Binding of Hg2+ with Phytochelatins: Study by Differential Pulse

Synopsis. Novel differential pulse voltammetric method on Au electrode, ESI-MS, and ITC are applied to study Hg2+ binding by phytochelatins (PC1−PC4...
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Environ. Sci. Technol. 2009, 43, 7010–7015

Binding of Hg2+ with Phytochelatins: Study by Differential Pulse Voltammetry on Rotating Au-Disk Electrode, Electrospray Ionization Mass-Spectrometry, and Isothermal Titration Calorimetry ELENA CHEKMENEVA, ´ M A N U E L D ´I A Z - C R U Z , JOSE CRISTINA ARIN ˜ O, AND MIQUEL ESTEBAN* Department of Analytical Chemistry. Faculty of Chemistry. University of Barcelona, Martı´ i Franque`s, 1-11, E - 08028, Barcelona, Spain

Received May 4, 2009. Revised manuscript received August 3, 2009. Accepted August 4, 2009.

The binding of Hg2+ with synthetic phytochelatins ((γ-Glu-Cys)nGly, PCn, n ) 2, 3, 4) was investigated by a recently proposed electroanalytical method, using differential pulse voltammetry on the rotating Au-disk electrode, Electrospray ionization massspectrometry (ESI-MS) and isothermal titration calorimetry (ITC). ESI-MS experiments provided the exact stoichiometries of the complexes formed at different PCn/Hg2+ ratios. Voltammetry provided more detailed information on the complexation processes through the use of multivariate curve resolution by alternating least squares of the data matrix obtained from titrations with fine increments of metal or ligand. The system Hg2+GSH-PC2 was investigated by voltammetry in order to obtain an estimation of the Hg2+ behavior in the presence of two related ligands. The additional assessment of the stability of Hg2+PCn complexes was achieved through ITC by using the therapeutic chelator sodium 2,3-dimercaptopropanesulfate (DMPS) over Hg2+-PCn systems. The stability of various Hg2+-PCn complexes and the ability of DMPS to replace PCn from these complexes were examined.

Introduction Mercury pollution is a global problem for humans and for the environment. The main Hg detoxification mechanism in ecosystems and organisms is based on thiol-containing molecules, and is due to their strong interactions with Hg (1). For example, extended X-ray absorption fine-structure spectroscopy (EXAFS) demonstrated that the reduced S groups in soil organic matter intensively participated in Hg binding (2), and that CH3Hg in fish was associated with Cys (3). Among the protective mechanisms in plants it is worth mentioning the synthesis of phytochelatins ((γ-Glu-Cys)nGly; PCn), which occurs under control of the enzyme PCsynthase from glutathione (γ-Glu-Cys-Gly; GSH or PC1) (4). The synthesis is induced by metals, such as Cd, Ag, Bi, Pb, Zn, Cu, Hg, and Au (4, 5). PCn can form complexes with Pb, *Corresponding author phone: (+34)93 403 91 17; fax: (+34) 93 402 12 33; e-mail: [email protected]. 7010

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Ag and Hg in vitro, but only the complexes with Cd, Ag, and Cu ions were detected in vivo (4). Little information is available on PCn-Hg complexes. Several studies of plants subjected to Hg stress showed an increase in Cys and PCn and a decrease in GSH contents as a result of PCn synthesis (6-8). In most studies in vivo only unbound PCn were identified, but not their Hg-complexes. The Hg accumulation mechanism was studied in Brassica napus, and only unbound PC2 was detected after the addition of the chelator DMPS (sodium 2,3-dimercaptopropanesulfonate monohydrate); the presence of polynuclear HgPCn complexes was suggested (9). However, PCn (n ) 2-4) and their Hg-complexes were detected in vivo in Brassica chinensis L. by LC-ESI-MS/MS (10). A more detailed knowledge of Hg-PCn binding characteristics in vitro is needed to understand the role of PCn in Hg detoxification in vivo as well as the nature of the species formed in plants subjected to Hg stress. As reported previously (11) a comparative study of the competitive binding of Cd2+ and Zn2+ with PC4 was carried out by the combination of differential pulse polarography with data analysis by multivariate curve resolution using alternating least-squares (DPP/MCR-ALS), ESI-MS and isothermal titration calorimetry (ITC). Here, we present a model study of Hg2+ binding with PC2, PC3 and PC4 using the same strategy. This was based on previous studies of these systems in vivo and in vitro (6-10, 12). The results confirm previous data, and new findings are reported, especially regarding the stability of the complexes, as revealed by assessment of ITC data. The use of a novel electrochemical approach using an Au-disk electrode (13), with data analyzed by a multivariate curve resolution method (MCR-ALS), provides a more precise picture of Hg2+ complexation by PCn.

