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Comparison of the zinc–cadmium exchange properties of the metallothionein related peptide {Lys–Cys–Thr–Cys–Cys–Ala} and a zinc-containing metallothionein: study by voltammetry and multivariate curve resolution
Journal of Electroanalytical Chemistry 523 (2002) 114– 125 www.elsevier.com/locate/jelechem
Comparison of the zinc–cadmium exchange properties of the metallothionein related peptide {Lys–Cys–Thr–Cys–Cys–Ala} and a zinc-containing metallothionein: study by voltammetry and multivariate curve resolution M.S. Dı´az-Cruz, M.J. Lo´pez, J.M. Dı´az-Cruz, M. Esteban * Departament de Quı´mica Analı´tica, Facultat de Quı´mica, Uni6ersitat de Barcelona, A6. Diagonal 647, E-08028 Barcelona, Spain Received 26 September 2001; received in revised form 28 January 2002; accepted 6 February 2002
Abstract The complexation of the hexapeptide Lys–Cys–Thr– Cys– Cys– Ala (FT), peptidic fragment {56– 61} in the a-domain of mouse liver metallothionein I (MT), in the simultaneous presence of Cd(II) and Zn(II) is compared with that of a Zn-containing mammalian MT (Zn–MT II) in the presence of Cd(II). The influence of Cd(II) is studied by square wave voltammetry and differential pulse polarography, at different Cd:Zn:FT molar ratios, which yield a rough picture of the metal exchange processes taking place. The application of multivariate curve resolution with alternating least squares optimization (MCR-ALS) allows both the unitary voltammograms and the concentration profiles to be obtained. Moreover, MCR-ALS is applied for the first time to the study of the complexing properties of a complete MT. On the basis of these results, the electrochemical reduction processes and some structures are depicted for the chemical species considered in the mixed metal system Cd– Zn– FT. In the case of Cd–Zn–MT II, additional information is required before proposing similar models. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Metallothioneins; Complexation; Metal exchange; Voltammetry; Cadmium; Zinc; Multivariate analysis; Deconvolution
1. Introduction Metallothioneins (MT) are of great biological interest due to their capability to bind metals with a d10 configuration, e.g. Zn, Cd or Hg [1,2]. MT can be induced by diverse stimuli such as metal uptake, stress, or some drugs. The characterization of MT presents several challenges that include the identification of metals involved in the complexes, the determination of the corresponding stoichiometries, the precise identification of the aminoacid sequence and the characterization of the chemical species present in solution. Some small peptides containing cysteine (Cys) residues at the proper positions are known to exhibit behavior similar to that of MT. This is the case for the peptidic fragment Lys – Cys – Thr – Cys – Cys – Ala {56 – * Corresponding author. Tel.: + 34-93-402-1277/1286; fax: +3493-402-1233. E-mail address: [email protected] (M. Esteban).
61} of mouse liver MT I (hereinafter denoted by FT), which has been widely studied as a simpler model of MT and of the a-domain in mammalian MT. The study of Cys-containing peptides such as glutathione (g-L-glutamyl-L-cysteinylglycine, GSH) and FT, in the absence and in the presence of Zn(II) or Cd(II), has recently been carried out by means of several electrochemical techniques such as differential pulse polarography (DPP), linear sweep voltammetry (LSV), cyclic voltammetry (CV), direct current polarography (DCP) and square wave voltammetry (SWV) [3–17] and, very recently, anodic stripping voltammetry [18]. The major advantages of SWV are its speed and its excellent sensitivity [19]. On the other hand, DPP represents a good alternative especially when very high sensitivity is not needed and the differences between the peak potentials are large enough to obtain well-resolved peaks. Because of all these reasons, both techniques have been widely used in the study of MT and related compounds. An additional reason to use DPP and
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SWV is that these techniques allow very low metal concentrations to be used, where potentiometry with ion-selective electrodes is not reliable enough, thus avoiding complications due to precipitation phenomena. Due to the rather complex voltammograms obtained in these studies, and the further difficulties in both data treatment and interpretation, the use of a multivariate data analysis method was recently proposed [5]. The multivariate curve resolution with alternating least squares optimization method (MCR-ALS) is based on the use of factor analysis techniques. It was originally developed for spectroscopic data [20], modified and adapted to voltammetric signals (current-potential sets of data) because of their specific characteristics [5]. Since then, it has been progressively improved by including new constraints in the ALS optimization [14,21,22]. In particular, MCR-ALS has been applied in the study of the Cd– GSH [5,6,17], Cd– FT [11,12], Zn –GSH [7] and Zn – FT [13] systems by DPP, LSV and CV, and in the study of the mixed metal Cd–Zn – FT system by DPP and SWV [15,16]. The last DPP study [16] reports the exchange experiments when Cd(II) is added to a 2:2 Zn:FT solution. At this concentration ratio the maximum stoichiometric proportion, Zn2FT2 is reached, and no free Zn(II) is initially present. Under these experimental conditions, the complex Cd2ZnFT2 is formed. Addition of Zn(II) to a 1:2 Cd:FT solution was also investigated, which led us to propose the formation of the CdZn2FT2 complex. These results indicate that the metal initially bound to the FT molecule cannot be totally released, even when a great excess of the other metal is added. In a previous paper [15], the total metal:FT ratios found for the complexes formed were higher than those reported here. In that study FT was added to a mixture of Cd and Zn at diverse initial proportions. Thus, the excess of metal initially present can provoke the saturation of sites in FT as soon as it is added, hindering further entrance of metals. In the present work, the metal binding behavior of FT in the simultaneous presence of Cd(II) and Zn(II), at several Cd/Zn molar ratios, has been studied by SWV and analyzed by MCR-ALS. Once the Cd–Zn – FT system has been understood in depth, a mammalian Zn-containing MT II is analyzed, to compare its behavior and to check the capabilities of MCR-ALS. In principle, ZnMT II represents a higher level of system complexity for the application of MCR-ALS to electrochemical data with respect to GSH and FT, where only 1 and 3 cysteines were involved, respectively. The DPP technique was selected for Zn-MT II studies in order to prevent the protein’s known adsorptive capacity onto the electrode surface (mainly due to its high cysteine content). SWV was preferred in Cd–Zn – FT studies because previous experience showed the
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poor resolution of polarographic techniques when diverse peaks are detected in the Zn-region and since only three thiol groups can lead to adsorption phenomena.
2. Experimental
2.1. Chemicals and instrumentation Lys –Cys –Thr –Cys –Cys –Ala acetate salt, containing mercaptoethanol as a stabilizer, and rabbit liver ZnMT II (7% Zn content, M= 9542 D) were obtained from Sigma Chemicals. Previous studies showed that the low mercaptoethanol content (below 5%) in the peptide sample does not interfere significantly [3,4]. Standard solutions of cadmium, lead and zinc (Titrisol), with a metal concentration of 1.000 g l − 1 9 2 mg l − 1, were purchased from Merck. All other reagents were of analytical grade. The samples were freshly prepared with ultrapure water (Millipore MilliQ Plus 185 system) and used immediately. A 0.13 M borate buffer solution at the desired pH was used as supporting electrolyte. The choice of this buffer solution followed the procedure outlined in previous studies [10–13,16]. Purified nitrogen was used for deareation. SWV and DPP measurements were carried out with an Autolab System potentiostat (Ecochemie, The Netherlands) coupled to a stand polarographic 663 VA (Metrohm, Switzerland) and attached to a personal computer implemented with a GPES version 2.0 software package (Ecochemie). The system was also connected to a Metrohm 665 dosimat for the automatic addition of standard solutions. The SWV instrumental parameters that ensure the best definition of the peaks are: amplitude of 15 mV, step potential of 2 mV and frequency of 75 Hz. The DPP characteristic parameters were: a pulse duration of 40 ms, pulse amplitude of 25 mV, drop time of 1 s and scan rate of 6 mV s − 1. In all experiments, the working, reference and auxiliary electrodes were the static mercury drop electrode (SMDE), Ag AgCl 3 M KCl and glassy carbon, respectively. pH was controlled with an Orion 920 A pH-meter (Orion, Boston, MA) equipped with a combined glass electrode.
2.2. Procedure 2.2.1. Experiments at fixed pH and different metal-to-ligand ratios An aliquot of 10 ml of the buffer solution was deaerated for 20 min with a N2 current and then a voltammogram (SWV or DPP) was recorded. Aliquots of the FT– or Zn –MT II-standard solutions were added to the cell in order to reach the desired initial concentration ratios. Then, the solution was stirred,
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deaerated for 1 min, and a new voltammogram was recorded. Replicate measurements were done to ensure the repetitivity of the voltammograms. Later, aliquots of the Cd(II)-stock solution are added to the cell in order to achieve a wide Cd-to-ligand concentration ratio range. When required, an aliquot of Zn(II)-standard solution is added (spiked) previous to Cd(II) additions. After each addition, the solution was stirred and deareated for 1 min, and a new voltammogram was recorded. During the measurements, a N2 stream was passed over the surface of the solutions to ensure an inert atmosphere. Very reproducible results were obtained after repetitive titrations, even after waiting periods (between the cadmium addition and recording of the voltammograms) greater than 1 min.
