Reactivity of an antimetastatic organometallicruthenium compound with metallothionein-2: relevance to the mechanism of action

Abstract

The reaction of metallothionein-2 (MT-2) with the organometallic antitumour compound [Ru(η⁶-p-cymene)Cl₂(pta)], RAPTA-C, was investigated using ESI MS and ICP AES. The studies were performed in comparison to cisplatin and significant differences in the binding of the two complexes were observed. RAPTA-C forms monoadducts with MT-2, at variance with cisplatin, that has been observed to form up to four adducts . These data, combined with ICP AES analysis, show that binding of both RAPTA-C and cisplatin to MT-2 requires the displacement of an equivalent amount of zinc, suggesting that Cys residues are the target binding sites for the two metallodrugs. The competitive binding of RAPTA-C and cisplatin towards a mixture of ubiquitin (Ub) and MT-2 was also studied, showing that MT-2 can abstract RAPTA-C from Ub more efficiently than it can abstract cisplatin. The mechanistic implications of these results are discussed.

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Introduction

Following the discovery of the anticancer properties of cis-diamminedichloridoplatinum(II) (cisplatin) in 1965,¹ many inorganic and organometallic compounds have now been evaluated for anticancer activity.^2,3 Among them Ru(III) and Ru(II) complexes are proving to be effective alternatives to those based on platinum. Two Ru(III) complexes, namely trans-[tetrachloro(DMSO)(imidazole)ruthenate(III)] (NAMI-A),⁴ and trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019)⁵ have passed phase I clinical trials.⁶ These compounds contain ruthenium(III) centres, which are thought to undergo reduction in vivo to form an active ruthenium(II) anticancer species. Accordingly, it was postulated that ruthenium(II) complexes might prove to be interesting drug candidates.

A number of ruthenium(II)–arene compounds have been developed as anticancer agents.⁷ In recent years, we have studied the cytotoxic and antimetastatic properties of a family of ruthenium(II)–arene complexes that include a 1,3,5-triaza-7-phosphaadamantane (pta) ligand, the prototype compound being [Ru(η⁶-p-cymene)Cl₂(pta)], termed RAPTA-C (Fig. 1).⁸ The studies have shown that although these compounds tend to be only moderately cytotoxic in vitro, they demonstrate a high selectivity toward cancer cells in comparison to non-tumourigenic cells. Moreover, a significant in vivo effect on the growth of lung metastases was established for RAPTA-C⁹ and subsequently [Ru(η⁶-toluene)Cl₂(pta)], RAPTA-T.¹⁰ Unlike cisplatin, which exhibits its main chemotherapeutic action through binding to the bases of DNA, the mechanism of action of the Ru(II) compounds may involve interactions with critical intracellular proteins.^10–12 This alternative protein-mediated mechanism of anticancer activity is particularly interesting, as a complementary therapeutic to cisplatin, as resistance to cisplatin remains a challenge in the clinics.

Fig. 1 Structures of the metal complexes employed in this study.

Despite the clinical success of cisplatin and related compounds, drug resistance, both intrinsic and acquired, is a major drawback limiting their applicability. Much progress had been made in understanding the multifactorial mechanisms involved in drug resistance,¹³ which include reduced cellular uptake, increased efflux of the drug from the cells, and inactivation through binding to cytosolic and nuclear proteins and glutathione.¹⁴ For drugs that damage DNA, such as cisplatin, increased ability of cancer cells to repair cisplatin damage can also occur.¹⁵ One possible mechanism to overcome resistance that has been demonstrated to be effective is to tether inhibitors of the inactivation proteins to the drug, for example, glutathione-S-transferase (GST) inhibitors covalently linked to platinum overcome GST-related platinum drug resistance.¹⁶ An advantage of ruthenium–arene compounds is the relative inertness of the metal–ligand scaffold to interaction with the deactivating cellular components; nevertheless, it has been shown that additional structural modifications of Ru(II) complexes can further suppress certain resistance mechanisms. For example, phenoxazine-type P-glycoprotein (Pgp) inhibitors attached to the Ru(II)–arene centre via imidazole linkages results in compounds that overcome multidrug resistance¹⁷ and GST inhibitors have been attached in a similar way,¹⁸ or to the arene via a cleavable group.¹⁹ Indeed, increased levels of both Pgp and GST have been correlated to drug resistance in certain tumours and inhibitors of both proteins have been used in combination therapies with, for example, cisplatin.²⁰

