose a cyclic, charge-neutralized structure for the monothio complex, and a linear ionic structure for the dithio complex. These results suggest that the type of complex formed is an important determinant of the mobility of cadmium. Published by Elsevier Science B.V.. Keywords: hydrophilic thiols; cadmium; 3-mercaptopropionic acid
1. Introduction Environmental contamination by heavy metals is a serious and growing concern worldwide. Cadmium q By acceptance of this article, the publisher andror recipient acknowledges the US Government’s right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper. ) Corresponding author. Tel.: q1-516-344-5337; fax: q1-516344-7905. E-mail address: [email protected] ŽM.A. Vairavamurthy..
0304-4203r00r$ - see front matter. Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 2 0 3 Ž 0 0 . 0 0 0 2 3 - 2
is among the primary metal contaminants at several US superfund sites and is listed as one of the US Environmental Agency’s priority pollutants. According to 1986 estimates, nearly 2.9 million pounds of cadmium were produced in the US, and nearly twice this amount was imported. Cadmium is used primarily in metal plating and coating, nickel–cadmium batteries, pigments, plastic stabilizers, pesticides, alloys, chemical reagents and nuclear rods ŽNriagu, 1980.. The fate and transport of cadmium in aquatic environments are important in environmental regula-
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tion. The toxicity of cadmium largely depends upon its speciation and not to its total concentration; free CdŽII. ion is considered to be the most toxic form ŽHare and Tessier, 1996.. Generally, the formation of organic complexes dramatically affects a metal’s behavior, not only changing its toxicity and bioavailability, but also its patterns of mobility and migration. Among the organic compounds, thiols Žsuch as glutathione and metallothioneins. play an important role in complexing cadmium in biological systems. This complexation is the primary mechanism of cadmium detoxification in organisms ŽPerrin, 1971; Dallinger, 1993; Stillman, 1995; Diaz-Cruz et al., 1997.. Biochemical thiols in the environment include those molecules originally present in organisms Že.g. glutathione., and those derived from the biodegradation of other sulfur-containing compounds, e.g. methane thiol ŽCH 3 SH. and 3-mercaptopropionic acid ŽMPA. ŽMopper and Taylor, 1986.. In anoxic environments, a well-known pathway for forming MPA is the degradation of dimethylsulfoniopropionate, a sulfur compound present at high concentrations in several types of algae and some higher plants Že.g. the salt-marsh grass Spartina alterniflora. ŽDacey et al., 1987.. Both biochemical and chemical pathways have been suggested for the formation of MPA from dimethylsulfoniopropionate ŽVairavamurthy and Mopper, 1987; Kiene and Taylor, 1988; Vairavamurthy et al., 1997.. The environmental importance of thiol interactions with cadmium mainly stems from the recognition that a variety of thiols are present in anaerobic systems, such as anoxic waters and organic-rich marine sediments ŽDyrssen et al., 1985; Luther et al., 1986; Mopper and Taylor, 1986; Vairavamurthy and Mopper, 1987; Shea and Maccrehan, 1988.. Among several low-molecular-weight and hydrophilic thiols reported in sediment pore waters from coastal and salt-marsh sediments, a dominant one is MPA ŽMopper and Taylor, 1986; Vairavamurthy and Mopper, 1987.. If low-molecular-weight hydrophilic thiols Žsuch as MPA. are the complexants, then the metal complexes may preferentially partition into the aqueous phase. The complexation with high-molecular-weight and particle-bound thiols will probably bind the metals to the particulate phase ŽVairavamurthy et al., 1997.. In an earlier study on the distributions of cadmium and other heavy-metal ions
in sediments from Chesapeake Bay sediments, the formation of soluble thiol complexes was thought to be the main reason for the large discrepancies between the measured levels in pore waters and the predicted values from thermodynamic data for metal–sulfide formation ŽShea and Maccrehan, 1988.. Inorganic polysulfide complexes have been recently shown to be important in increasing the solubility of mercuric sulfide ŽPaquette and Helz, 1997., and HgSŽaq. has been suggested to be an important bioavailable species of mercury ŽBenoit et al., 1999.. The importance of such inorganic sulfur compounds for cadmium complexation is not known. The primary aim of this study was to seek a better understanding of the complexation chemistry of cadmium with a simple hydrophilic thiol, MPA. This information will help us to understand and predict the behavior of cadmium in biological systems where hydrophilic thiols such as glutathione and cysteine play a major role in detoxification mechanisms. Since MPA is a dominant hydrophilic thiol in organic-rich marine sediments, we can assess its role in binding cadmium in coastal and salt-marsh sediments, which have increasingly become polluted with heavymetals. A significant portion of the anthropogenic wastes containing heavy-metal contaminants, including industrial effluents and municipal wastes, is discharged into coastal, estuarine, and near-shore waters where they are scavenged rapidly by the underlying sediments. We used mainly a potentiometric approach ŽBreshnan et al., 1978. to measure the free-ion concentrations of either cadmium or thiol in mixtures containing both species. Electrospray mass spectrometry and extended X-ray absorption fine structure ŽEXAFS. spectroscopy were used to elucidate the structure of Cd–MPA complexes.