2.2.2. Experiments at different pH and fixed metal-to-MT ratio Ten milliliters of buffer solution at pH 4.0 were placed in the cell, deaerated for 20 min and the DPP scan was recorded. The Zn– MT II stock solution was added to the cell and a new voltammogram was registered. Further measurements were performed after every addition of a standard NaOH solution in order to increase the pH value. Two initial Zn:Zn –MT II concentration ratios were selected: the one as supplied (native rabbit liver MT II), which corresponds to 7:1 Zn:MT, and a Zn-spiked sample which assures an initial excess of Zn(II) in the medium (corresponding to a 7:1 Zn:Zn– MT II). 2.3. Data treatment Voltammograms are analyzed by two different approaches. One is based on multivariate analysis by means of MCR-ALS, and it is applied to experiments performed at fixed pH. The other approach is an individual voltammogram deconvolution, which is applied to experiments at fixed metal-to-ligand concentration ratio but changing pH. In both approaches, as a preliminary step, the voltammograms are smoothed and a point-by-point subtraction of the background current (obtained for the supporting electrolyte) is accomplished. In the MCR-ALS approach, after this preliminary treatment, the set of voltammograms is arranged in a data matrix of currents. This matrix contains as many rows as the number of voltammograms recorded at each metal-to-ligand ratio and as many columns as potentials scanned in every run. Once the matrix is composed, the data treatment follows the MCR-ALS method (described elsewhere [5,21]) implemented using the MATLAB computer graphics environment program [23]. MATLAB programs for MCR-ALS are available at the web site http://www.ub.es/gesq/ mcr/mcr.htm.
In the second approach, which is a univariate method, all voltammograms are deconvoluted (one by one) by using PeakFit software [24]. Inside PeakFit options, method I (based on the analysis of residuals) and the power peak equation (for signal shape) are used. The reason for using this simple univariate method instead of the more complex and powerful MCR-ALS is that the data matrices resulting for pHvariation experiments do not follow the linear model required for the application of MCR-ALS [6].
3. Results and discussion
3.1. Study of the Cd–Zn –FT system as analyzed by MCR-ALS 3.1.1. Additions of Cd(II) to a 0.75:1 Zn:FT solution The voltammograms corresponding to the titration of a 0.75:1 Zn:FT solution with Cd(II) are shown in Fig. 1a. The voltammogram before the addition of Cd(II) presents two broad peaks at ca. − 0.5 and −1.1 V. According to previous studies [3,4,16], the very broad peak at ca. − 0.5 V is related to the electrochemical oxidation of the mercury from the electrode in the presence of either free thiol groups of FT or thiol groups complexed with Zn, and the further reduction. The peak at ca. −1.1 V is attributed to the reduction of Zn-containing species. After the progressive addition of Cd(II), the initial peak at −0.5 V decreases steadily, and a new peak at even less negative potential begins to grow. This peak is related to the oxidation of the mercury in the presence of the new Cd–Zn –FT complex and its further reduction [25]. Thus, the sequence of potentials is in agreement with the apparent stability constants (K%Hg – SH \ K%Cd – SH \ K%Zn – SH) previously established [25]. Given the aim of the present study, we will focus our attention on the metal reduction regions, and further mention of these Hg related peaks will be made only when strictly necessary to understand the behaviour of the systems studied. In the Zn– region (potentials from −1.0 to −1.4 V), the broad peak initially present is not modified significantly after a few Cd(II) additions. Simultaneously, in the Cd-region (from −0.55 to − 1.0 V) some new peaks appear successively from the most negative (at ca. −0.9 V) region until they reach that due to the reduction of free Cd(II) (at ca. −0.59 V). Because of the highly changing number and shape of the peaks, this behavior cannot be explained directly, and the help of MCR-ALS is needed. The application of the singular value decomposition (SVD) method, which is the first step in the MCR-ALS approach [5], shows that an unreasonably high number of components is required to explain the experimental data varia-
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tion. Despite this fact, the full MCR-ALS data analysis was completed, but the results reached (data not shown) were not consistent enough.
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likely due to the high number of components needed, as happened in the 0.75:1 Zn:FT case.
3.2. Simultaneous MCR-ALS analysis of both series 3.1.2. Additions of Cd(II) to a 1.1:1 Zn:FT solution When the Zn-FT system contains an excess of Zn, i.e. free Zn(II) is present, the direct interpretation of the voltammograms (Fig. 1b) is even more chancy. Now, at least two peaks related to Zn, and three peaks related to Cd can be distinguished. The Zn-region is not significantly modified during Cd(II) additions. Again, the MCR-ALS analysis yields unsatisfactory results to explain the experimentally observed behavior,
The voltammograms of each titration have several common points. At least six peaks from both experiments seem to be related directly to each other. Thus, data can be joined in a column-wise augmented data matrix and treated simultaneously to overcome the uncertainties found in the individual analysis. Nine mathematical components were concluded after SVD analysis (results not shown). Starting at this esti-
Fig. 1. SWV data set for the titration of a 4.8 × 10 – 6 M Zn(II) and 6.6 ×10 – 6 M FT solution (a) and a 7.2 ×10 – 6 M Zn(II) and 6.6 ×10 – 6 M FT solution (b) with a 10 − 4 M Cd(II) solution, in borate buffer at pH 8.0, until a final Cd(II) excess. First voltammogram in each titration is shown separately.
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Fig. 2. Normalized individual voltammograms (a) and concentration profiles (b and c), obtained in the MCR-ALS decomposition of the augmented data matrix composed of data shown in Fig. 1 by imposing selectivity and non-negativity constraints for concentrations, and non-negativity and signal-shape constraints for voltammograms. (b) and (c) visualize the individual matrices obtained after the constrained ALS optimization for the experiments at 0.75:1 and 1:1 Zn:FT, respectively. For the sake of simplicity, concentration profiles of components 1 and 2 are omitted.
mation, ALS decomposes the current data matrix into one matrix containing the normalized individual voltammograms for each mathematical component (Fig. 2a), and one augmented matrix containing the concentration profiles (currents vs. Cd-to-FT ratios) for such components. This augmented matrix is visualized through the individual matrices from each experiment (Fig. 2b and c). In order to simplify the visualization of the unitary voltammograms, they are normalized to one. As a consequence, the y-axis values in the concentration profile plots have arbitrary units. As described elsewhere [5,21], some constraints must be included along the ALS optimization in order to achieve (electro)chemically satisfactory results. The constraints were: selectivity and non-negativity for concentrations; non-negativity and peak-shape-constraint for voltam-
mograms. The lack of fit after the constrained ALS optimization is 14.78%. The peak potential values of the normalized individual voltammograms (Fig. 2a) seem to be fully coherent with the experimentally observed global signals (Fig. 1). It must be remarked that component 7 is attributed to the reduction of free Zn(II) even when small differences in the peak potential, with respect to the signal for a solution of free Zn(II) (Fig. 1b), are detected. This situation has been also found in previous studies of FT in the presence of Zn [9,16,25] and of Zn–MT [27–30]. Results in Fig. 2b show that after Cd(II) addition, component 6 increases quickly until a plateau at a Cd:FT ratio of ca. 1 is reached. From Cd-to-FT concentration ratios of 0.2 and 0.5, new components 5 and 4 appear, increasing their contributions until the concentration ratios of 0.6 and 1.2, respectively, from
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which they maintain their values. Component 3, associated with the reduction of free Cd(II), can be detected at ratios higher than 1. For the sake of simplicity, components 1 and 2, corresponding to Hg related peaks, are omitted. Fig. 2c (from the 1.1:1 Zn:FT experiment) shows that components 4 and 5 (corresponding to Cd complexes) grow simultaneously and reach a plateau value at a metal-to-ligand ratio of 1, component 7 (due to free Zn(II)) is clearly detected throughout the experiment, and free Cd(II) (component 3) is detected at ratios above 1. A different behaviour is shown by component 9. When Zn(II) is in excess (1.1:1 Zn:FT) component 9 is detected clearly from the beginning of the experiment (Fig. 2c). However, in the absence of free Zn(II) (0.75:1 Zn:FT) it is not detected (its contribution lies in the noise level). Both components 8 and 9 are associated with the reduction processes of differently complexed Zn(II). In the interpretation of previous voltammetric results [5– 7,11–13] it was clearly proved that every component resolved by MCR-ALS must be associated with an electrochemical process and not with a chemical component (species), as happens when spectroscopic signals are analyzed. As a consequence, the number of components resolved by MCR-ALS is not necessarily the same as the number of chemical components because more than one electrochemical process can take place in a single individual chemical component. The position and evolution of the signals described so far is consistent, in principle, with different complexation models. Only techniques providing direct structural information can confirm the geometrical distribution of the atoms inside the complexes formed. But nowadays none of these techniques can provide reliable information at the concentration levels used in this study. In previous work [12,30] some information was obtained by circular dichroism, 113Cd-NMR and EXAFS at much higher concentrations, but the lack of sensitivity and the precipitation of the species dramatically lowered the quality of the results and restricted the metal-to-ligand ratios to be studied. Thus, in the absence of reliable structural information, the unitary voltammograms and the concentration profiles obtained by the combined use of voltammetry and MCR-ALS give information about the number of different complexation sites involved (through the number of different electrochemical processes detected), the association/dissociation kinetics (in electrochemically labile complexes a single signal is observed which moves progressively towards negative potentials as complexation takes place, whereas in electrochemically inert complexes separated signals are observed for free metal and complexes remaining at constant potential) and the evolution of the stoichiometries of the com-