Experimental Section Chemicals and Instrumentation. Phytochelatins (PCn, (γGlu-Cys)n-Gly) PC2, PC3 and PC4 as trifluoroacetates were provided by DiverDrugs S.L. (Barcelona, Spain), with purities of 91.8, 91.4, and 90.5% respectively (HPLC-MS). The impurities were some minor fragments of PCn without reduced thiol groups, with negligible impact on complexation. Reduced glutathione (GSH) and sodium 2,3-dimercaptopropanesulfate (DMPS) were from Sigma/Aldrich. Hg2+-stock solution, 0.01 ( 0.00015 M, was prepared from Hg(NO3)2 (Merck) and standardized. Borate buffer 0.02 M was prepared from Na2B4O7 · 10H2O (Sigma), and pH was adjusted to 7.5 with ultrapure HClO4 (Hg < 5 10-7%; Fluka). Ultrapure water (Millipore Milli-Q Plus 185) was used. All solutions were deoxygenated with nitrogen and subsequently stored in a tightly closed manner to prevent the oxidation of thiol compounds. Differential pulse voltammetric, DPV, measurements were performed in VA Stand 663 (Metrohm, Herisau, Switzerland) connected via IME-663 module to µAutolab type III (Eco Chemie, Utrecht, The Netherlands) with GPES software, using a rotating Au-disk electrode of 3 mm diameter (rotating speed 1500 rpm), an Ag|AgCl|3 M KCl reference electrode and a Pt auxiliary electrode (Metrohm). All measurements were carried out using a glass cell at thermostatted room temperature (20 °C). ESI-MS experiments were performed using an LC/MSD TOF (Agilent Technologies, Inc.) instrument with direct injection of the sample. Isothermal titration calorimetry (ITC) measurements were carried out with a VP-ITC MicroCal titration calorimeter (MicroCal, Inc., Northampton, MA). 10.1021/es901325f CCC: $40.75