Another major class of proteins responsible for metallodrug resistance are metallothioneins (MTs).²¹ Human metallothioneins (MTs) are a class of small (∼7000 Da) cysteine- and metal-rich proteins, abundant in most human tissues. They are represented by four highly conserved isoforms (MT-1/-2/-3/-4). Not much is known about the function of MT-4, which is exclusively expressed in certain squamous epithelia. MT-1 and MT-2 occur ubiquitously in high amounts in mammalian cells, and in contrast to MT-3/-4, the biosynthesis of MT-1/-2 is induced by a variety of compounds including hormones, cytokines, and metal compounds such as cisplatin.^21–24 The suggested functions of MT-1 and MT-2 include, among others, homeostasis and transport of physiologically essential metals (Zn, Cu),²¹ detoxification of toxic metals (Cd, Hg, Pt), protection against oxidative stress²⁵ and regulation of cell proliferation and apoptosis.²⁶ Although the expression of MT-3, also known as the neuronal inhibitory factor, is mainly confined to the brain, this protein is over-expressed in a number of cancer tissues. It has been shown, moreover, that over-expression of the MT-1/-2/-3 isoforms confers resistance to platinum anticancer drugs in many cancer cell lines .^27–29 In fact, high levels of MT expression have been associated with the poor clinical prognosis and a low survival rate of cancer patients undergoing platinum-based chemotherapy.³⁰

Human MTs are composed of a single polypeptide chain of 61–68 amino acids including 20 cysteines. The cysteine thiolates are involved in the binding of seven divalent metal ions forming two independent metal–thiolate clusters in which each metal is tetrahedrally coordinated by both terminal and bridging thiolate ligands (Fig. 2a). An M^II₃CysS₉ cluster is located in the β-domain and an M^II₄CysS₁₁ cluster in the α-domain of the protein.^31,32 The M^II₇MT structures are highly dynamic, allowing facile metal exchange, mainly in the β-domain. Even though thiolate ligands are involved in metal binding they retain a substantial degree of nucleophilicity seen with the metal-free protein. Naturally occurring MTs usually contain seven Zn(II) ions. However, these metals can be displaced by other metal ionsin vivo that have a higher affinity for thiolates such as Cd(II), Hg(II), and Pt(II), and the molecular mechanisms of the Pt(II) interactions with MT-1/-2 have been the subject of numerous studies.^33–36

Fig. 2 (a) Three-dimensional structure of Cd₇MT-2 obtained by NMR (generated with the program PyMOL).⁴⁸ (b) Sequence alignment of human and rabbit MT-2 (human MT-2, entry P02795; rabbit MT2a, entry P18055). Calculated identity is 90%.

As far as we are aware, studies on the interaction of MTs with ruthenium compounds are lacking, despite the growing interest in ruthenium anticancer drugs and their ability to function in platinum-resistant tumours. Herein, we describe the use of electrospray ionisation mass spectrometry (ESI MS) to probe the interactions of RAPTA-C with rabbit MT-2, using cisplatin as a reference compound. The amino acid sequence of rabbit MT-2 has a high degree of identity with that of human MT-2, see Fig. 2. The competitive binding of RAPTA-C towards MT-2 and the model proteinubiquitin (Ub) has also been studied.