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plexes along the voltammetric titration. Moreover, for electrochemically inert complexes, it can be considered that, usually, the more negative the peak potential of the bound metal (as compared to that of the free metal) the more stable is the complex. These do not provide unambiguous structures but restrict the large number of possibilities to those consistent with such information. In the case of the Cd–Zn – FT system, the MCR-ALS results described here and in previous work [15,16] allowed us to propose the complexation model summarized in Figs. 3 and 4. According to previous studies [15,16], the species expected in the Cd–Zn – FT exchange experiments are ZnFT2, Zn2FT2, CdZnFT2, CdZn2FT2 and Cd2ZnFT2 (charges are omitted for the sake of simplicity). Fig. 3 shows the reduction processes that we propose for each species and their correspondence with the signals of Fig. 2. This model has been based on the idea that in a molecule containing both Cd and Zn, after the reduction of Cd(II) and the breaking of the respective bonds, the remaining Zn(II) can be reorganized before their reduction. Another important point is that Zn(II) displaced by Cd(II) along the titration could remain more weakly bound to the molecule. Thus, when Cd(II) is reduced, such Zn(II) can bind again the sites vacated by cadmium. This is consistent with the absence of a clear increase in the signals of Zn while Cd(II) is being introduced into the molecule. It is also consistent with some exchange experiments with metallothioneins [29], which suggested the presence of weak complexes of Zn with MT. The structures proposed for ZnFT2 and Zn2FT2 are consistent with previous experiments [13]. Taking into account that, at the beginning of the titration at a Zn:FT ratio of 0.75, only one signal was detected for Zn (signal 8 in Fig. 3), this may mean that such a signal is an average of all labile Zn atoms bound in different ways to FT. Another possibility (not shown in the graph) is that the more strongly bonded Zn atoms are not electrochemically reduced. The structure proposed for CdZnFT2 is similar to that of Zn2FT2. In this complex, Cd(II) is reduced before Zn(II) and produces signal 6 (the further reduction of Zn(II) would contribute to signal 8). In the complexes containing three ions there is no way to guess the exact position of Cd and Zn. Indeed, there could be an equilibrium between different combinations. In Figs. 3 and 4 we have chosen one of the possibilities in an arbitrary way. This means that, for instance, the CdZn2FT2 complex is depicted in the order Cd–Zn –Zn, but it could equally be Zn–Cd –Zn or Zn –Zn –Cd. Keeping this in mind, when Cd(II) of the CdZn2FT2 complex is reduced, it produces a new signal 5 which appears at potentials less negative than signal 6 because the binding of this Cd is less stable than that of the Cd
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in CdZnFT2 producing signal 6. After this process, Zn(II) can recover its previous configuration to produce signal 8 again. In the case of the Cd2ZnFT2 complex, the reduction of the first Cd(II) (breaking the less stable bond) produces signal 4, which is less negative than signal 5 but still more negative than the reduction signal of free Cd(II) (component 3). The release of this Cd can produce two limiting situations. In the absence of an excess of free Zn(II), the molecule can be reorganized so that the second Cd(II) can be reduced from a situation similar to that of CdZnFT2 to yield signal 6. In contrast, if a large excess of free Zn(II) is present, the first outcoming cadmium can be replaced by a
Zn(II) to yield the same structure as in the CdZn2FT2 complex, thus producing signal 5 in the reduction of the second Cd(II). Of course both ways can be competitive so that it is likely that mixtures of all the mentioned species will occur. Fig. 4 summarizes how this reduction scheme can explain the evolution of the concentration profiles in Fig. 2. In the titration at 0.75 Zn:FT ratio (Fig. 4A), there is initially an equimolar mixture of ZnFT2 and Zn2FT2 complexes. Thus, the first Cd(II) added are surely introduced in the empty sites of ZnFT2 to form CdZnFT2 until such sites are saturated, which happens at a 0.25 M/L ratio. As these Cd(II) produce signal 6, this would explain the rapid increase of component 6 in
Fig. 3. Electrochemical reduction processes proposed for each chemical species present in the mixed metal system Cd,ZnFT. The structures depicted show only one of the various possibilities compatible with the experimental results (see text).
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Fig. 4. Evolution of the species along the titration with Cd(II) of a 0.75:1 Zn:FT solution (A) and a 1.1:1 Zn:FT solution (B), according to the proposed electrochemical model.
Fig. 2b until a M/L ratio of 0.25 is reached. The next Cd(II) introduced into the complexes probably transform Zn2FT2 into CdZn2FT2, so that they would produce the increase of process 5 mostly noticed between M/L ratios of 0.25 and 0.5 in Fig. 2b. Finally, Cd(II) added between the M/L ratios of 0.5 and 1.0 would introduce a second Cd into the molecule by addition to CdZnFT2 or by substitution of one Zn in CdZn2FT2. These last Cd(II) make process 4 appear and increase, while process 6 increases and process 5 decreases, which is consistent with the profiles of Fig. 2b. The further Cd(II) additions cannot displace more Zn(II), as is proved by the appearance of signal 3 of the free Cd(II). In the titration at 1.1 Zn:FT ratio (Fig. 4B), it is likely that all FT is as Zn2FT2 and that 0.1 mol of Zn(II) remain free per mole of FT. A first Cd(II) can then be added to form CdZn2FT2 and produce signal 5. A second one can be added later to form Cd2ZnFT2 and produce signal 4. The fact that in Fig. 2c both processes 4 and 5 increase monotonically from a M/L ratio of 0 to 1 suggests that the second cadmium starts
to be introduced long before all the Zn2FT2 molecules have been transformed into CdZn2FT2. Although the higher plateau value reached by component 4 in Fig. 2c suggests that it predominates with respect to component 5, this is not necessarily true, since the concentration profiles are deduced by assuming the same height of the corresponding pure signals, and phenomena like electrochemical irreversibility or electrodic adsorption can cause them to be quite different. According to the previous discussion, the presence of an initial excess of Zn(II) could be responsible for the absence of component 6 (associated with CdZnFT2 species) in this experiment. As in the previous titration, the Cd(II) added for M/L ratios higher than 1 remain as free Cd(II) (signal 3).
3.3. Study of the Zn–MT II 3.3.1. Influence of pH. Analysis by decon6olution approach When the pH of a metal-ligand solution is changed, the slope of the plot E versus pH allows the determina-
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tion of the ratio between the number of protons (of the ligand) substituted by the metal ion and the transferred electrons, as derived from the Nernst equation. Fig. 5 shows the evolution of the peak potentials (E), obtained through the individual deconvolution by using PeakFit software, as a function of pH for a Zn(II) spiked native Zn–MT II solution. Results obtained for a native Zn– MT II solution only differ in the absence of the peak corresponding to free Zn2 + at pH \6.0, when Zn2 + has been fully complexed. These results are in total agreement with those in HEPES-phosphate media earlier published by other authors [28]. The signal corresponding to the reduction of the electrochemical labile Zn(II) (line a) changes slightly until pH ca. 8.0, according to that found in previous work [7]. At pH \8.0 the formation of hydroxocomplexes shifts the peak potentials to more negative values. At pH ca. 6.0 complexation with thiol groups begins and a new signal (b) is detected at more negative potentials. Its slope is of −0.069 V/log unit, which corresponds to 1.17 H+/e−, i.e. the transference of two protons and two electrons. At pH ca. 8.0, the signal splits into two new signals of complexed Zn(II) (lines c and d). Component c maintains an almost constant peak potential value of − 1.2 V, while d is considerably influenced by pH (slope of − 0.125 V/log unit, i.e. 2.12 H+/e−), corresponding to the transference of four protons and two electrons. The small deviation with respect to the theoretical values (1 and 2 H+/e−, respectively) are mainly due to the coexistence of other minority species with different degrees of protonation. In the pH range 6– 8 only one type of binding site is involved. However, at pH\8 the deprotonation of other groups (the total deprotonation of thiol groups is achieved at pH 9.0) can provoke a structural reorgani-
Fig. 5. Influence of pH on the DPP peak potentials corresponding to a 1 × 10 − 5 M Zn– MT II solution spiked with Zn(II) until a 7 × 10 − 5 M Zn(II), in 0.13 M borate buffer. (a) Denotes the peak due to the reduction of electrochemically labile Zn(II); (b – d) Denotes the reduction of differently bound Zn(II).
zation of the molecule. As a consequence, Zn can be found in different environments, as implied by the diverse slope values. Experiments at changing pH allow selection of the proper working pH in order to choose the complexity of the system under study. Hence, for the first application of the MCR-ALS analysis it seems more reasonable to analyze a simple system. In addition, because of the physiological pH values, 7.5 was the value chosen to perform the exchange experiments with Zn–MT II.