 2009 American Chemical Society

Published on Web 08/17/2009

Procedures. DPV titrations were performed as follows: 25 mL of buffer solution was poured into the cell and purged with nitrogen for 40 min, and the DPV curve was recorded (blank). Afterward, Hg2+ or ligand (direct or inverse mode, respectively) were added to obtain a final concentration of 1 × 10-5 M, and the corresponding voltammograms were recorded to ensure reproducibility. The aliquots of ligand or Hg2+ (stock solution 1 × 10-3 M) were added to the initial solution. After every addition the solution in the cell was purged with nitrogen for 1 min, and the DPV curve for each addition was registered. For all measurements the DPV parameters were as follows: voltage step 0.005 V s-1 and modulation amplitude 0.05 V. The electrode was electrochemically cleaned in situ by applying a potential of 1.2 V for each ligand/Hg2+ ratio for 60 s, which was high enough to ensure Hg2+ desorption. This was checked with reproducible blank experiments with Hg2+ solutions. After cleaning the electrode, the deposition potential 0.2 V was applied for 60 s followed by 5 s of equilibrium period and cathodic potential scan between 0.2 and -1.5 V. In positive ESI-MS experiments, direct sample injections (10-50 µL) in pH 7.5 ammonium acetate/ammonium hydroxide buffer were carried out at a flow rate of 0.2 mL min-1 at a source temperature of 300 °C. The applied potential was fixed at 4000 V for the capillary and at 250 V for the fragmentor. MS-spectra were collected throughout an m/z range of 100-1500. A 1:9 acetonitrile (HPLC grade): ammonium acetate (5 mM) mixture was adjusted at pH 7.5 and used as mobile phase. These buffer and mobile phase solutions proved to be adequate for this kind of experiments with metal complexes (11). The mixtures of thiols and Hg2+ (1 × 10-5 M) were prepared in buffer medium with a range of M/L proportions (1:2, 1:1, 2:1 and 2:3). ITC measurements were carried out at 25 °C as follows: the mix of Hg2+ with PCn or DMPS in a certain proportion (2 10-5 M) in 0.02 M borate buffer pH 7.5 was placed in the cell, and the automatic titration was then performed with DMPS or PCn, respectively, (2 10-4 M) in the same buffer. A typical ITC measurement was carried out as reported elsewhere (11). More detailed experimental information on the preparation of Au-disk electrode is given in the Supporting Information (SI). Data Treatment. Mass-spectra deconvolution was performed by means of Analyst QS software (Applied Biosystems). Electrochemical data was prepared following the procedure, as described elsewhere (11). The Multivariate curve resolution by alternating least squares (MCR-ALS) method is summarized elsewhere (14) and available at http://www.ub.edu/mcr/ndownload.html. All programs are written in Matlab (15). The fitting of ITC data to different binding models by nonlinear least-squares approach (Levenberg-Marquardt algorithm) was done with MicroCal Origin software (16).

Results and Discussion ESI-MS Experiments. Direct-injection ESI-MS is a powerful technique to obtain the stoichiometries of Cd- and Zn-PC4 complexes (11). The soft energies applied in ESI-MS do not allow significant fragmentation of metal-ligand units, and facilitate the identification of complexes of biological molecules (17-19). Table 1 summarizes the complexes detected for Hg-PCn (n ) 2-4) systems at different Hg2+/PCn ratios: only one complex for PC2, and two for PC3 and PC4. SI Figure S1 shows representative spectra. For PC2, the same Hg(PC2) complex previously reported (10) was detected, but here free PC2 was detected at both PC2

TABLE 1. Hg-phytochelatin Complexes Signals Observed in ESI-MS Spectra. Hg2+ (1 × 10-5 M) in Ammonium Acetate/ Ammonium Hydroxide Buffer pH 7.5 phytochelatin

Hg: phytochelatin

complexes (m/z)

PC2

2:1 1:2 1:1

Hg(PC2) (740,09) PC2 (538,12) Hg(PC2) (740,09)

PC3

2:1 1:1 1:2

Hg2(PC3) (1170,09) Hg(PC3) (972,14)

PC4

2:1 1:1 1:2

PC4 (1004,24) Hg(PC4) (1204,19) Hg2(PC4) (1402,14)