Results and discussion

Recently, ESI MS was used to study the interactions of MT-2 with several new generation anticancer Pt(II) complexes,³⁶ revealing that MTs can scavenge a Pt(II) drug fraction already bound to DNA, thus reversing Pt(II)–DNAadducts or converting them into stable ternary complexes.³⁷ Consequently, the reaction of the organometallic ruthenium(II) compound, RAPTA-C, with MT-2 was monitored using ESI MS. In a typical experiment, three molar equivalents of RAPTA-C were added to an aqueous solution of MT-2 buffered at pH 7.4 and the mixture was maintained at 37 °C until analysis. Fig. 3 shows the ESI MS multicharged spectra of the sample recorded at different times over a period of 48 hours, focusing on the +6 ions. At 24 hours, in addition to the peaks of the unbound protein at m/z 1021, new signals appear corresponding to ruthenium adducts of MT-2. Fragments in which either the pta and cymene ligands, i.e.[(η⁶-cymene)(pta)Ru] at m/z 1087, or just the cymene ligands, i.e. [(η⁶-cymene)Ru] at m/z 1061, bound to MT-2 are observed. In the control experiments with cisplatin, adducts with various Pt(II) : MT-2 ratios were detected. These data show that cisplatin, when bound to MT-2, has both carrier and leaving ligands replaced by thiolates or maintains only one bound NH₃ (or H₂O/OH) molecule (Fig. 3), in agreement with previously reported results.³⁶ Notably, in contrast to cisplatin/MT-2 samples, only ruthenium mono-adducts are formed in the case of RAPTA-C.

Fig. 3 ESI mass spectra (+6 ions) of MT-2 treated with cisplatin or RAPTA-C (3 : 1, metal : protein ratio) in buffer TMeAmAc (pH 7.4) after 48 hours incubation at 37 °C. The mass peaks were assigned to MT-2 species containing Pt- or Ru-containing fragments as follows: (top spectrum) m/z 1021 = MT-2; m/z 1053 = MT-2 + Pt; m/z 1088 = MT-2 + 2Pt; m/z 1121 = MT-2 + 3Pt; m/z 1153 = MT-2 + 4Pt; (bottom spectrum) m/z 1021 = MT-2; m/z 1060 = MT-2 + [Ru(η⁶-p-cymene)]; m/z 1087 = MT-2 + [Ru(η⁶-p-cymene)(pta)].

It is worth noting that on binding of the Ru(II) complex to MT-2 protein Zn(II) ions are released. However, it was not possible to determine the exact number of Zn(II) ions displaced from the protein by ESI MS analysis. Therefore, the MT-2 samples incubated with RAPTA-C and cisplatin were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP AES) to quantify the metals present (see Table 1). The results confirm that RAPTA-C has a lower affinity for MT-2 than cisplatin and, as expected, displaces less zinc ions. Combined, these data show that binding of both RAPTA-C and cisplatin to MT-2 requires the displacement of an equivalent amount of Zn, therefore suggesting that Cys residues are the target binding sites for the two metallodrugs. The lower amount of RAPTA-C that binds to MT-2 compared to cisplatin is probably due to the presence of the η⁶-arene ligand, which is not displaced. In fact, in all the MS studies of RAPTA-C binding to proteins, the chloride ligands and to a lesser extent the pta ligand are lost, whereas the arene always remains attached to the Ru(II) center.^38,39 Even in a crystal structure of a RAPTA-type compound embedded within human GST P1-1 the arene (and the pta ligand) remain coordinated with the two chloride ligands substituted by two Cys residues.¹⁹ The hydrophobic nature of the arene combined with its steric demand and rigidity over three coordination sites of the Ru(II) center presumably reduce the capacity of RAPTA-C for MT-2 binding.

Table 1 Metal : MT-2 ratios determined by ICP AES analysis. The metallodrug–proteinadducts were established at a protein concentration of 100 μM and metal to protein ratio of 3 : 1. All the reaction mixtures were incubated for 48 hours at 37 °C and samples analyzed after separation of any unbound metal by ultracentrifugation with microconcentrators (Centricon YM-3, Amicon). Fully-loaded Zn₇MT-2 was used for the preparation of the samples (Zn : protein ratio of 7.0 ± 0.3)

RAPTA-C		Cisplatin
Ru : MT-2	Zn : MT-2	Pt : MT-2	Zn : MT-2
1.5 ± 0.3	5.6 ± 0.7	2.9 ± 0.5	3.0 ± 0.5

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The ability of MT-2 to cleave Ru–proteinadducts was investigated by incubating RAPTA-C with Ub and subsequently adding MT-2 at differrent MT-2 : Ub ratios, namely 1 : 1 and 2 : 1. It is worth noting that Ub has been selected as a model system since it is particularly suitable for MS analysis. In fact, it has favourable properties such as moderate size (M_w of ca. 8500 Da) and has a high stability in solution under physiological-like conditions.