3.3.2. Additions of Cd(II) to a Zn(II) -spiked Zn–MT II solution: analysis by MCR-ALS Some Zn(II) was added to a native Zn–MT II solution in order to assure an excess of free Zn(II) in the system. No significant variation in the morphology of the voltammogram (with respect to that of native Zn– MT II) was detected after the addition, from the current increase for free Zn(II). This is in total agreement with the metal content specified by Sigma (7% Zn, protein saturated). The initial voltammogram is characterized by two signals, as Fig. 6 shows. That located at potentials more negative than −1.0 V has a high baseline in the more negative region. The other at ca. −0.5 V is broad and, as was explained before, related to the oxidation of the mercury electrode. After Cd(II) additions, signals related to that latter process are found inside the potential range between − 0.35 V and − 0.55 V. In the Cd-region, only two peaks are detected: one centered at ca. − 0.59 V, corresponding to free Cd(II) reduction, and the other one, at more negative potentials, related to the reduction of some complexed Cd(II). After a few Cd(II) additions a new peak at ca. −1.15 V develops in the Zn-region. Following the usual MCR-ALS procedure, the SVD analysis estimates six components as being responsible for the data variation summarized in Fig. 6. The unitary voltammograms and the concentration profiles recovered after the constrained ALS optimization, applying the same constraints as before, are shown in Fig. 7a and b, respectively. The lack of fit is 12.86%. Components 1 and 2 are clearly related to the oxidation of the mercury electrode (and the further reduction) in the presence of Cd–Zn –MT II (component 1) and of Zn–MT II (component 2). Component 3 appears when an excess of Cd(II) has been added, from Cd-to-ZnMT concentration ratios of ca. 2.2 (Fig. 7b), and its peak potential is at ca. −0.59 V. As a consequence, this component is associated with the reduction of the free Cd(II) present in solution. Component 4, at more negative potentials than component 3, also increases as Cd(II) is added, but not so sharply. Thus, the increase must be related to the
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Fig. 6. DPP data set for the titration of a 1.16 × 10 – 6 M Zn(II) and 1.17 ×10 – 6 M Zn – MT II solution with 1 × 10 − 4 M Cd(II) solution, in borate buffer at pH 7.5, until a final Cd-to-MT concentration ratio of 8. The first voltammogram of the titration is shown separately.
reduction of some complexed Cd(II) in the Cd–Zn – MT II compound that is formed. Similarly, component 5 increases when Cd(II) is added, and its peak potential corresponds to that of the reduction of free Zn(II) in the medium used. As a consequence, this component must be associated with the reduction of the free Zn(II). Finally, component 6, at the most negative potentials, increases slowly as Cd(II) is added. It is presumably related to the reduction of some complexed Zn(II) in the Cd– Zn – MT II compound formed. However, before the addition of Cd(II), no peak is detected in this region, although there is a peak in the free Zn(II) region. As specified by the supplier and verified in our work, the ZnMT II is fully saturated with Zn (7:1 Zn:MT). This would indicate, then, that under these experimental conditions the Zn –MT complex initially present is not electroactive. This could be the consequence of a very stable or very occluded structure of the complex, preventing the reduction of the metals. However, as Cd(II) is added the signal due to bound Zn appears and increases progressively. So, it seems that the presence of Cd in the complex favors the reduction of complexed Zn. In order to investigate whether these current increases are related to the intrinsic nature of the newly incorporated metal ion or mostly to its dimensions, an experiment with successive additions of Pb(II) was performed (data not shown). The covalent radii for Pb (1.47× 10 − 10 m) is very similar to that of Cd (1.48× 10 − 10 m) and somewhat larger than that of Zn (1.25×10 − 10 m). The addition of Pb(II) also produced an increase of all Zn-related signals, suggesting that the incorporation of new metals
with high covalent radii in the molecule should open or destabilize the structure of the complex allowing an easier reduction of the bound metals. Also, the previous reduction of Pb or Cd as compared to Zn can destabilize the complex when Zn is reduced. Moreover, the increase of the signal of the free (or weakly complexed) Zn suggests that when Cd displaces Zn from Zn –MT II it could remain weakly bound to the molecule but cannot recover its native binding when the bound Cd is reduced. Such behavior, which contrasts with the situation in the FT system, may be due to the difficulty of establishing the higher degree of order achieved by the MT complex quickly. The fact that only one signal is noticed for either the reduction of bound Zn or Cd suggests that all Cd is introduced into MT in very similar positions, and that the Zn which becomes electroactive is bound to equivalent chemical environments, too.
3.4. Comparison between MT and FT complexation Metal exchange experiments with Zn-MT II at pH 8.5 and 7.0 have been previously analyzed, but under an univariate approach [26–28]. In spite of the lack of resolution reported at pH 7.0, the number of signals and their relative locations are in accord in both approaches, and the Cd-to-MT II concentration ratio at which free Cd(II) is detected (ca. 2.5 mol Cd/mol MT II) agrees perfectly (see profile 3 in Fig. 7b). Since in Zn-MT II most Zn ions are electroinactive and some of them become reducible in the presence of Cd, no definitive conclusions about the stoichiometries
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of the complexes formed can be proposed. It is possible only to conclude that free Cd(II) begins to be in excess at a Cd-to-MT II concentration ratio of ca. 2.2, which is double that in the FT system. This is a reasonable value, taking into account that FT and Zn– MT II have three and seven thiol groups, respectively, and means that ca. of 35% of sites are occupied by Cd. The electrochemical behavior of MT, as compared to that of FT, is relatively simple. FT can be more dynamic structurally taking several three-dimensional conformations, while Zn–MT II is more compact, acquiring a closer structure around the metal ions that complicates the substitution of Zn by Cd. This makes their reduction even more difficult. Thus, Cd is able to replace only those Zn ions that are in the proper spatial positions, and simultaneously open the entire structure, thus enabling the reduction of some native Zn in Zn– MT II. So, signals inside the Zn-region are modified as
Fig. 7. Normalized pure voltammograms (a) and concentration profiles (b) obtained in the MCR-ALS decomposition of data shown in Fig. 6, by imposing selectivity and non-negativity constraints for concentrations and non-negativity and signal shape constraints for voltammograms. For the sake of simplicity, concentration profiles of components 1 and 2 are omitted.
Cd(II) is added. However, for FT, Zn-related signals hardly change, since its molecule (smaller and more open) lets the Zn rearrange itself inside the complex after the reduction of Cd and before its own reduction. At the considered pH, the reduction process involves two protons and two electrons, which means two thiol groups per metal ion. This is in agreement with the reduction of any Zn bound to two shared – SH groups (bridging) and to the other two thiol groups not shared (terminals).
4. Conclusions The present work constitutes the first application of the MCR-ALS soft modeling approach to electrochemical characterization of involved natural biomolecules such as Zn–MT II. The combined use of voltammetry and MCR-ALS provides a powerful tool in the study of metal complexation equilibria of metalloproteins. It has been proved that the peptidic fragment Lys–Cys – Thr – Cys –Cys – Ala, although it has been used widely as a reliable simple model for MT, presents electroanalytical behavior in mixed metal systems even more complex than that of MT. This can be due to the different structures of FT and MT. FT has a very flexible and dynamic structure and a small size that allow the hypothetical formation of different electroactive complexes. In contrast, the much more rigid structure of MT has a lower number of possibilities, acquiring more compact and closer conformations around the metal ions and making more difficult the electrochemical reduction of the bound metals. The results presented here are of interest for a better interpretation of solving metal equilibria of MT and may prove useful for a better understanding of some structural features. Thus, two identical metal ions bound in different chemical environments produce different electrochemical signals that can be distinguished with the support of MCR-ALS. For the present cases, voltammetry joined to MCR-ALS resolution can be considered as a complementary tool to spectral means for a better understanding of the metal binding properties (especially structural features) of molecules. On the basis of these results, some compatible structures are proposed and the electrochemical reduction processes are depicted for the chemical species considered in the mixed metal system Cd–Zn –FT. In the case of Cd– Zn – MT II, additional information is required before proposing similar models.
Acknowledgements The authors gratefully acknowledge financial support from the Spanish Ministerio de Educacio´ n y Cultura
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(DGICYT project PB96-379-C03), from the Ministerio de Ciencia y Tecnologı´a (project BQU2000-0642-C03), and from the Generalitat of Catalonia (1999SGR00048).
References [1] M.J. Stillman, C.F. Shaw III, K.T. Suzuki, Metallothioneins, Synthesis, Structure and Properties of Metallothioneins, Phytochelatins and Metal –Thiolate Complexes, VCH, New York, 1992. [2] C. Klaassen, Metallothionein IV, Birkha¨ user Verlag, Basel, 1999. [3] J. Mendieta, J. Chivot, A. Mun˜ oz, A.R. Rodrı´guez, Electroanalysis 7 (1995) 663. [4] J. Mendieta, A.R. Rodrı´guez, Electroanalysis 8 (1996) 473. [5] J. Mendieta, M.S. Dı´az-Cruz, R. Tauler, M. Esteban, Anal. Biochem. 240 (1996) 134. [6] M.S. Dı´az-Cruz, J. Mendieta, R. Tauler, M. Esteban, J. Inorg. Biochem. 66 (1997) 29. [7] M.S. Dı´az-Cruz, J. Mendieta, R. Tauler, M. Esteban, J. Inorg. Biochem. 70 (1998) 91. [8] C. Harlyk, G. Bordin, O. Nieto, A.R. Rodrı´guez, J. Electroanal. Chem. 446 (1998) 139. [9] C. Harlyk, G. Bordin, O. Nieto, A.R. Rodrı´guez, J. Electroanal. Chem. 451 (1998) 267. [10] O. Nieto, A.R. Rodrı´guez, Electroanalysis 11 (1999) 175. [11] M. Esteban, C. Harlyk, A.R. Rodrı´guez, J. Electroanal. Chem. 468 (1999) 202. [12] M.S. Dı´az-Cruz, J. Mendieta, A. Monjonell, R. Tauler, M. Esteban, Anal. Chim. Acta 390 (1999) 15.