2. Experimental All potentiometric measurements were taken at room temperature Ž25 " 18C. with an Orion model 420A pHrmV meter ŽBeverly, MA. equipped with either an ion-selective electrode for cadmium ŽOrion model 9648 BN. or a combination AgrS electrode for free MPA ŽOrion model 9616 BN.. With these ion-selective electrodes, the concentrations are mea-
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sured on the basis that the electrode potential measured in millivolts correlates linearly with the logarithmic values of the concentrations over a wide working range from 10y6 to 10y2 M for the free ions. The AgrS electrode exhibits a linear response to thiols at pH values above p K a when the organic sulfhydryl group is completely dissociated Ž –SH –Sy. ŽTseng and Gutknecht, 1975.; typically, we used a pH of 12 for MPA measurements. For MPA, the electrode showed a linear response in the concentration range from 10y6 to 10y3 M. The ionic strength of the experimental solutions was always adjusted by adding sodium nitrate to a concentration of 0.1 M to optimize the response of the electrodes. Because thiols are oxidized rapidly in air, the experiments were conducted with argon flowing on the surface of the solution. The required chemicals Žincluding cadmium nitrate, MPA. were purchased from Aldrich ŽMilwaukee, WI.. Deionized water Žproduced by a Milli-Q ultra-pure water system, Millipore, Bedford, MA. was used in all experiments. The mass spectra were collected in negative-ion full-scan mode with a Finnigan MAT LCQ ion trap mass spectrometer fitted with an electrospray ionization source. Samples were directly infused using a 250-ml microsyringe at a flow rate of 3 mlrmin. The following instrumental conditions were used: spray voltages y4.3 kV, flow rate for nitrogen sheath gas s 50 unitsrmin; temperature of heated capillary s 200 C; lens voltages y30 V; limit for the maximum number of ions trapped was set at 2 = 10 7 counts; automatic gain control ŽAGC. was turned on with a maximum injection time of 400 ms. At the start of the experiment, the instrument was calibrated and tuned using the procedures recommended by the manufacturer. For each sample, a full-scan mass spectrum was collected for 3 min and averaged. The Cd K-edge X-ray absorption spectroscopy was performed at the X18-B beamline at the National Synchrotron Light Source, Brookhaven National Laboratory ŽUpton, NY.. An X-ray absorption spectrum is produced by the photoexcitation of an electron from a core level Že.g. 1s. to symmetryavailable outer empty states and to the continuum, i.e. beyond the sphere of influence of the atom. When the absorbing atom is surrounded by other atoms, as in any condensed phase, the outgoing photoelectron wave can be backscattered by the sur-
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rounding atoms. The backscattered waves interfere either constructively or destructively with the outgoing waves to produce either peaks or troughs with increasing X-ray energy. These oscillations constitute the EXAFS portion of the X-ray absorption spectrum and allow the determination of the local atomic environment of the absorbing element in terms of the type and number of neighboring atoms, their interatomic distances, and their disorder. The EXAFS spectra of our samples and the reference compounds ŽCdŽClO4 . 2 and CdS. were measured in fluorescence mode using a liquid nitrogen cooled, 13-element germanium detector. In order to improve the signal-to-noise ratio, the samples were cooled down to 150 K using a Displex refrigerator. The EXAFS data analysis was performed using the UWEXAFS suite of programs consisting of AUTOBK and FEFFIT ŽNewville et al., 1993; Stern et al., 1995..
3. Results and discussion Organic molecules complex with metal ions through a variety of functional groups containing heteroatoms such as oxygen Že.g. the carboxylic group., nitrogen Že.g. the amino group., and sulfur Že.g. the sulfhydryl group.. Since MPA contains both a carboxylic group and sulfhydryl group, we first established the relative importance of these functional groups in complexing with cadmium. To verify whether the carboxylic group can complex cadmium, we compared the complexing ability of MPA with that of propionic acid, a molecule similar to MPA but without a sulfhydryl group.
Cadmium ion was gradually added to a 5 mM solution of each ligand, and the extent of complexation was determined by measuring the concentration of free cadmium-ion in solution with a cadmium ionselective electrode. The pH was maintained at 7.5 " 0.1, which is representative of the values found in
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Fig. 1. Comparison of the reactivities of MPA and propionic acid with cadmium. Cadmium ion was added in 0.5 mM aliquots to 5 mM of each organic compound and the free ion was measured after each addition. Conditions: temperatures 25"0.18C; ionic strengths 0.1; pH s 7.5"0.2.
natural waters. Fig. 1 plots the measured values vs. the added cadmium concentrations in propionic acid and in MPA. With propionic acid, the measured concentrations of cadmium ion increased linearly, matching the added levels suggesting that there is no complexation between cadmium and propionic acid. However, with MPA, the measured level was nearly zero up to 5 mM of the added cadmium, and increased linearly afterwards. Therefore, the carboxylic group alone cannot complex with the cadmium ion, and the primary functional group in MPA that binds the metal ion is the sulfhydryl group. The ability of the thiol group to strongly complex with cadmium can be explained from the ease of polarizability of the ‘‘electron cloud’’ of the ligand group by the central metal cation. A common approach to recognizing the reactivity between a metal ion and an organic ligand is a classification based on their polarizing power Žchargerradius ratio. as ‘‘hard’’ and ‘‘soft’’ acids Želectron acceptors., and ‘‘hard’’ and ‘‘soft’’ bases Želectron donors. ŽPearson, 1963.. Generally, hard acids preferentially bind to hard bases, and soft acids to soft bases. Metal ions that are small and firmly retain their outer electrons are hard acids Že.g. Mg 2q and Al 3q .; they form the
strongest bonds with hard bases, which have small, highly electronegative ligand atoms with low polarizability Že.g. O-ligands.. Metal ions that are large with many filled d-orbitals are soft acids, and interact strongly with soft bases, which contain large, easily polarizable ligand atoms Že.g. S-ligands.. Cadmium is a typical soft acid; hence, it binds strongly with sulfur-ligands, such as thiols. We studied the stoichiometry of the Cd–MPA complexes formed in solution by adding either the thiol or cadmium in excess and following its free ion concentration. Fig. 2 plots the measured vs. the added concentration of MPA to a 25 mM aqueous solution of cadmium nitrate. We used a concentrated solution of the thiol, and added it in small aliquots so that the final volume was not changed significantly. As Fig. 2 shows, the measured concentration of the thiol remained nearly zero up to the addition of 50 mM, but increased linearly afterwards with the added levels. The consumption of nearly 50 mM thiol for complexing with 25 mM of the cadmium suggests that the dominant aqueous complex had a 1:2 cadmium-to-thiol ratio. The formation of the Cd–dithio complex Ž1. was further substantiated by electrospray mass spectrometry, which is increasingly used in the characteriza-
Fig. 2. Complexation of cadmium with excess MPA; plot of the measured vs. the added concentration of MPA to a 25 mM solution of cadmium nitrate. Conditions: temperatures 25"0.18C; ionic strengths 0.1; pH s12.0"0.2.