and Hg2+ excesses (see SI Figure S1). The presence of PC2 signal, even at very low proportion, when Hg2+ is in excess, is surprising. Although ESI-MS provides precise information on the complex stoichiometries, the transfer of species from the liquid to the gas phase may lead to the rupture of several bonds. At Hg2+ excess, the less stable Hg2(PC2) species is formed predominantly, as can also be seen from voltammetric data. In Hg2(PC2) each Hg2+ ion is bound to one thiol group. The Hgsthiol bonds are highly labile and Hg2+ is continuously interchanged between thiols, as observed by NMR studies (20). Moreover, a toxicological study of different Hg-thiol complexes also pointed out this reactivity: 1:1 Hg2+-thiol complexes with Cys, Cys-Gly, GSH, and albumin were as toxic as uncomplexed Hg2+ for the LLC-PK1 renal cells (21). In Hg2(PC2), Hg2+ conserves a great deal of reactivity, as ITC results demonstrate (see below). Thus, the presence of a small fraction of PC2 at Hg2+ excess could be a consequence of a partial rupture of Hg-thiol bonds in Hg2(PC2). For PC3, Hg(PC3) was observed as the predominant species at 1:1 and 1:2 Hg2+/PC3 ratios, and Hg2(PC3) only at Hg2+ excess (SI Figure S1b). Both Hg(PC3) and Hg2(PC3) have been reported elsewhere (10, 12). For PC4, both Hg(PC4) and Hg2(PC4) were reported (12), in agreement with present results (SI Figure S1c). Moreover, both one- ((MnLm+1H+)/1) and two-charged ((MnLm+2H+)/ 2) species were detected. The free PC4 was also observed in these spectra (as in the case of PC2) in a low proportion. For Hg(PC2), Hg2(PC3), and Hg2(PC4) complexes, the digonal Hg2+ coordination was suggested (12). On the basis of UV/vis spectra, trigonal and tetrahedral coordination was proposed for Hg(PC3) and Hg(PC4), respectively (12). It is worth mentioning that, for equimolar PCn mixtures, at fixed Hg2+ concentration, the most prominent species were Hg(PC3) (m/z ) 972.14) and, to a lesser extent, Hg(PC4) (m/z ) 1204.19). This is coherent with HPLC results on Hg2+transfer from shorter- to longer-chain PCn (12): when Hg2(PC3) was added to PC4, Hg2+ was transferred to PC4 giving rise to its Hg2+ complex, while Hg(PC3) persisted in solution. This could mean that PC3 provides trigonal conformation to Hg2+ and that Hg(PC3) should be stable. Voltammetric Experiments. The experimental scheme of voltammetric measurements includes the deposition step during which Hg2+-complexes are adsorbed onto the electrode at a potential (0.2 V) which does not allow reduction of free Hg2+ (13). Both thiols and Hg2+ are readily adsorbed on an Au electrode (22). Moreover, higher affinity of Hg2+ for thiols ensures the primary formation of Hg-thiol complexes followed by their adsorption on the Au electrode (23). Here the method is applied to study the Hg2+-PCn systems. Hg-PC2 System. In this method, the initial Hg2+-reduction signal is split (Figure 1). The first signal (at ca. 0.1 V) should correspond to the adsorbed Hg2+ reduction, whereas the second (at ca. -0.1 V) should represent the reduction of Hg2+VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Differential pulse voltammograms obtained during the titration of 1 × 10-5 M Hg2+ with 1 × 10-3 M PC2 in borate buffer at pH 7.5 (PC2/Hg2+ from 0 to 1.6). Deposition potential: 0.2 V during 60 s; scan rate: 5 mV s-1; equilibration time: 5 s. ions from the solution. The higher the concentration, the greater the split (13). It seems that at 0.2 V the layer of adsorbed Hg2+ ions is formed on the Au electrode surface, and then Hg2+ from the solution is reduced. With PC2 additions, Hg2+-reduction peaks gradually decrease. Although it is difficult to perceive, a small new peak appears at ca. -1 V (Figure 1). At [PC2]/[Hg2+] > 0.5 this peak decreases while new large peaks develop at more negative potentials. These signals are related with cathodic reductive desorption of thiols from Au-disk electrodes (24). They are taken into account because their changes depend on the complexation process, and help in data interpretation. In order to extract the maximum information from these data, the MCR-ALS optimization method was applied to data from Figure 1. For the satisfactory explanation of the system, six components are considered after singular value decomposition (SVD) analysis. Figure 2 shows the normalized individual voltammograms (a) and concentration profiles (b) obtained. A satisfactory mathematical resolution was reached, with a lack of fit (lof) of 6.76%. Components I and 2+ (comII are related to the free Hg2+ reduction. The Hgsoln ponent II) is almost totally bound at ratio of 0.5, whereas 2+ (component I) disappears at ratios slightly higher than Hgads 1. This coincides with the Hg(PC2) complex revealed by UV/ vis spectroscopy (12), i.e., Hg2+ bonded to two sSH groups in the same PC2 molecule. Although the concentration profile of component III is not very well-defined, it reaches a plateau in the ratio region of ca. 0.5, that is, when there is one sSH group per Hg2+. Component III is coherent with the hypothetical formation of Hg2(PC2), which was not detected from either our ESI-MS data or previous data (10). Component IV stabilizes at the PC2/Hg2+ ratio of ca. 1.2. This is coherent with the formation of the more stable Hg(PC2), as its more negative unitary voltammogram demonstrates. Formation of Hg(PC2) was detected by ESI-MS (Table 1). Components V and VI, whose signals stabilize at the same ratio of ca. 1.2, may be responsible for the cathodic reductive desorption (24) of Hg-PC2 complexes and the excess of PC2. When PC2 is titrated with Hg2+, initially only a large peak at ca. -1.4 V was observed. It persisted up to the Hg2+/PC2 ratio of 0.5, when it began to decrease. Until the Hg2+/PC2 ratio of 1 a stable Hg(PC2) complex should be present in solution. SI Figure S2 shows the signals observed at the ratios higher than 1. With more Hg2+ additions, a new small peak appeared, at -1 V, due to Hg2(PC2) formation. Free Hg2+ reduction was also observed at these ratios, which again shows the lability of Hgsthiol bond. 7012