Fig. 4 shows the ESI MSspectra obtained for RAPTA-C samples. Prior to the addition of MT-2, Ub was identified as one of the main peaks at charge states >+8 at m/z 1071 [Ub + 8H]⁸⁺ and RAPTA-Cadducts were identified at m/z 1100 [Ub + Ru(η⁶-p-cymene) + 6H]⁸⁺ and at m/z 1120 [Ub + (η⁶-cymene)Ru(pta) + 6H]⁸⁺ (Fig. 4, bottom spectrum and Table 2), in agreement with previously reported results.³⁹ In order to avoid the formation of artefacts, due to the presence of unbound complex in solution during the analysis, any excess RAPTA-C present was removed by centrifugation through a molecular weight cut-off filter (3 kDa) prior to the addition of MT-2. This experimental approach follows a recently reported study from our group, in which the reactivity of representative metallodrugs with a mixture of proteins was probed without using any chromatographic separation prior to analysis.⁴⁰

Fig. 4 ESI mass spectra (+8 ions) of Ub treated with RAPTA-C (3 : 1, metal : protein ratio) in buffer TMeAmAc (pH 7.4) before (bottom spectrum) and after addition of MT-2 (middle and top spectra).

Table 2 Main peaks present in the ESI mass spectra (+8 ions) of cisplatin or RAPTA-C-treated Ub in buffer TMeAmAc (pH 7.4) after 48 hours incubation at 37 °C^a

Compound	m/z Measured	m/z Theoretical	Protein–metal adduct
a 1071 m/z = Ub.
RAPTA-C	1100	1100.37	Ub-[Ru(η^6-p-cymene)]
	1120	1120.01	Ub-[Ru(η^6-p-cymene)(pta)]
	1125	1124.38	Ub-[Ru(η^6-p-cymene)(pta)Cl]
	1149	1149.38	Ub-[Ru(η^6-p-cymene)(pta)] + Ub-[Ru(η^6-p-cymene)]
	1154	1153.75	Ub-[Ru(η^6-p-cymene)(pta)Cl] + Ub-[Ru(η^6-p-cymene)]
Cisplatin	1100	1099.62	Ub-Pt(NH3)2
	1128	1128.24	Ub- 2 × [Pt(NH3)2]
	1154	1156.86	Ub- 3 × [Pt(NH3)2]

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After addition of a stoichiometric amount of MT-2 to the sample, significant changes were detected by ESI MS. Fig. 4 (middle and upper spectra) shows representative spectra recorded over 48 hours incubation. Notably, the multicharged spectra of the sample show only the signals of Ub, indicating that Ub is more easily ionized with respect to MT-2 under the experimental conditions used, facilitating the changes in the relative intensities (r.i.) of the peaks corresponding to the Ub–RAPTA-Cadducts , in comparison to the one of the unbound Ub, to be discerned. Notably, it has already been shown that metallothioneins produce very weak responses to ionization.⁴¹

After 24 hours the multicharged spectra of the sample (Fig. 4) shows that the peak at ca. m/z 1100 corresponding to the (Ru(η⁶-p-cymene)-Ubadduct decreased dramatically from 50% (immediately after addition of MT-2) to about 10%, with the relative intensities of the peaks remaining essentially the same thereafter. Notably, the intensity of the peaks corresponding to adducts in which ruthenium has retained the pta ligand is less perturbed, possibly due to the hydrophilic pta ligand repelling the MT-2. It has previously been shown that adducts formed between RAPTA-C and Ub may be reversed by the addition of GSH, but the process was much slower than with MT-2.³⁹

To ascertain the concomitant formation of MT-2-ruthenium adducts in the sample we also separated the two proteins in the mixture by size exclusion chromatography prior to MS analysis. Remarkably, we could identify ruthenium fragments bound to MT-2 (see ESI† ).

A similar competition experiment was undertaken with cisplatin for comparison purposes. Cisplatin–Ubadducts have been previously characterized and the data obtained are in good agreement with the literature data, i.e. showing the main Pt-bound species as the [Pt(NH₃)₂]²⁺fragment at ca. m/z 1099 for the +6 charge state (Fig. 5, Table 2). Notably, the cisplatin–Ubadducts were less readily removed by MT-2 compared to RAPTA-C. This difference is clear from the spectrum recorded after 24 hours in which the decrease in relative intensity of the main peak at m/z 1099 is only ca. 50%, with essentially no further change after this time.