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tion of polar and hydrophilic species, including metal–ion complexes ŽD’Agostino, 1996; Alvarez et al., 1997.. In this technique, gas-phase molecular ions are generated under mild conditions at ambient temperature by applying a high voltage and are then analyzed in a mass spectrometer. We determined the negative-ion mass spectrum by directly injecting a sample of a cadmium thiol mixture containing 1 mM Cd 2q and 3 mM MPA into our Finnigan MAT LCQ bench-top mass spectrometer with an electrospray interface. For cadmium, the presence of several isotopes generally complicates the mass spectra of its compounds, but also gives much compound-specific information. The Cd–dithio complex gave a group of peaks with the dominant peak at a mass to charge ratio of 323; the distribution of peaks arises from the presence of different isotopes for the constituent atoms in the molecule. The peaks clustered at mrz 323 can be attributed to a singly charged Cd–dithio complex formed from the deprotonation of one of the carboxylic groups in the molecule Ž2..
It is important to note that in electrospray mass spectrometry, even when a molecule contains several ionizable groups Že.g. a polycarboxylic acid., the dominant peak usually results from a single ionization of the molecule transferred to the gas phase in the electrospraying process. The expected distribution of isotopic masses of this structure can easily be calculated from the isotopic composition of the constituent elements. A comparison of the observed isotopic mass distribution for the complex with the calculated values Žsee Fig. 3. is in excellent agreement, confirming the stoichiometry of the complex. An important parameter in evaluating the reactivity of thiols is the dissociation constant of the sulfhydryl group as a weak acid ŽRSH RSyq Hq .. A p K a value of 10.27 for the dissociation of the –SH group in MPA implies that the concentration of
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Fig. 3. Electrospray mass spectrum of the cadmium dithio-complex with MPA ŽInstrument: Finnigan LCQ; spray voltage: 4 kV..
the thiolate ion will be very low at acidic and neutral pH values ŽTseng and Gutknecht, 1975.. Despite such low thiolate ion concentration, the complexation of cadmium with MPA proceeds readily and stoichiometrically at this pH, suggesting that the cadmium ion has a high affinity for the thiolate ion. The reaction between Cd 2q and MPA forming the dithio complex probably occurs in two consecutive steps. In the first step, the thiolate ion reacts with Cd 2q to form a monothio complex ŽEq. 1., which then reacts with another thiolate ion to form a dithio complex ŽEq. 2.. The constants K 1 and K 2 refer to the first- and second-formation constants with the dissociated thiolate ion as the complexant; K a is the dissociation constant for the sulfhydryl group of the thiol.
| Ž Cd–SR.
Cd 2qq RSy
q
q
q
Ž Cd–SR. Ž Cd–SR. w Hq x K1 s s w Cd 2q xw RSy x w Cd 2q xw RSHx K a q
Ž 1.
|
Ž Cd–SR. q RSy Cd Ž SR. 2 Cd Ž SR . 2 K2 s q Ž Cd–SR. w RSy x s
Cd Ž SR . 2 w Hq x
Ž Cd–SR.
q
w RSHx K a
Ž 2.
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Experimental evidence for this mechanism is given in Fig. 4, which presents the results of the ion-selective electrode measurements for the concentrations of complexed cadmium vs. MPA. The values for the complexed cadmium were obtained by subtracting the amount of free cadmium-ion concentration Žmeasured with ion-selective electrode. from the original concentration of 2 mM. The slope of the curve is equal to one, suggesting that only the monothio complex was formed with excess cadmium and a limiting thiol concentration. When the thiol is in excess of the cadmium ion, the complexation proceeds to the second step forming the dithio complex. We measured K 1 experimentally by an ion-selective electrode titration of a 50 mM solution of MPA with cadmium ion at pH 7.5 " 0.1 ŽFig. 5.. The free cadmium concentration remained nearly zero up to 50.0 mM of added cadmium ion, but then increased linearly as more was added. This again demonstrates the formation of a 1:1 complex when the concentration of cadmium ion is equal to or in excess of that of MPA. At the point of 1:1 addition of Cd 2q to MPA, the concentration of free MPA should be equal to that of free cadmium, which was measured with the ion-selective electrode. The amount of cadmium ion complexed with MPA was calculated by subtracting the free cadmium-ion concentration from the total amount of cadmium added. From these values, we calculated a value of 8.8 for log K 1 using Eq. 1 at the 1:1 titration point.
Fig. 4. Complexation of excess cadmium with MPA. The plot shows a 1:1 correlation between cadmium ion and the thiol due to the formation of the monothio-complex.
Fig. 5. Complexation of excess cadmium with MPA; plot of the measured vs. the added concentration of cadmium ion to a 50 mM solution of MPA. Conditions: temperatures 25"0.18C; ionic strengths 0.1; pH s 7.5"0.2. The inset is an expanded view near the titration point.