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FIGURE 2. Normalized individual voltammograms (a) and concentration profiles (b) obtained from MCR-ALS optimization of the experimental data set from Figure 1 applying the non-negativity, signal shape and selectivity constraints. The concentration profiles are plotted versus [PC2]/[Hg2+] (lower axis) and versus [SH]/[Hg2+] (upper axis) molar ratios. Hg2+-PC3 and Hg2+-PC4 Systems. The system Hg2+-PC3 was analyzed through DPV titrations of Hg2+ with PC3 (SI Figure S3) and opposite (results not shown). Further MCRALS analysis of data yielded quite satisfactory results (SI Figure S4). These data allow us to estimate when the free Hg2+ disappears and then calculate the complex stoichiometries. Moreover, the evolution of the signals appearing at more negative potentials also allows analysis of the complexation process. Free Hg2+ disappears almost totally when there are two thiol groups for each Hg2+ ion (see the concentration profiles in SI Figure S4). Again, this is coherent with the high stability of Hg(thiol)2 complexes. Analogies can be drawn between Hg-PC2 and Hg-PC3 for the signals appearing at the negative potentials region. For PC3 these signals are more negative. This can be explained by the larger size, and the higher number of thiol groups, of PC3 as compared to PC2. PC3 molecules can block the electrode surface and hinder the transfer of metal ions to the electrode. Experimental data seem to support the initial formation of Hg3(PC3), where each Hg2+ is bound to one sSH group. This statement is based on MCR-ALS analysis (see SI Figure S4): component III, assigned to the peak at ca. -1.2 V, has similar characteristics to component III (at ca. -1 V) in Hg-PC2 systems. Although evolution of its concentration profile is not so well-defined, it may be related to Hg3(PC3). Two other components (IV and V, SI Figure S4), as in Hg2+-PC2, stabilize when Hg2+ can

FIGURE 3. Differential pulse voltammograms registered in the titration of 1 × 10-5 M PC2 with Hg2+ (a), 1 × 10-5 M GSH with Hg2+ (b) and 1 × 10-5 M GSH -1 × 10-5 M PC2 mixture with Hg2+(c). In (a) and (b), the numbers correspond to the Hg2+/PC2 or Hg2+/GSH ratios. In (c) the shown curves correspond to the Hg2+/GSH/PC2 mixture ratios of 1:1:1 (1), 2:1:1 (2), 2.75:1:1 (3), 3:1:1 (4), and 3.5:1:1 (5). In insert in (c), [mixture] ) 1 × 10-5 M. In all cases: borate buffer pH 7.4; deposition potential: 0.2 V during 60 s; scan rate: 5 mV s-1; equilibration time: 5 s. be bound by more than one thiol groups. From their concentration profiles the formation of Hg2(PC3) complex can be deduced, which was also detected by Mehra et al. (12) and in our ESI-MS measurements (Table 1). The formation of Hg(PC3), which was detected in either our ESI-MS measurements or elsewhere (12), cannot be deduced from DPV data. The titration of Hg2+ with PC4 showed that free Hg2+ disappeared when the PC4/Hg2+ ratio was 0.5, which points to the formation of Hg2(PC4). Besides this complex, Hg(PC4) and PC4 were also detected by ESI-MS (Table 1). The global DPV behavior was similar to that of PC3. Hg-PC2-GSH System. Voltammetric measurements of an equimolar GSH-PC2 mixture were done to obtain an additional assessment of their Hg2+-complexing ability. Previous HPLC measurements reported that the stability of Hg2+ complexes with GSH and PCn follows the sequence GSH < PC2 < PC3 < PC4 (12). A recently proposed electroanalytical method (13) provides the opportunity to study the complexation processes that could take place in a mixture of several ligands. We chose GSH and PC2, as they present the