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[13] M.S. Dı´az-Cruz, J. Mendieta, A. Monjonell, R. Tauler, M. Esteban, Anal. Chim. Acta 385 (1999) 353. [14] M.S. Dı´az-Cruz, J.M. Dı´az-Cruz, J. Mendieta, R. Tauler, M. Esteban, Anal. Biochem. 279 (2000) 189. [15] M.S. Dı´az-Cruz, M. Esteban, A.R. Rodrı´guez, Anal. Chim. Acta 428 (2001) 285. [16] M.S. Dı´az-Cruz, J. Mendieta, M. Esteban, Electroanalysis 14 (2002) 50. [17] M.S. Dı´az-Cruz, J. Mendieta, R. Tauler, M. Esteban, Anal. Chem. 71 (1999) 4629. [18] M. Erk, B. Raspor, J. Electroanal. Chem. 502 (2001) 174. [19] J. Osteryoung, J.J. O’Dea, Electroanalytical Chemistry, vol. 14, Marcel Dekker, New York, 1986, pp. 209 – 308. [20] R. Tauler, A.K. Smilde, B.R. Kowalski, J. Chemom. 9 (1995) 31. [21] M. Esteban, C. Arin˜ o, J.M. Dı´az-Cruz, M.S. Dı´az-Cruz, R. Tauler, Trends Anal. Chem. 19 (2000) 49. [22] J.M. Dı´az-Cruz, J. Agullo´ , M.S. Dı´az-Cruz, C. Arin˜ o, R. Tauler, M. Esteban, Analyst 126 (2001) 371. [23] Matlab version 4.2, Math Works Inc., Cochituate Place, MA, 1994. [24] Peak Fit, Peak Separation and Analysis Software version 4.0 Jandel Scientific Software, Erkrath, Germany, 1995. [25] C. Ruiz, J. Mendieta, A.R. Rodrı´guez, Anal. Chim. Acta 305 (1995) 285. [26] O. Nieto, G. Hellemans, G. Bordin, M. De Ley, A.R. Rodrı´guez, Bioelectrochem. Bioenergetics 46 (1998) 45. [27] C. Ruiz, A.R. Rodrı´guez, Anal. Chim. Acta 350 (1997) 305. [28] O. Nieto, G. Hellemans, G. Bordin, M. De Ley, A.R. Rodrı´guez, Talanta 46 (1998) 315. [29] M. Dabrio, A.R. Rodrı´guez, Anal. Chim. Acta 385 (1999) 295 – 306. [30] M.S. Dı´az-Cruz, J.M. Dı´az-Cruz, M. Esteban, Electroanalysis 14 (2002) in press.
Comparison of the zinc–cadmium exchange properties of the metallothionein related peptide {Lys–Cys–Thr–Cys–Cys–Ala} and a zinc-containing metallothionein: study by voltammetry and multivariate curve resolution M.S. Dı´az-Cruz, M.J. Lo´pez, J.M. Dı´az-Cruz, M. Esteban * Departament de Quı´mica Analı´tica, Facultat de Quı´mica, Uni6ersitat de Barcelona, A6. Diagonal 647, E-08028 Barcelona, Spain Received 26 September 2001; received in revised form 28 January 2002; accepted 6 February 2002
Abstract The complexation of the hexapeptide Lys–Cys–Thr– Cys– Cys– Ala (FT), peptidic fragment {56– 61} in the a-domain of mouse liver metallothionein I (MT), in the simultaneous presence of Cd(II) and Zn(II) is compared with that of a Zn-containing mammalian MT (Zn–MT II) in the presence of Cd(II). The influence of Cd(II) is studied by square wave voltammetry and differential pulse polarography, at different Cd:Zn:FT molar ratios, which yield a rough picture of the metal exchange processes taking place. The application of multivariate curve resolution with alternating least squares optimization (MCR-ALS) allows both the unitary voltammograms and the concentration profiles to be obtained. Moreover, MCR-ALS is applied for the first time to the study of the complexing properties of a complete MT. On the basis of these results, the electrochemical reduction processes and some structures are depicted for the chemical species considered in the mixed metal system Cd– Zn– FT. In the case of Cd–Zn–MT II, additional information is required before proposing similar models. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Metallothioneins; Complexation; Metal exchange; Voltammetry; Cadmium; Zinc; Multivariate analysis; Deconvolution
1. Introduction Metallothioneins (MT) are of great biological interest due to their capability to bind metals with a d10 configuration, e.g. Zn, Cd or Hg [1,2]. MT can be induced by diverse stimuli such as metal uptake, stress, or some drugs. The characterization of MT presents several challenges that include the identification of metals involved in the complexes, the determination of the corresponding stoichiometries, the precise identification of the aminoacid sequence and the characterization of the chemical species present in solution. Some small peptides containing cysteine (Cys) residues at the proper positions are known to exhibit behavior similar to that of MT. This is the case for the peptidic fragment Lys – Cys – Thr – Cys – Cys – Ala {56 – * Corresponding author. Tel.: + 34-93-402-1277/1286; fax: +3493-402-1233. E-mail address: [email protected] (M. Esteban).
61} of mouse liver MT I (hereinafter denoted by FT), which has been widely studied as a simpler model of MT and of the a-domain in mammalian MT. The study of Cys-containing peptides such as glutathione (g-L-glutamyl-L-cysteinylglycine, GSH) and FT, in the absence and in the presence of Zn(II) or Cd(II), has recently been carried out by means of several electrochemical techniques such as differential pulse polarography (DPP), linear sweep voltammetry (LSV), cyclic voltammetry (CV), direct current polarography (DCP) and square wave voltammetry (SWV) [3–17] and, very recently, anodic stripping voltammetry [18]. The major advantages of SWV are its speed and its excellent sensitivity [19]. On the other hand, DPP represents a good alternative especially when very high sensitivity is not needed and the differences between the peak potentials are large enough to obtain well-resolved peaks. Because of all these reasons, both techniques have been widely used in the study of MT and related compounds. An additional reason to use DPP and
0022-0728/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 7 2 8 ( 0 2 ) 0 0 7 3 8 - 6
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SWV is that these techniques allow very low metal concentrations to be used, where potentiometry with ion-selective electrodes is not reliable enough, thus avoiding complications due to precipitation phenomena. Due to the rather complex voltammograms obtained in these studies, and the further difficulties in both data treatment and interpretation, the use of a multivariate data analysis method was recently proposed [5]. The multivariate curve resolution with alternating least squares optimization method (MCR-ALS) is based on the use of factor analysis techniques. It was originally developed for spectroscopic data [20], modified and adapted to voltammetric signals (current-potential sets of data) because of their specific characteristics [5]. Since then, it has been progressively improved by including new constraints in the ALS optimization [14,21,22]. In particular, MCR-ALS has been applied in the study of the Cd– GSH [5,6,17], Cd– FT [11,12], Zn –GSH [7] and Zn – FT [13] systems by DPP, LSV and CV, and in the study of the mixed metal Cd–Zn – FT system by DPP and SWV [15,16]. The last DPP study [16] reports the exchange experiments when Cd(II) is added to a 2:2 Zn:FT solution. At this concentration ratio the maximum stoichiometric proportion, Zn2FT2 is reached, and no free Zn(II) is initially present. Under these experimental conditions, the complex Cd2ZnFT2 is formed. Addition of Zn(II) to a 1:2 Cd:FT solution was also investigated, which led us to propose the formation of the CdZn2FT2 complex. These results indicate that the metal initially bound to the FT molecule cannot be totally released, even when a great excess of the other metal is added. In a previous paper [15], the total metal:FT ratios found for the complexes formed were higher than those reported here. In that study FT was added to a mixture of Cd and Zn at diverse initial proportions. Thus, the excess of metal initially present can provoke the saturation of sites in FT as soon as it is added, hindering further entrance of metals. In the present work, the metal binding behavior of FT in the simultaneous presence of Cd(II) and Zn(II), at several Cd/Zn molar ratios, has been studied by SWV and analyzed by MCR-ALS. Once the Cd–Zn – FT system has been understood in depth, a mammalian Zn-containing MT II is analyzed, to compare its behavior and to check the capabilities of MCR-ALS. In principle, ZnMT II represents a higher level of system complexity for the application of MCR-ALS to electrochemical data with respect to GSH and FT, where only 1 and 3 cysteines were involved, respectively. The DPP technique was selected for Zn-MT II studies in order to prevent the protein’s known adsorptive capacity onto the electrode surface (mainly due to its high cysteine content). SWV was preferred in Cd–Zn – FT studies because previous experience showed the
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poor resolution of polarographic techniques when diverse peaks are detected in the Zn-region and since only three thiol groups can lead to adsorption phenomena.
2. Experimental
2.1. Chemicals and instrumentation Lys –Cys –Thr –Cys –Cys –Ala acetate salt, containing mercaptoethanol as a stabilizer, and rabbit liver ZnMT II (7% Zn content, M= 9542 D) were obtained from Sigma Chemicals. Previous studies showed that the low mercaptoethanol content (below 5%) in the peptide sample does not interfere significantly [3,4]. Standard solutions of cadmium, lead and zinc (Titrisol), with a metal concentration of 1.000 g l − 1 9 2 mg l − 1, were purchased from Merck. All other reagents were of analytical grade. The samples were freshly prepared with ultrapure water (Millipore MilliQ Plus 185 system) and used immediately. A 0.13 M borate buffer solution at the desired pH was used as supporting electrolyte. The choice of this buffer solution followed the procedure outlined in previous studies [10–13,16]. Purified nitrogen was used for deareation. SWV and DPP measurements were carried out with an Autolab System potentiostat (Ecochemie, The Netherlands) coupled to a stand polarographic 663 VA (Metrohm, Switzerland) and attached to a personal computer implemented with a GPES version 2.0 software package (Ecochemie). The system was also connected to a Metrohm 665 dosimat for the automatic addition of standard solutions. The SWV instrumental parameters that ensure the best definition of the peaks are: amplitude of 15 mV, step potential of 2 mV and frequency of 75 Hz. The DPP characteristic parameters were: a pulse duration of 40 ms, pulse amplitude of 25 mV, drop time of 1 s and scan rate of 6 mV s − 1. In all experiments, the working, reference and auxiliary electrodes were the static mercury drop electrode (SMDE), Ag AgCl 3 M KCl and glassy carbon, respectively. pH was controlled with an Orion 920 A pH-meter (Orion, Boston, MA) equipped with a combined glass electrode.