We similarly determined K 2 by titrating a known concentration of cadmium with an excess of thiol. Because the AgrS ion-selective electrode is sensitive only to the thiolate ion and not to the undissociated thiol, the experimental quantity measured was the thiolate ion. Thus, we carried out the measurements at pH 12.0 " 0.2 when almost all the thiol exists as thiolate ions. At the titrations’ end point, the thiolate ion measured should equal the concentration of cadmium–monothio complex. The dithio complex concentration was determined by subtracting the monothio complex concentration from the initial cadmium concentration. From these results, we determined a value of 4.8 for log K 2 . The equilibrium constant for the first step in forming the Ca–monothio complex is about 4 logarithm units larger than that for the second step forming the dithio complex. These results are comparable to those reported for the complexes of glutathione with zinc, which essentially behave similarly to form dithio complexes in solution ŽPerrin and Watt, 1971.. The formation constants described for zinc–glutathione complexes were 7.94 and 4.47 for log K 1 and log K 2 , respectively. For cadmium, the large difference between the constants K 1 and K 2 suggests that the first ligand is more strongly bound to the metal ion than the second one.
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On the basis that the sulfhydryl group alone in MPA binds with cadmium ion, the monothio complex is expected to be present as a zwitterion Ž3. in aqueous solutions at ca. neutral pH because the carboxylic group should be ionized at this pH. However, we observed that this species rapidly precipitated from the solution at a pH of 7.5 in experiments
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where cadmium and thiol were initially present at millimolar concentrations. For example, in a mixture with thiol and cadmium, each at 50 mM initial concentrations, the monothio complex formed mainly precipitated, with less than 0.1 mM of the complex remaining in solution. Because the monothio complex is nearly insoluble, we think that it does not
Fig. 6. ŽA. k 2-weighted x Ž k . of Cd K-edge EXAFS in Cd–MPA Ž1:1. at 150 K. ŽB. Data, fit, and individual Cd–O and Cd–S contributions for Cd–MPA Ž1:1. at 150 K.
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exist as a zwitterion but as a charge-neutralized species, probably produced from a bonding interaction between the carboxylate group and the cadmium ion, resulting in a cyclic structure Ž4..
The nondestructive technique of X-ray absorption spectroscopy gave further information for the proposed cyclic structure for the Cd–monothio complex. Fig. 6A gives the extracted EXAFS data Ž k 2weighted x Ž k ., where k is the photoelectron wave vector. for the Cd–monothio complex. We obtained this EXAFS data from the averaged absorption coefficient after removing a smooth background function Žcharacteristic of isolated-atom absorption. using the program AUTOBK. It is well established that when x Ž k . is Fourier transformed over a finite k range from k min to k max , the result is a radial structure function exhibiting a series of peaks whose positions and amplitudes are related to the interatomic distances and the number of atoms in different coordination shells, respectively. To obtain such structural parameters, we performed a nonlinear least-squares fit on the Fourier-transformed data using the program FEFFIT. We used a superposition of Cd–O and Cd–S contributions to model the total EXAFS spectrum; the coordination numbers of S and O, their distances to the central Cd atom, and the disorder in these distances were allowed to vary during fitting. We used the scattering amplitudes and phases, characteristic for Cd–S and Cd–O interactions, that were extracted from the experimental standards, CdS and CdŽClO4 . 2 , respectively, measured at the same temperature Ž150 K. as the unknown sample. The results of the best fit gives values of 1.3 " 0.4 and 1.3 " 0.2 for Cd–O and Cd–S coordination numbers, respectively ŽFig. 6B.. The presence of one sulfur and one oxygen atoms in the first neighboring shell of cadmium as revealed by the EXAFS data strongly supports structure 4 for the monothio complex. Hence, we suggest that the carboxylic group may become involved as a secondary ligand in a chelating mode of complexation once a bond is formed between cadmium and the
sulfhydryl group. In contrast to the monothio complex, the dithio complex was completely soluble at pH 7.5. It appears that in this complex, the carboxylic groups are free, rendering it a hydrophilic structure. We presented mass spectrometric evidence in support of this structure for the dithio complex earlier. The differential solubility of the monothio and dithio complexes suggests that the type of complex formed is a crucial factor in controlling the mobility and transport of cadmium. Because the dithio complex is highly soluble, its formation will enhance the mobility of cadmium in environments in which such complex form. On the other hand, the formation of the monothio complex will immobilize the metal. These results clearly suggest that chemical structure is an important factor in controlling the mobility of cadmium in biological and environmental systems containing thiol complexants. Further studies are in progress to understand the effect of pH in influencing the structure and speciation of these complexes.
Acknowledgements This material is based upon the work supported by the NABIR program of the Office of Biological and Environmental Research, US Department of Energy and Engineering and Geosciences Program, Office of Basic Energy Sciences, US Department of Energy under contract No. DE-AC02-98CH10886 to Brookhaven National Laboratory. We thank Anatoly Frenkel for the assistance with EXAFS data analysis. Avril Woodhead and Jennifer Ayla Jay are acknowledged for the constructive comments on the manuscript.