voltammetric signals of Hg2+ complexes at less negative potentials, and also due to their involvement in Hg2+ detoxification in plants (6, 7, 9). Individual titrations of GSH and PC2 by Hg2+ (Figures 3a and 3b) show Hg2+ reduction signals at -0.815 and -1.017 V from the Hg(GSH) and Hg2(PC2) complexes respectively. These complexes are formed when there are less than two thiol groups for each Hg2+. As examples, Figure 3a and b show characteristic DPV curves for Hg2+/GSH and Hg2+/PC2 ratios of 1.05 and 1.69, respectively, which guarantee the formation of above complexes. For an equimolar GSH-PC2 mixture (Figure 3c), which has three thiol groups per mixture unit, multiple Hg2+additions were needed to decrease the desorptive signals appearing at potentials more negative than -1.2 V. Initially, the stable Hg(GSH)2 and Hg(PC2) species, detected by ESIMS (Table 1), are formed. In both species Hg2+ is strongly bound by two thiol groups. Simultaneously, the Hg2+reduction peak drastically increases from the [Hg2+]:[GSH]: [PC2] ratio of 2:1:1, that is, from more than one Hg2+-ion for two thiol groups (0.67 Hg2+ per thiol group). At this moment VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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the formation of less stable Hg(GSH) and Hg2(PC2) species should occur. When the ratio is higher than 3 (i.e., more than one Hg2+-ion for each thiol group of the mixture; curve 5 in Figure 3c), two separate peaks are clearly observed at the -0.8/-1.1 V potential region, as expected. The fine analysis of the data from Figure 3c reveals that Hg(GSH) complex forms ahead of Hg2(PC2), as the [Hg2+]/[mixture] scale in the insert in the Figure 3c shows. Fewer Hg2+ additions are needed to transform Hg(GSH)2 into Hg(GSH) than to transform Hg(PC2) into Hg2(PC2), as the peak at -0.8 V appears earlier than that at ca. -1 V. Thus, Hg(GSH)2 seems to be less stable than Hg(PC2). These results were further confirmed by the results obtained in the titration of Hg2+ with an equimolar mixture of GSH and PC2 (SI Figure S5). The larger thiol-containing ligands may provide extra intramolecular stability to the complexes (e.g., Hg(PC2)), in comparison with the complexes which involve more than one ligand moiety to achieve the same Hg conformation (e.g., Hg(GSH)2). The analysis of Hg2+ interactions with PC3 and PC4, explained below, also provides evidence in favor of this hypothesis. ITC Experiments. The main advantage of ITC is its ability to characterize any type of interaction providing, in one experiment, the values of complex stoichiometry (n), stability constant (K), and enthalpy (∆H). However, it has some limitations related to the order of magnitude of the interaction measured. Thus, extremely high stability constants, such as those associated with many Hg-thiol interactions, cannot be measured directly. Then, competitive binding, using proper reactants, is the common approach for such cases. In the present case, in order to try to characterize Hg-PCn complexes, DMPS was used as competitive ligand. This choice was based on previous studies for the determination of unbound and complexed PCn by Hg2+ in plant cultures, using HPLC-MS/MS, where DMPS (one of the most effective chelators for heavy metals in animals and humans) was used, resulting in the detection of only PC2 (9). On the basis of these results, the authors affirm that only PC2 is responsible for Hg2+ detoxification in the plants studied (9). The replacement of PC2 by an excess of DMPS could be explained by the relatively similar stability of PC2 and DMPS complexes with Hg2+, as both provide linear geometry with two thiol groups for Hg2+ ion. It is also well documented that DMPS is not an optimal chelator because only species with participation of several DMPS units are formed (25). When 1:1 Hg2+/DMPS ratio, the Hg2(DMPS)2 complex is formed, where Hg2+ reaches practically linear two-thiol coordination. In the present study, owing to the high stability of the species formed, ITC allowed us to characterize the type of interaction and to obtain only an estimation of the parameters. Hg-PC2 System. Figure 4 presents the experimental curves obtained during the ITC titration of Hg2+-PC2 mixtures, at various proportions, with DMPS. This plot shows that DMPS efficiency increases with Hg2+ proportions over PC2. When 2:1 Hg2+/PC2 (i.e., when the less stable Hg2(PC2) species is formed as it was commented above,) the first process is very exothermic (curve 3), with a stoichiometric value of ca. 0.25 (by assuming a binding model of two set of sites). The second process, occurring at higher molar ratios, is less exothermic, and the stoichiometric value is ca. 1, which points to the formation of the 1:1 Hg-DMPS species. At 1:1 Hg2+/PC2, curve 2 shows that Hg(PC2) (which must be formed at this M/L proportion) is only partially decomposed by DMPS, which can only partially replace PC2 from its Hg2+-complexes. When PC2 is in excess over Hg2+(curve 1), the interaction observed is too weak as compared with previous cases, and presumably a large excess of DMPS is needed to replace PC2. Hg-PC3 System. When 1:1 Hg2+/PC3 solution was titrated with DMPS, no interaction was observed (results not shown). 7014