2.2. Procedure 2.2.1. Experiments at fixed pH and different metal-to-ligand ratios An aliquot of 10 ml of the buffer solution was deaerated for 20 min with a N2 current and then a voltammogram (SWV or DPP) was recorded. Aliquots of the FT– or Zn –MT II-standard solutions were added to the cell in order to reach the desired initial concentration ratios. Then, the solution was stirred,
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deaerated for 1 min, and a new voltammogram was recorded. Replicate measurements were done to ensure the repetitivity of the voltammograms. Later, aliquots of the Cd(II)-stock solution are added to the cell in order to achieve a wide Cd-to-ligand concentration ratio range. When required, an aliquot of Zn(II)-standard solution is added (spiked) previous to Cd(II) additions. After each addition, the solution was stirred and deareated for 1 min, and a new voltammogram was recorded. During the measurements, a N2 stream was passed over the surface of the solutions to ensure an inert atmosphere. Very reproducible results were obtained after repetitive titrations, even after waiting periods (between the cadmium addition and recording of the voltammograms) greater than 1 min.
2.2.2. Experiments at different pH and fixed metal-to-MT ratio Ten milliliters of buffer solution at pH 4.0 were placed in the cell, deaerated for 20 min and the DPP scan was recorded. The Zn– MT II stock solution was added to the cell and a new voltammogram was registered. Further measurements were performed after every addition of a standard NaOH solution in order to increase the pH value. Two initial Zn:Zn –MT II concentration ratios were selected: the one as supplied (native rabbit liver MT II), which corresponds to 7:1 Zn:MT, and a Zn-spiked sample which assures an initial excess of Zn(II) in the medium (corresponding to a 7:1 Zn:Zn– MT II). 2.3. Data treatment Voltammograms are analyzed by two different approaches. One is based on multivariate analysis by means of MCR-ALS, and it is applied to experiments performed at fixed pH. The other approach is an individual voltammogram deconvolution, which is applied to experiments at fixed metal-to-ligand concentration ratio but changing pH. In both approaches, as a preliminary step, the voltammograms are smoothed and a point-by-point subtraction of the background current (obtained for the supporting electrolyte) is accomplished. In the MCR-ALS approach, after this preliminary treatment, the set of voltammograms is arranged in a data matrix of currents. This matrix contains as many rows as the number of voltammograms recorded at each metal-to-ligand ratio and as many columns as potentials scanned in every run. Once the matrix is composed, the data treatment follows the MCR-ALS method (described elsewhere [5,21]) implemented using the MATLAB computer graphics environment program [23]. MATLAB programs for MCR-ALS are available at the web site http://www.ub.es/gesq/ mcr/mcr.htm.
In the second approach, which is a univariate method, all voltammograms are deconvoluted (one by one) by using PeakFit software [24]. Inside PeakFit options, method I (based on the analysis of residuals) and the power peak equation (for signal shape) are used. The reason for using this simple univariate method instead of the more complex and powerful MCR-ALS is that the data matrices resulting for pHvariation experiments do not follow the linear model required for the application of MCR-ALS [6].
3. Results and discussion
3.1. Study of the Cd–Zn –FT system as analyzed by MCR-ALS 3.1.1. Additions of Cd(II) to a 0.75:1 Zn:FT solution The voltammograms corresponding to the titration of a 0.75:1 Zn:FT solution with Cd(II) are shown in Fig. 1a. The voltammogram before the addition of Cd(II) presents two broad peaks at ca. − 0.5 and −1.1 V. According to previous studies [3,4,16], the very broad peak at ca. − 0.5 V is related to the electrochemical oxidation of the mercury from the electrode in the presence of either free thiol groups of FT or thiol groups complexed with Zn, and the further reduction. The peak at ca. −1.1 V is attributed to the reduction of Zn-containing species. After the progressive addition of Cd(II), the initial peak at −0.5 V decreases steadily, and a new peak at even less negative potential begins to grow. This peak is related to the oxidation of the mercury in the presence of the new Cd–Zn –FT complex and its further reduction [25]. Thus, the sequence of potentials is in agreement with the apparent stability constants (K%Hg – SH \ K%Cd – SH \ K%Zn – SH) previously established [25]. Given the aim of the present study, we will focus our attention on the metal reduction regions, and further mention of these Hg related peaks will be made only when strictly necessary to understand the behaviour of the systems studied. In the Zn– region (potentials from −1.0 to −1.4 V), the broad peak initially present is not modified significantly after a few Cd(II) additions. Simultaneously, in the Cd-region (from −0.55 to − 1.0 V) some new peaks appear successively from the most negative (at ca. −0.9 V) region until they reach that due to the reduction of free Cd(II) (at ca. −0.59 V). Because of the highly changing number and shape of the peaks, this behavior cannot be explained directly, and the help of MCR-ALS is needed. The application of the singular value decomposition (SVD) method, which is the first step in the MCR-ALS approach [5], shows that an unreasonably high number of components is required to explain the experimental data varia-
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tion. Despite this fact, the full MCR-ALS data analysis was completed, but the results reached (data not shown) were not consistent enough.
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likely due to the high number of components needed, as happened in the 0.75:1 Zn:FT case.
3.2. Simultaneous MCR-ALS analysis of both series 3.1.2. Additions of Cd(II) to a 1.1:1 Zn:FT solution When the Zn-FT system contains an excess of Zn, i.e. free Zn(II) is present, the direct interpretation of the voltammograms (Fig. 1b) is even more chancy. Now, at least two peaks related to Zn, and three peaks related to Cd can be distinguished. The Zn-region is not significantly modified during Cd(II) additions. Again, the MCR-ALS analysis yields unsatisfactory results to explain the experimentally observed behavior,
The voltammograms of each titration have several common points. At least six peaks from both experiments seem to be related directly to each other. Thus, data can be joined in a column-wise augmented data matrix and treated simultaneously to overcome the uncertainties found in the individual analysis. Nine mathematical components were concluded after SVD analysis (results not shown). Starting at this esti-
Fig. 1. SWV data set for the titration of a 4.8 × 10 – 6 M Zn(II) and 6.6 ×10 – 6 M FT solution (a) and a 7.2 ×10 – 6 M Zn(II) and 6.6 ×10 – 6 M FT solution (b) with a 10 − 4 M Cd(II) solution, in borate buffer at pH 8.0, until a final Cd(II) excess. First voltammogram in each titration is shown separately.
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Fig. 2. Normalized individual voltammograms (a) and concentration profiles (b and c), obtained in the MCR-ALS decomposition of the augmented data matrix composed of data shown in Fig. 1 by imposing selectivity and non-negativity constraints for concentrations, and non-negativity and signal-shape constraints for voltammograms. (b) and (c) visualize the individual matrices obtained after the constrained ALS optimization for the experiments at 0.75:1 and 1:1 Zn:FT, respectively. For the sake of simplicity, concentration profiles of components 1 and 2 are omitted.