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The interaction of hydrophilic thiols with cadmium: investigation with a simple model, 3-mercaptopropionic acid q Murthy A. Vairavamurthy a,) , Wayne S. Goldenberg a , Shi Ouyang a , Syed Khalid b a
Department of EnÕironmental Sciences, BrookhaÕen National Laboratory, Upton, NY 11973, USA b National Synchotron Light Source, BrookhaÕen National Laboratory, Upton, NY 11973, USA Received 15 May 1999; received in revised form 22 September 1999; accepted 27 October 1999
Abstract Environmental contamination by cadmium is widely recognized as a serious problem. All cadmium compounds are considered toxic and are regulated by the US Environmental Protection Agency. In organisms, thiols or organic sulfhydryl compounds are primarily involved in mobilizing and detoxifying cadmium through the formation of cadmium–thiol complexes inside the cell. This complexation is also of environmental importance because a variety of thiols are generated by biotic and abiotic processes in anaerobic environments, such as estuarine wetlands and coastal sediments, which have increasingly become the targets of metal pollution. We investigated the complexation of cadmium by 3-mercaptopropionic acid ŽMPA., using it as a simple model to better understand the potential pathways of cadmium transformations by hydrophilic thiols. Our results show that cadmium forms primarily 1:1 and 1:2 complexes with MPA in aqueous solutions at near-neutral pH values. While the dithio complex was highly soluble in water, the monothio complex rapidly precipitated from solution. Based on mass spectrometric and X-ray absorption spectroscopic data, we propose a cyclic, charge-neutralized structure for the monothio complex, and a linear ionic structure for the dithio complex. These results suggest that the type of complex formed is an important determinant of the mobility of cadmium. Published by Elsevier Science B.V.. Keywords: hydrophilic thiols; cadmium; 3-mercaptopropionic acid
1. Introduction Environmental contamination by heavy metals is a serious and growing concern worldwide. Cadmium q By acceptance of this article, the publisher andror recipient acknowledges the US Government’s right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper. ) Corresponding author. Tel.: q1-516-344-5337; fax: q1-516344-7905. E-mail address: [email protected] ŽM.A. Vairavamurthy..
0304-4203r00r$ - see front matter. Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 2 0 3 Ž 0 0 . 0 0 0 2 3 - 2
is among the primary metal contaminants at several US superfund sites and is listed as one of the US Environmental Agency’s priority pollutants. According to 1986 estimates, nearly 2.9 million pounds of cadmium were produced in the US, and nearly twice this amount was imported. Cadmium is used primarily in metal plating and coating, nickel–cadmium batteries, pigments, plastic stabilizers, pesticides, alloys, chemical reagents and nuclear rods ŽNriagu, 1980.. The fate and transport of cadmium in aquatic environments are important in environmental regula-
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tion. The toxicity of cadmium largely depends upon its speciation and not to its total concentration; free CdŽII. ion is considered to be the most toxic form ŽHare and Tessier, 1996.. Generally, the formation of organic complexes dramatically affects a metal’s behavior, not only changing its toxicity and bioavailability, but also its patterns of mobility and migration. Among the organic compounds, thiols Žsuch as glutathione and metallothioneins. play an important role in complexing cadmium in biological systems. This complexation is the primary mechanism of cadmium detoxification in organisms ŽPerrin, 1971; Dallinger, 1993; Stillman, 1995; Diaz-Cruz et al., 1997.. Biochemical thiols in the environment include those molecules originally present in organisms Že.g. glutathione., and those derived from the biodegradation of other sulfur-containing compounds, e.g. methane thiol ŽCH 3 SH. and 3-mercaptopropionic acid ŽMPA. ŽMopper and Taylor, 1986.. In anoxic environments, a well-known pathway for forming MPA is the degradation of dimethylsulfoniopropionate, a sulfur compound present at high concentrations in several types of algae and some higher plants Že.g. the salt-marsh grass Spartina alterniflora. ŽDacey et al., 1987.. Both biochemical and chemical pathways have been suggested for the formation of MPA from dimethylsulfoniopropionate ŽVairavamurthy and Mopper, 1987; Kiene and Taylor, 1988; Vairavamurthy et al., 1997.. The environmental importance of thiol interactions with cadmium mainly stems from the recognition that a variety of thiols are present in anaerobic systems, such as anoxic waters and organic-rich marine sediments ŽDyrssen et al., 1985; Luther et al., 1986; Mopper and Taylor, 1986; Vairavamurthy and Mopper, 1987; Shea and Maccrehan, 1988.. Among several low-molecular-weight and hydrophilic thiols reported in sediment pore waters from coastal and salt-marsh sediments, a dominant one is MPA ŽMopper and Taylor, 1986; Vairavamurthy and Mopper, 1987.. If low-molecular-weight hydrophilic thiols Žsuch as MPA. are the complexants, then the metal complexes may preferentially partition into the aqueous phase. The complexation with high-molecular-weight and particle-bound thiols will probably bind the metals to the particulate phase ŽVairavamurthy et al., 1997.. In an earlier study on the distributions of cadmium and other heavy-metal ions
in sediments from Chesapeake Bay sediments, the formation of soluble thiol complexes was thought to be the main reason for the large discrepancies between the measured levels in pore waters and the predicted values from thermodynamic data for metal–sulfide formation ŽShea and Maccrehan, 1988.. Inorganic polysulfide complexes have been recently shown to be important in increasing the solubility of mercuric sulfide ŽPaquette and Helz, 1997., and HgSŽaq. has been suggested to be an important bioavailable species of mercury ŽBenoit et al., 1999.. The importance of such inorganic sulfur compounds for cadmium complexation is not known. The primary aim of this study was to seek a better understanding of the complexation chemistry of cadmium with a simple hydrophilic thiol, MPA. This information will help us to understand and predict the behavior of cadmium in biological systems where hydrophilic thiols such as glutathione and cysteine play a major role in detoxification mechanisms. Since MPA is a dominant hydrophilic thiol in organic-rich marine sediments, we can assess its role in binding cadmium in coastal and salt-marsh sediments, which have increasingly become polluted with heavymetals. A significant portion of the anthropogenic wastes containing heavy-metal contaminants, including industrial effluents and municipal wastes, is discharged into coastal, estuarine, and near-shore waters where they are scavenged rapidly by the underlying sediments. We used mainly a potentiometric approach ŽBreshnan et al., 1978. to measure the free-ion concentrations of either cadmium or thiol in mixtures containing both species. Electrospray mass spectrometry and extended X-ray absorption fine structure ŽEXAFS. spectroscopy were used to elucidate the structure of Cd–MPA complexes.