Fig. 5 ESI mass spectra (+8 ions) of Ub treated with cisplatin (3 : 1, metal : protein ratio) in buffer TMeAmAc (pH 7.4) before (bottom spectrum) and after addition of MT-2 (middle and top spectra).

When an excess of MT-2 with respect to Ub (2 : 1) was added to the RAPTA-C sample, only modest variations were observed with respect to the 1 : 1 ratio. It is worth mentioning that the almost complete “stripping” of the metallofragments from Ub was achieved, for both MT-2 : Ub ratios, only after 24 hours, while less significant changes were observed during the first 6 hours of incubation (Fig. 6).

Fig. 6 ESI mass spectra (+8 ions) of Ub treated with RAPTA-C (3 : 1, metal : protein ratio) in buffer TMeAmAc (pH 7.4) before and at different times after addition of MT-2 (MT-2 : Ub = 2 : 1). The m/z peaks were assigned to Ub species containing Ru-fragments as follows: Ub = m/z 1071; a: m/z 1100 = Ub + [Ru(η⁶-p-cymene)]; b: m/z 1120 = Ub + [Ru(η⁶-p-cymene)(pta)]. The observed relative intensities are indicated in brackets for adduct a.

At variance with RAPTA-C, cisplatinadducts were more rapidly removed from Ub using an excess of MT-2 and a marked decrease was observed after 1 hour of incubation (see Fig. S2 in the ESI† ). However, even upon addition of an excess of MT-2, they were never completely removed from Ub. Overall, these results suggest that while in the case of cisplatin the reactivity is dependent on the relative concentrations of the two proteins in solution, for RAPTA-T other factors may contribute, such as the steric hindrance around the ruthenium center that might prevent the concomitant binding of the proteins.

The competitive binding of RAPTA-C and cisplatin towards a mixture of Ub and MT-2 was also studied. Samples were prepared by mixing Ub and MT-2 concomitantly with an excess of RAPTA-C or cisplatin. Through this approach it was possible to establish the binding preference of the compounds. The incubation mixture was analyzed after 24 hours by ESI MS and the spectra of the protein mixtures, focusing on the region of Ubadducts , are shown in Fig. 7 (for RAPTA-C) and in Fig. 8 (for cisplatin). For comparison purposes, the spectrum of Ub alone, treated with the metal complex under the same conditions, is also shown (upper spectrum in Fig. 7 and 8). In all cases, adducts of Ub with both RAPTA-C and cisplatin are detected but with much lower relative intensity compared to samples in which MT-2 is not present. Notably, this feature is markedly more pronounced for RAPTA-C.

Fig. 7 ESI mass spectra (+8 ions) of Ub (top spectrum) and of a mixture of Ub and MT-2 (1 : 1) treated with RAPTA-C (3 : 1, metal : protein ratio) in buffer TMeAmAc pH 7.4 after 24 h incubation at 37 °C.

Fig. 8 ESI mass spectra (+8 ions) of Ub (top spectrum) and of a mixture of Ub and MT-2 (1 : 1) treated with cisplatin (3 : 1, metal : protein ratio) in buffer TMeAmAc (pH 7.4) after 24 h incubation at 37 °C.

The greater ability of MT-2 to strip RAPTA-C from Ub relative to cisplatin, and to absorb free RAPTA-C from solution, could be important with respect to the pharmacological properties of this new putative metallodrug. It has already been shown that RAPTA-C can be tolerated in vivo at much higher doses than cisplatin without observable side effects, although it is important to note that RAPTA-C is less active than cisplatin in certain respects.⁹ It is also believed that enzymatic targets are more relevant than DNA for RAPTA-C. Indeed, both compounds bind extensively to proteins within the cell cytoplasm although with cisplatin the ultimate biological target is DNA,^42,43 whereas for RAPTA-C it is thought to be an enzyme/protein target. One possible protein target identified is cathepsin B,¹² and once RAPTA-C or other RAPTA compounds are buried inside this protease it is difficult to envisage that it could be displaced by MT-2. In contrast, RAPTA-C indiscriminately bound to appropriate surface residues on other proteins, that would otherwise lead to unwanted toxic side-effects, could be readily removed and detoxified by MT-2 and potentially other metallothioneins. In the case of cisplatin, the release of Pt-adducts from Ub upon addition of MT-2 was much less pronounced, and could explain the higher general toxicity of this compound. However, the fact that lower ratios of RAPTA-C : MT-2 compared to cisplatin : MT-2 are observed also suggests that RAPTA-C is more likely to be active in tumours containing high levels of metallothioneins although MT-2 is nevertheless able to mop up (bind) free circulating RAPTA-C forming covalently modified adducts , as shown here.