mation, ALS decomposes the current data matrix into one matrix containing the normalized individual voltammograms for each mathematical component (Fig. 2a), and one augmented matrix containing the concentration profiles (currents vs. Cd-to-FT ratios) for such components. This augmented matrix is visualized through the individual matrices from each experiment (Fig. 2b and c). In order to simplify the visualization of the unitary voltammograms, they are normalized to one. As a consequence, the y-axis values in the concentration profile plots have arbitrary units. As described elsewhere [5,21], some constraints must be included along the ALS optimization in order to achieve (electro)chemically satisfactory results. The constraints were: selectivity and non-negativity for concentrations; non-negativity and peak-shape-constraint for voltam-
mograms. The lack of fit after the constrained ALS optimization is 14.78%. The peak potential values of the normalized individual voltammograms (Fig. 2a) seem to be fully coherent with the experimentally observed global signals (Fig. 1). It must be remarked that component 7 is attributed to the reduction of free Zn(II) even when small differences in the peak potential, with respect to the signal for a solution of free Zn(II) (Fig. 1b), are detected. This situation has been also found in previous studies of FT in the presence of Zn [9,16,25] and of Zn–MT [27–30]. Results in Fig. 2b show that after Cd(II) addition, component 6 increases quickly until a plateau at a Cd:FT ratio of ca. 1 is reached. From Cd-to-FT concentration ratios of 0.2 and 0.5, new components 5 and 4 appear, increasing their contributions until the concentration ratios of 0.6 and 1.2, respectively, from
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which they maintain their values. Component 3, associated with the reduction of free Cd(II), can be detected at ratios higher than 1. For the sake of simplicity, components 1 and 2, corresponding to Hg related peaks, are omitted. Fig. 2c (from the 1.1:1 Zn:FT experiment) shows that components 4 and 5 (corresponding to Cd complexes) grow simultaneously and reach a plateau value at a metal-to-ligand ratio of 1, component 7 (due to free Zn(II)) is clearly detected throughout the experiment, and free Cd(II) (component 3) is detected at ratios above 1. A different behaviour is shown by component 9. When Zn(II) is in excess (1.1:1 Zn:FT) component 9 is detected clearly from the beginning of the experiment (Fig. 2c). However, in the absence of free Zn(II) (0.75:1 Zn:FT) it is not detected (its contribution lies in the noise level). Both components 8 and 9 are associated with the reduction processes of differently complexed Zn(II). In the interpretation of previous voltammetric results [5– 7,11–13] it was clearly proved that every component resolved by MCR-ALS must be associated with an electrochemical process and not with a chemical component (species), as happens when spectroscopic signals are analyzed. As a consequence, the number of components resolved by MCR-ALS is not necessarily the same as the number of chemical components because more than one electrochemical process can take place in a single individual chemical component. The position and evolution of the signals described so far is consistent, in principle, with different complexation models. Only techniques providing direct structural information can confirm the geometrical distribution of the atoms inside the complexes formed. But nowadays none of these techniques can provide reliable information at the concentration levels used in this study. In previous work [12,30] some information was obtained by circular dichroism, 113Cd-NMR and EXAFS at much higher concentrations, but the lack of sensitivity and the precipitation of the species dramatically lowered the quality of the results and restricted the metal-to-ligand ratios to be studied. Thus, in the absence of reliable structural information, the unitary voltammograms and the concentration profiles obtained by the combined use of voltammetry and MCR-ALS give information about the number of different complexation sites involved (through the number of different electrochemical processes detected), the association/dissociation kinetics (in electrochemically labile complexes a single signal is observed which moves progressively towards negative potentials as complexation takes place, whereas in electrochemically inert complexes separated signals are observed for free metal and complexes remaining at constant potential) and the evolution of the stoichiometries of the com-
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plexes along the voltammetric titration. Moreover, for electrochemically inert complexes, it can be considered that, usually, the more negative the peak potential of the bound metal (as compared to that of the free metal) the more stable is the complex. These do not provide unambiguous structures but restrict the large number of possibilities to those consistent with such information. In the case of the Cd–Zn – FT system, the MCR-ALS results described here and in previous work [15,16] allowed us to propose the complexation model summarized in Figs. 3 and 4. According to previous studies [15,16], the species expected in the Cd–Zn – FT exchange experiments are ZnFT2, Zn2FT2, CdZnFT2, CdZn2FT2 and Cd2ZnFT2 (charges are omitted for the sake of simplicity). Fig. 3 shows the reduction processes that we propose for each species and their correspondence with the signals of Fig. 2. This model has been based on the idea that in a molecule containing both Cd and Zn, after the reduction of Cd(II) and the breaking of the respective bonds, the remaining Zn(II) can be reorganized before their reduction. Another important point is that Zn(II) displaced by Cd(II) along the titration could remain more weakly bound to the molecule. Thus, when Cd(II) is reduced, such Zn(II) can bind again the sites vacated by cadmium. This is consistent with the absence of a clear increase in the signals of Zn while Cd(II) is being introduced into the molecule. It is also consistent with some exchange experiments with metallothioneins [29], which suggested the presence of weak complexes of Zn with MT. The structures proposed for ZnFT2 and Zn2FT2 are consistent with previous experiments [13]. Taking into account that, at the beginning of the titration at a Zn:FT ratio of 0.75, only one signal was detected for Zn (signal 8 in Fig. 3), this may mean that such a signal is an average of all labile Zn atoms bound in different ways to FT. Another possibility (not shown in the graph) is that the more strongly bonded Zn atoms are not electrochemically reduced. The structure proposed for CdZnFT2 is similar to that of Zn2FT2. In this complex, Cd(II) is reduced before Zn(II) and produces signal 6 (the further reduction of Zn(II) would contribute to signal 8). In the complexes containing three ions there is no way to guess the exact position of Cd and Zn. Indeed, there could be an equilibrium between different combinations. In Figs. 3 and 4 we have chosen one of the possibilities in an arbitrary way. This means that, for instance, the CdZn2FT2 complex is depicted in the order Cd–Zn –Zn, but it could equally be Zn–Cd –Zn or Zn –Zn –Cd. Keeping this in mind, when Cd(II) of the CdZn2FT2 complex is reduced, it produces a new signal 5 which appears at potentials less negative than signal 6 because the binding of this Cd is less stable than that of the Cd
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in CdZnFT2 producing signal 6. After this process, Zn(II) can recover its previous configuration to produce signal 8 again. In the case of the Cd2ZnFT2 complex, the reduction of the first Cd(II) (breaking the less stable bond) produces signal 4, which is less negative than signal 5 but still more negative than the reduction signal of free Cd(II) (component 3). The release of this Cd can produce two limiting situations. In the absence of an excess of free Zn(II), the molecule can be reorganized so that the second Cd(II) can be reduced from a situation similar to that of CdZnFT2 to yield signal 6. In contrast, if a large excess of free Zn(II) is present, the first outcoming cadmium can be replaced by a
Zn(II) to yield the same structure as in the CdZn2FT2 complex, thus producing signal 5 in the reduction of the second Cd(II). Of course both ways can be competitive so that it is likely that mixtures of all the mentioned species will occur. Fig. 4 summarizes how this reduction scheme can explain the evolution of the concentration profiles in Fig. 2. In the titration at 0.75 Zn:FT ratio (Fig. 4A), there is initially an equimolar mixture of ZnFT2 and Zn2FT2 complexes. Thus, the first Cd(II) added are surely introduced in the empty sites of ZnFT2 to form CdZnFT2 until such sites are saturated, which happens at a 0.25 M/L ratio. As these Cd(II) produce signal 6, this would explain the rapid increase of component 6 in
Fig. 3. Electrochemical reduction processes proposed for each chemical species present in the mixed metal system Cd,ZnFT. The structures depicted show only one of the various possibilities compatible with the experimental results (see text).
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Fig. 4. Evolution of the species along the titration with Cd(II) of a 0.75:1 Zn:FT solution (A) and a 1.1:1 Zn:FT solution (B), according to the proposed electrochemical model.
Fig. 2b until a M/L ratio of 0.25 is reached. The next Cd(II) introduced into the complexes probably transform Zn2FT2 into CdZn2FT2, so that they would produce the increase of process 5 mostly noticed between M/L ratios of 0.25 and 0.5 in Fig. 2b. Finally, Cd(II) added between the M/L ratios of 0.5 and 1.0 would introduce a second Cd into the molecule by addition to CdZnFT2 or by substitution of one Zn in CdZn2FT2. These last Cd(II) make process 4 appear and increase, while process 6 increases and process 5 decreases, which is consistent with the profiles of Fig. 2b. The further Cd(II) additions cannot displace more Zn(II), as is proved by the appearance of signal 3 of the free Cd(II). In the titration at 1.1 Zn:FT ratio (Fig. 4B), it is likely that all FT is as Zn2FT2 and that 0.1 mol of Zn(II) remain free per mole of FT. A first Cd(II) can then be added to form CdZn2FT2 and produce signal 5. A second one can be added later to form Cd2ZnFT2 and produce signal 4. The fact that in Fig. 2c both processes 4 and 5 increase monotonically from a M/L ratio of 0 to 1 suggests that the second cadmium starts
to be introduced long before all the Zn2FT2 molecules have been transformed into CdZn2FT2. Although the higher plateau value reached by component 4 in Fig. 2c suggests that it predominates with respect to component 5, this is not necessarily true, since the concentration profiles are deduced by assuming the same height of the corresponding pure signals, and phenomena like electrochemical irreversibility or electrodic adsorption can cause them to be quite different. According to the previous discussion, the presence of an initial excess of Zn(II) could be responsible for the absence of component 6 (associated with CdZnFT2 species) in this experiment. As in the previous titration, the Cd(II) added for M/L ratios higher than 1 remain as free Cd(II) (signal 3).
3.3. Study of the Zn–MT II 3.3.1. Influence of pH. Analysis by decon6olution approach When the pH of a metal-ligand solution is changed, the slope of the plot E versus pH allows the determina-
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tion of the ratio between the number of protons (of the ligand) substituted by the metal ion and the transferred electrons, as derived from the Nernst equation. Fig. 5 shows the evolution of the peak potentials (E), obtained through the individual deconvolution by using PeakFit software, as a function of pH for a Zn(II) spiked native Zn–MT II solution. Results obtained for a native Zn– MT II solution only differ in the absence of the peak corresponding to free Zn2 + at pH \6.0, when Zn2 + has been fully complexed. These results are in total agreement with those in HEPES-phosphate media earlier published by other authors [28]. The signal corresponding to the reduction of the electrochemical labile Zn(II) (line a) changes slightly until pH ca. 8.0, according to that found in previous work [7]. At pH \8.0 the formation of hydroxocomplexes shifts the peak potentials to more negative values. At pH ca. 6.0 complexation with thiol groups begins and a new signal (b) is detected at more negative potentials. Its slope is of −0.069 V/log unit, which corresponds to 1.17 H+/e−, i.e. the transference of two protons and two electrons. At pH ca. 8.0, the signal splits into two new signals of complexed Zn(II) (lines c and d). Component c maintains an almost constant peak potential value of − 1.2 V, while d is considerably influenced by pH (slope of − 0.125 V/log unit, i.e. 2.12 H+/e−), corresponding to the transference of four protons and two electrons. The small deviation with respect to the theoretical values (1 and 2 H+/e−, respectively) are mainly due to the coexistence of other minority species with different degrees of protonation. In the pH range 6– 8 only one type of binding site is involved. However, at pH\8 the deprotonation of other groups (the total deprotonation of thiol groups is achieved at pH 9.0) can provoke a structural reorgani-
Fig. 5. Influence of pH on the DPP peak potentials corresponding to a 1 × 10 − 5 M Zn– MT II solution spiked with Zn(II) until a 7 × 10 − 5 M Zn(II), in 0.13 M borate buffer. (a) Denotes the peak due to the reduction of electrochemically labile Zn(II); (b – d) Denotes the reduction of differently bound Zn(II).
zation of the molecule. As a consequence, Zn can be found in different environments, as implied by the diverse slope values. Experiments at changing pH allow selection of the proper working pH in order to choose the complexity of the system under study. Hence, for the first application of the MCR-ALS analysis it seems more reasonable to analyze a simple system. In addition, because of the physiological pH values, 7.5 was the value chosen to perform the exchange experiments with Zn–MT II.