2. Experimental All potentiometric measurements were taken at room temperature Ž25 " 18C. with an Orion model 420A pHrmV meter ŽBeverly, MA. equipped with either an ion-selective electrode for cadmium ŽOrion model 9648 BN. or a combination AgrS electrode for free MPA ŽOrion model 9616 BN.. With these ion-selective electrodes, the concentrations are mea-
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sured on the basis that the electrode potential measured in millivolts correlates linearly with the logarithmic values of the concentrations over a wide working range from 10y6 to 10y2 M for the free ions. The AgrS electrode exhibits a linear response to thiols at pH values above p K a when the organic sulfhydryl group is completely dissociated Ž –SH –Sy. ŽTseng and Gutknecht, 1975.; typically, we used a pH of 12 for MPA measurements. For MPA, the electrode showed a linear response in the concentration range from 10y6 to 10y3 M. The ionic strength of the experimental solutions was always adjusted by adding sodium nitrate to a concentration of 0.1 M to optimize the response of the electrodes. Because thiols are oxidized rapidly in air, the experiments were conducted with argon flowing on the surface of the solution. The required chemicals Žincluding cadmium nitrate, MPA. were purchased from Aldrich ŽMilwaukee, WI.. Deionized water Žproduced by a Milli-Q ultra-pure water system, Millipore, Bedford, MA. was used in all experiments. The mass spectra were collected in negative-ion full-scan mode with a Finnigan MAT LCQ ion trap mass spectrometer fitted with an electrospray ionization source. Samples were directly infused using a 250-ml microsyringe at a flow rate of 3 mlrmin. The following instrumental conditions were used: spray voltages y4.3 kV, flow rate for nitrogen sheath gas s 50 unitsrmin; temperature of heated capillary s 200 C; lens voltages y30 V; limit for the maximum number of ions trapped was set at 2 = 10 7 counts; automatic gain control ŽAGC. was turned on with a maximum injection time of 400 ms. At the start of the experiment, the instrument was calibrated and tuned using the procedures recommended by the manufacturer. For each sample, a full-scan mass spectrum was collected for 3 min and averaged. The Cd K-edge X-ray absorption spectroscopy was performed at the X18-B beamline at the National Synchrotron Light Source, Brookhaven National Laboratory ŽUpton, NY.. An X-ray absorption spectrum is produced by the photoexcitation of an electron from a core level Že.g. 1s. to symmetryavailable outer empty states and to the continuum, i.e. beyond the sphere of influence of the atom. When the absorbing atom is surrounded by other atoms, as in any condensed phase, the outgoing photoelectron wave can be backscattered by the sur-
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rounding atoms. The backscattered waves interfere either constructively or destructively with the outgoing waves to produce either peaks or troughs with increasing X-ray energy. These oscillations constitute the EXAFS portion of the X-ray absorption spectrum and allow the determination of the local atomic environment of the absorbing element in terms of the type and number of neighboring atoms, their interatomic distances, and their disorder. The EXAFS spectra of our samples and the reference compounds ŽCdŽClO4 . 2 and CdS. were measured in fluorescence mode using a liquid nitrogen cooled, 13-element germanium detector. In order to improve the signal-to-noise ratio, the samples were cooled down to 150 K using a Displex refrigerator. The EXAFS data analysis was performed using the UWEXAFS suite of programs consisting of AUTOBK and FEFFIT ŽNewville et al., 1993; Stern et al., 1995..
3. Results and discussion Organic molecules complex with metal ions through a variety of functional groups containing heteroatoms such as oxygen Že.g. the carboxylic group., nitrogen Že.g. the amino group., and sulfur Že.g. the sulfhydryl group.. Since MPA contains both a carboxylic group and sulfhydryl group, we first established the relative importance of these functional groups in complexing with cadmium. To verify whether the carboxylic group can complex cadmium, we compared the complexing ability of MPA with that of propionic acid, a molecule similar to MPA but without a sulfhydryl group.
Cadmium ion was gradually added to a 5 mM solution of each ligand, and the extent of complexation was determined by measuring the concentration of free cadmium-ion in solution with a cadmium ionselective electrode. The pH was maintained at 7.5 " 0.1, which is representative of the values found in
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Fig. 1. Comparison of the reactivities of MPA and propionic acid with cadmium. Cadmium ion was added in 0.5 mM aliquots to 5 mM of each organic compound and the free ion was measured after each addition. Conditions: temperatures 25"0.18C; ionic strengths 0.1; pH s 7.5"0.2.