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FIGURE 4. Isothermal titration calorimetric curves obtained in the titration of Hg2+-PC2 solutions, at different proportions, with DMPS at 25 °C in borate buffer pH 7.5. Proportions Hg2+/ PC2: 1:3 (triangles, 1), 1:1 (circles, 2), 2:1 (squares, 3). Syringe: 2 10-4 M of DMPS; calorimetric cell: 2 × 10-5 M of Hg2+. The calorimetric titration was performed for 10 µL injections with 240 s of spacing between successive additions. This finding confirms the assumption about the high stability of the complex. When the ratio Hg2+/PC3 is 2:1, an interaction between Hg2+ and DMPS takes place (see SI Figure S6). The reaction is highly exothermic. As for PC2, the obtained stoichiometric value of ca. 0.25 indicates that only part of Hg2+ bound with PC3 interacts with DMPS. The absence of interaction in the first case indicates that PC3 may be a better chelator than DMPS. Thus, inverse titrations were carried out. 1:1 and 1:4 Hg2+/DMPS mixtures were titrated with PC3. For the 4-fold excess of DMPS over Hg2+, the Hg(DMPS)4 complex was reported (25). This structure is very stable, and no interaction was observed when PC3 was added to this solution (results not shown). In contrast, when the 1:1 Hg2+/ DMPS solution was titrated with PC3, the interaction occurred (see SI Figure S7). From ITC findings it can be assumed that DMPS cannot liberate Hg2+ from its PC3 complexes. Thus, they may have remained unchanged when DMPS was used to detect unbound PCn.

Acknowledgments We gratefully acknowledge financial support from the Spanish Ministerio de Ciencia e Innovacio´n (CTQ2006-14385-C0201). E.C. acknowledges the University of Barcelona for a Ph.D. grant. We also acknowledge the assistance of Dra. Irene Ferna´ndez and Sra. Laura Ortiz in ESI-MS experiments. We thank anonymous reviewers for their extremely valuable recommendations.

Supporting Information Available Description of some experimental procedures is available along with additional experimental data concerning ESI-MS experiments, electrochemical studies and ITC measurements. This material is available free of charge via the Internet at http://pubs.acs.org.

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