3.3.2. Additions of Cd(II) to a Zn(II) -spiked Zn–MT II solution: analysis by MCR-ALS Some Zn(II) was added to a native Zn–MT II solution in order to assure an excess of free Zn(II) in the system. No significant variation in the morphology of the voltammogram (with respect to that of native Zn– MT II) was detected after the addition, from the current increase for free Zn(II). This is in total agreement with the metal content specified by Sigma (7% Zn, protein saturated). The initial voltammogram is characterized by two signals, as Fig. 6 shows. That located at potentials more negative than −1.0 V has a high baseline in the more negative region. The other at ca. −0.5 V is broad and, as was explained before, related to the oxidation of the mercury electrode. After Cd(II) additions, signals related to that latter process are found inside the potential range between − 0.35 V and − 0.55 V. In the Cd-region, only two peaks are detected: one centered at ca. − 0.59 V, corresponding to free Cd(II) reduction, and the other one, at more negative potentials, related to the reduction of some complexed Cd(II). After a few Cd(II) additions a new peak at ca. −1.15 V develops in the Zn-region. Following the usual MCR-ALS procedure, the SVD analysis estimates six components as being responsible for the data variation summarized in Fig. 6. The unitary voltammograms and the concentration profiles recovered after the constrained ALS optimization, applying the same constraints as before, are shown in Fig. 7a and b, respectively. The lack of fit is 12.86%. Components 1 and 2 are clearly related to the oxidation of the mercury electrode (and the further reduction) in the presence of Cd–Zn –MT II (component 1) and of Zn–MT II (component 2). Component 3 appears when an excess of Cd(II) has been added, from Cd-to-ZnMT concentration ratios of ca. 2.2 (Fig. 7b), and its peak potential is at ca. −0.59 V. As a consequence, this component is associated with the reduction of the free Cd(II) present in solution. Component 4, at more negative potentials than component 3, also increases as Cd(II) is added, but not so sharply. Thus, the increase must be related to the
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Fig. 6. DPP data set for the titration of a 1.16 × 10 – 6 M Zn(II) and 1.17 ×10 – 6 M Zn – MT II solution with 1 × 10 − 4 M Cd(II) solution, in borate buffer at pH 7.5, until a final Cd-to-MT concentration ratio of 8. The first voltammogram of the titration is shown separately.
reduction of some complexed Cd(II) in the Cd–Zn – MT II compound that is formed. Similarly, component 5 increases when Cd(II) is added, and its peak potential corresponds to that of the reduction of free Zn(II) in the medium used. As a consequence, this component must be associated with the reduction of the free Zn(II). Finally, component 6, at the most negative potentials, increases slowly as Cd(II) is added. It is presumably related to the reduction of some complexed Zn(II) in the Cd– Zn – MT II compound formed. However, before the addition of Cd(II), no peak is detected in this region, although there is a peak in the free Zn(II) region. As specified by the supplier and verified in our work, the ZnMT II is fully saturated with Zn (7:1 Zn:MT). This would indicate, then, that under these experimental conditions the Zn –MT complex initially present is not electroactive. This could be the consequence of a very stable or very occluded structure of the complex, preventing the reduction of the metals. However, as Cd(II) is added the signal due to bound Zn appears and increases progressively. So, it seems that the presence of Cd in the complex favors the reduction of complexed Zn. In order to investigate whether these current increases are related to the intrinsic nature of the newly incorporated metal ion or mostly to its dimensions, an experiment with successive additions of Pb(II) was performed (data not shown). The covalent radii for Pb (1.47× 10 − 10 m) is very similar to that of Cd (1.48× 10 − 10 m) and somewhat larger than that of Zn (1.25×10 − 10 m). The addition of Pb(II) also produced an increase of all Zn-related signals, suggesting that the incorporation of new metals
with high covalent radii in the molecule should open or destabilize the structure of the complex allowing an easier reduction of the bound metals. Also, the previous reduction of Pb or Cd as compared to Zn can destabilize the complex when Zn is reduced. Moreover, the increase of the signal of the free (or weakly complexed) Zn suggests that when Cd displaces Zn from Zn –MT II it could remain weakly bound to the molecule but cannot recover its native binding when the bound Cd is reduced. Such behavior, which contrasts with the situation in the FT system, may be due to the difficulty of establishing the higher degree of order achieved by the MT complex quickly. The fact that only one signal is noticed for either the reduction of bound Zn or Cd suggests that all Cd is introduced into MT in very similar positions, and that the Zn which becomes electroactive is bound to equivalent chemical environments, too.
3.4. Comparison between MT and FT complexation Metal exchange experiments with Zn-MT II at pH 8.5 and 7.0 have been previously analyzed, but under an univariate approach [26–28]. In spite of the lack of resolution reported at pH 7.0, the number of signals and their relative locations are in accord in both approaches, and the Cd-to-MT II concentration ratio at which free Cd(II) is detected (ca. 2.5 mol Cd/mol MT II) agrees perfectly (see profile 3 in Fig. 7b). Since in Zn-MT II most Zn ions are electroinactive and some of them become reducible in the presence of Cd, no definitive conclusions about the stoichiometries
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of the complexes formed can be proposed. It is possible only to conclude that free Cd(II) begins to be in excess at a Cd-to-MT II concentration ratio of ca. 2.2, which is double that in the FT system. This is a reasonable value, taking into account that FT and Zn– MT II have three and seven thiol groups, respectively, and means that ca. of 35% of sites are occupied by Cd. The electrochemical behavior of MT, as compared to that of FT, is relatively simple. FT can be more dynamic structurally taking several three-dimensional conformations, while Zn–MT II is more compact, acquiring a closer structure around the metal ions that complicates the substitution of Zn by Cd. This makes their reduction even more difficult. Thus, Cd is able to replace only those Zn ions that are in the proper spatial positions, and simultaneously open the entire structure, thus enabling the reduction of some native Zn in Zn– MT II. So, signals inside the Zn-region are modified as
Fig. 7. Normalized pure voltammograms (a) and concentration profiles (b) obtained in the MCR-ALS decomposition of data shown in Fig. 6, by imposing selectivity and non-negativity constraints for concentrations and non-negativity and signal shape constraints for voltammograms. For the sake of simplicity, concentration profiles of components 1 and 2 are omitted.
Cd(II) is added. However, for FT, Zn-related signals hardly change, since its molecule (smaller and more open) lets the Zn rearrange itself inside the complex after the reduction of Cd and before its own reduction. At the considered pH, the reduction process involves two protons and two electrons, which means two thiol groups per metal ion. This is in agreement with the reduction of any Zn bound to two shared – SH groups (bridging) and to the other two thiol groups not shared (terminals).
4. Conclusions The present work constitutes the first application of the MCR-ALS soft modeling approach to electrochemical characterization of involved natural biomolecules such as Zn–MT II. The combined use of voltammetry and MCR-ALS provides a powerful tool in the study of metal complexation equilibria of metalloproteins. It has been proved that the peptidic fragment Lys–Cys – Thr – Cys –Cys – Ala, although it has been used widely as a reliable simple model for MT, presents electroanalytical behavior in mixed metal systems even more complex than that of MT. This can be due to the different structures of FT and MT. FT has a very flexible and dynamic structure and a small size that allow the hypothetical formation of different electroactive complexes. In contrast, the much more rigid structure of MT has a lower number of possibilities, acquiring more compact and closer conformations around the metal ions and making more difficult the electrochemical reduction of the bound metals. The results presented here are of interest for a better interpretation of solving metal equilibria of MT and may prove useful for a better understanding of some structural features. Thus, two identical metal ions bound in different chemical environments produce different electrochemical signals that can be distinguished with the support of MCR-ALS. For the present cases, voltammetry joined to MCR-ALS resolution can be considered as a complementary tool to spectral means for a better understanding of the metal binding properties (especially structural features) of molecules. On the basis of these results, some compatible structures are proposed and the electrochemical reduction processes are depicted for the chemical species considered in the mixed metal system Cd–Zn –FT. In the case of Cd– Zn – MT II, additional information is required before proposing similar models.
Acknowledgements The authors gratefully acknowledge financial support from the Spanish Ministerio de Educacio´ n y Cultura
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(DGICYT project PB96-379-C03), from the Ministerio de Ciencia y Tecnologı´a (project BQU2000-0642-C03), and from the Generalitat of Catalonia (1999SGR00048).
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