natural waters. Fig. 1 plots the measured values vs. the added cadmium concentrations in propionic acid and in MPA. With propionic acid, the measured concentrations of cadmium ion increased linearly, matching the added levels suggesting that there is no complexation between cadmium and propionic acid. However, with MPA, the measured level was nearly zero up to 5 mM of the added cadmium, and increased linearly afterwards. Therefore, the carboxylic group alone cannot complex with the cadmium ion, and the primary functional group in MPA that binds the metal ion is the sulfhydryl group. The ability of the thiol group to strongly complex with cadmium can be explained from the ease of polarizability of the ‘‘electron cloud’’ of the ligand group by the central metal cation. A common approach to recognizing the reactivity between a metal ion and an organic ligand is a classification based on their polarizing power Žchargerradius ratio. as ‘‘hard’’ and ‘‘soft’’ acids Želectron acceptors., and ‘‘hard’’ and ‘‘soft’’ bases Želectron donors. ŽPearson, 1963.. Generally, hard acids preferentially bind to hard bases, and soft acids to soft bases. Metal ions that are small and firmly retain their outer electrons are hard acids Že.g. Mg 2q and Al 3q .; they form the
strongest bonds with hard bases, which have small, highly electronegative ligand atoms with low polarizability Že.g. O-ligands.. Metal ions that are large with many filled d-orbitals are soft acids, and interact strongly with soft bases, which contain large, easily polarizable ligand atoms Že.g. S-ligands.. Cadmium is a typical soft acid; hence, it binds strongly with sulfur-ligands, such as thiols. We studied the stoichiometry of the Cd–MPA complexes formed in solution by adding either the thiol or cadmium in excess and following its free ion concentration. Fig. 2 plots the measured vs. the added concentration of MPA to a 25 mM aqueous solution of cadmium nitrate. We used a concentrated solution of the thiol, and added it in small aliquots so that the final volume was not changed significantly. As Fig. 2 shows, the measured concentration of the thiol remained nearly zero up to the addition of 50 mM, but increased linearly afterwards with the added levels. The consumption of nearly 50 mM thiol for complexing with 25 mM of the cadmium suggests that the dominant aqueous complex had a 1:2 cadmium-to-thiol ratio. The formation of the Cd–dithio complex Ž1. was further substantiated by electrospray mass spectrometry, which is increasingly used in the characteriza-
Fig. 2. Complexation of cadmium with excess MPA; plot of the measured vs. the added concentration of MPA to a 25 mM solution of cadmium nitrate. Conditions: temperatures 25"0.18C; ionic strengths 0.1; pH s12.0"0.2.
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tion of polar and hydrophilic species, including metal–ion complexes ŽD’Agostino, 1996; Alvarez et al., 1997.. In this technique, gas-phase molecular ions are generated under mild conditions at ambient temperature by applying a high voltage and are then analyzed in a mass spectrometer. We determined the negative-ion mass spectrum by directly injecting a sample of a cadmium thiol mixture containing 1 mM Cd 2q and 3 mM MPA into our Finnigan MAT LCQ bench-top mass spectrometer with an electrospray interface. For cadmium, the presence of several isotopes generally complicates the mass spectra of its compounds, but also gives much compound-specific information. The Cd–dithio complex gave a group of peaks with the dominant peak at a mass to charge ratio of 323; the distribution of peaks arises from the presence of different isotopes for the constituent atoms in the molecule. The peaks clustered at mrz 323 can be attributed to a singly charged Cd–dithio complex formed from the deprotonation of one of the carboxylic groups in the molecule Ž2..
It is important to note that in electrospray mass spectrometry, even when a molecule contains several ionizable groups Že.g. a polycarboxylic acid., the dominant peak usually results from a single ionization of the molecule transferred to the gas phase in the electrospraying process. The expected distribution of isotopic masses of this structure can easily be calculated from the isotopic composition of the constituent elements. A comparison of the observed isotopic mass distribution for the complex with the calculated values Žsee Fig. 3. is in excellent agreement, confirming the stoichiometry of the complex. An important parameter in evaluating the reactivity of thiols is the dissociation constant of the sulfhydryl group as a weak acid ŽRSH RSyq Hq .. A p K a value of 10.27 for the dissociation of the –SH group in MPA implies that the concentration of
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Fig. 3. Electrospray mass spectrum of the cadmium dithio-complex with MPA ŽInstrument: Finnigan LCQ; spray voltage: 4 kV..
the thiolate ion will be very low at acidic and neutral pH values ŽTseng and Gutknecht, 1975.. Despite such low thiolate ion concentration, the complexation of cadmium with MPA proceeds readily and stoichiometrically at this pH, suggesting that the cadmium ion has a high affinity for the thiolate ion. The reaction between Cd 2q and MPA forming the dithio complex probably occurs in two consecutive steps. In the first step, the thiolate ion reacts with Cd 2q to form a monothio complex ŽEq. 1., which then reacts with another thiolate ion to form a dithio complex ŽEq. 2.. The constants K 1 and K 2 refer to the first- and second-formation constants with the dissociated thiolate ion as the complexant; K a is the dissociation constant for the sulfhydryl group of the thiol.
| Ž Cd–SR.
Cd 2qq RSy
q
q
q
Ž Cd–SR. Ž Cd–SR. w Hq x K1 s s w Cd 2q xw RSy x w Cd 2q xw RSHx K a q
Ž 1.
|
Ž Cd–SR. q RSy Cd Ž SR. 2 Cd Ž SR . 2 K2 s q Ž Cd–SR. w RSy x s
Cd Ž SR . 2 w Hq x
Ž Cd–SR.
q
w RSHx K a
Ž 2.
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Experimental evidence for this mechanism is given in Fig. 4, which presents the results of the ion-selective electrode measurements for the concentrations of complexed cadmium vs. MPA. The values for the complexed cadmium were obtained by subtracting the amount of free cadmium-ion concentration Žmeasured with ion-selective electrode. from the original concentration of 2 mM. The slope of the curve is equal to one, suggesting that only the monothio complex was formed with excess cadmium and a limiting thiol concentration. When the thiol is in excess of the cadmium ion, the complexation proceeds to the second step forming the dithio complex. We measured K 1 experimentally by an ion-selective electrode titration of a 50 mM solution of MPA with cadmium ion at pH 7.5 " 0.1 ŽFig. 5.. The free cadmium concentration remained nearly zero up to 50.0 mM of added cadmium ion, but then increased linearly as more was added. This again demonstrates the formation of a 1:1 complex when the concentration of cadmium ion is equal to or in excess of that of MPA. At the point of 1:1 addition of Cd 2q to MPA, the concentration of free MPA should be equal to that of free cadmium, which was measured with the ion-selective electrode. The amount of cadmium ion complexed with MPA was calculated by subtracting the free cadmium-ion concentration from the total amount of cadmium added. From these values, we calculated a value of 8.8 for log K 1 using Eq. 1 at the 1:1 titration point.
Fig. 4. Complexation of excess cadmium with MPA. The plot shows a 1:1 correlation between cadmium ion and the thiol due to the formation of the monothio-complex.
Fig. 5. Complexation of excess cadmium with MPA; plot of the measured vs. the added concentration of cadmium ion to a 50 mM solution of MPA. Conditions: temperatures 25"0.18C; ionic strengths 0.1; pH s 7.5"0.2. The inset is an expanded view near the titration point.
We similarly determined K 2 by titrating a known concentration of cadmium with an excess of thiol. Because the AgrS ion-selective electrode is sensitive only to the thiolate ion and not to the undissociated thiol, the experimental quantity measured was the thiolate ion. Thus, we carried out the measurements at pH 12.0 " 0.2 when almost all the thiol exists as thiolate ions. At the titrations’ end point, the thiolate ion measured should equal the concentration of cadmium–monothio complex. The dithio complex concentration was determined by subtracting the monothio complex concentration from the initial cadmium concentration. From these results, we determined a value of 4.8 for log K 2 . The equilibrium constant for the first step in forming the Ca–monothio complex is about 4 logarithm units larger than that for the second step forming the dithio complex. These results are comparable to those reported for the complexes of glutathione with zinc, which essentially behave similarly to form dithio complexes in solution ŽPerrin and Watt, 1971.. The formation constants described for zinc–glutathione complexes were 7.94 and 4.47 for log K 1 and log K 2 , respectively. For cadmium, the large difference between the constants K 1 and K 2 suggests that the first ligand is more strongly bound to the metal ion than the second one.
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On the basis that the sulfhydryl group alone in MPA binds with cadmium ion, the monothio complex is expected to be present as a zwitterion Ž3. in aqueous solutions at ca. neutral pH because the carboxylic group should be ionized at this pH. However, we observed that this species rapidly precipitated from the solution at a pH of 7.5 in experiments
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where cadmium and thiol were initially present at millimolar concentrations. For example, in a mixture with thiol and cadmium, each at 50 mM initial concentrations, the monothio complex formed mainly precipitated, with less than 0.1 mM of the complex remaining in solution. Because the monothio complex is nearly insoluble, we think that it does not
Fig. 6. ŽA. k 2-weighted x Ž k . of Cd K-edge EXAFS in Cd–MPA Ž1:1. at 150 K. ŽB. Data, fit, and individual Cd–O and Cd–S contributions for Cd–MPA Ž1:1. at 150 K.
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exist as a zwitterion but as a charge-neutralized species, probably produced from a bonding interaction between the carboxylate group and the cadmium ion, resulting in a cyclic structure Ž4..
The nondestructive technique of X-ray absorption spectroscopy gave further information for the proposed cyclic structure for the Cd–monothio complex. Fig. 6A gives the extracted EXAFS data Ž k 2weighted x Ž k ., where k is the photoelectron wave vector. for the Cd–monothio complex. We obtained this EXAFS data from the averaged absorption coefficient after removing a smooth background function Žcharacteristic of isolated-atom absorption. using the program AUTOBK. It is well established that when x Ž k . is Fourier transformed over a finite k range from k min to k max , the result is a radial structure function exhibiting a series of peaks whose positions and amplitudes are related to the interatomic distances and the number of atoms in different coordination shells, respectively. To obtain such structural parameters, we performed a nonlinear least-squares fit on the Fourier-transformed data using the program FEFFIT. We used a superposition of Cd–O and Cd–S contributions to model the total EXAFS spectrum; the coordination numbers of S and O, their distances to the central Cd atom, and the disorder in these distances were allowed to vary during fitting. We used the scattering amplitudes and phases, characteristic for Cd–S and Cd–O interactions, that were extracted from the experimental standards, CdS and CdŽClO4 . 2 , respectively, measured at the same temperature Ž150 K. as the unknown sample. The results of the best fit gives values of 1.3 " 0.4 and 1.3 " 0.2 for Cd–O and Cd–S coordination numbers, respectively ŽFig. 6B.. The presence of one sulfur and one oxygen atoms in the first neighboring shell of cadmium as revealed by the EXAFS data strongly supports structure 4 for the monothio complex. Hence, we suggest that the carboxylic group may become involved as a secondary ligand in a chelating mode of complexation once a bond is formed between cadmium and the
sulfhydryl group. In contrast to the monothio complex, the dithio complex was completely soluble at pH 7.5. It appears that in this complex, the carboxylic groups are free, rendering it a hydrophilic structure. We presented mass spectrometric evidence in support of this structure for the dithio complex earlier. The differential solubility of the monothio and dithio complexes suggests that the type of complex formed is a crucial factor in controlling the mobility and transport of cadmium. Because the dithio complex is highly soluble, its formation will enhance the mobility of cadmium in environments in which such complex form. On the other hand, the formation of the monothio complex will immobilize the metal. These results clearly suggest that chemical structure is an important factor in controlling the mobility of cadmium in biological and environmental systems containing thiol complexants. Further studies are in progress to understand the effect of pH in influencing the structure and speciation of these complexes.
Acknowledgements This material is based upon the work supported by the NABIR program of the Office of Biological and Environmental Research, US Department of Energy and Engineering and Geosciences Program, Office of Basic Energy Sciences, US Department of Energy under contract No. DE-AC02-98CH10886 to Brookhaven National Laboratory. We thank Anatoly Frenkel for the assistance with EXAFS data analysis. Avril Woodhead and Jennifer Ayla Jay are acknowledged for the constructive comments on the manuscript.
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