To conclude, the potential range of applications of ESI MS has been extended to study competitive binding of metallodrugs to proteins, in this case studying a system of relevance to drug resistance and accordingly general drug toxicity. It was shown that MT-2 can abstract RAPTA-C from Ub more efficiently than it can cleave and abstract cisplatin–Ubadducts , and this process may have important pharmacological consequences accounting, at least in part, for the different pharmacological profiles of the two compounds.

Experimental

Proteins

Red blood cell ubiquitin was obtained from Sigma (U6253). MT-2 was isolated from the liver of New Zealand White rabbits and purified as described.⁴⁴Protein purity was further assessed by SDS-PAGE.⁴⁵ The concentration of reduced thiols was determined by 2,2′-dithiodipyridineassay .⁴⁴ In all cases, a cysteine to protein ratio of 20.0 ± 0.5 was obtained. Fully Zn(II)-loaded MT-2 was prepared by a reconstitution method previously described in the literature.⁴⁶ Metal-to-protein ratios were determined by measuring the metal content by flame atomic absorption spectrometry (SpectrAA-110, Varian Inc.) in 15 mM HNO₃⁴⁷ and that of the protein spectrophotometrically in 0.1 M HCl (ε₂₂₀ = 48 200 M⁻¹ cm⁻¹).⁴⁴ In all cases, a metal-to-protein ratio of 7.0 ± 0.3 was obtained.

Metal complexes

Cisplatin was obtained from Sigma and Ru(η⁶-p-cymene)(pta)Cl₂ (RAPTA-C) was prepared using a literature method.⁸

Mass spectrometry

Samples were prepared by mixing MT-2 100 μM (freshly dissolved and pre-reduced with DTT to avoid oxidation of the cysteines) with an excess of the appropriate metal complex (3 : 1, metal : protein ratio) in tetramethylammonium acetate buffer (TMeAmAc) 25 mM (pH 7.4) and incubated over 48 h at 37 °C. Prior to analysis samples were extensively ultrafiltered using a Centricon YM-3 filter (Amicon Bioseparations, Millipore Corporation) in order to remove the unbound complex. In a second series of experiments, 100 μM Ub was first reacted with an excess of metal complex (3 : 1 ratio) and after 24 hours incubation at 37 °C ultrafiltered. Then addition of MT-2 at different ratios with respect to Ub (1 : 1 or 2 : 1) was performed and the mixture incubated at 37 °C for different time intervals (1, 3, 6, 24, and 48 hours) prior to MS analysis. For the competition experiments, protein samples containing both Ub, MT-2 and RAPTA-C (3 : 1 metal : protein ratio) were prepared as described above and analysed by MS over a period of 24 h. Each sample was analyzed in triplicate.

Electrospray-ionisation MS data were acquired on a Q-Tof Ultima mass spectrometer (Waters) fitted with a standard Z-spray ion source and operated in the positive ionization mode. Experimental parameters were set as follows: capillary voltage 3.5 kV, source temperature 80 °C, desolvation temperature 120 °C, sample cone voltage 100 V, desolvation gas flow 400 L h⁻¹, acquisition window 300–2000 m/z in 1 s. The samples were diluted 1 : 10 in water and 5 μl were introduced into the mass spectrometer by infusion at a flow rate of 20 μl min⁻¹ with a solution of ACN/H₂O/HCOOH 50 : 49.8 : 0.2 (v : v : v). External calibration was carried out with a solution of phosphoric acid at 0.01%. Data were processed using the MassLynx 4.1 software.

Metal content quantification by ICP AES analysis

The determination of Zn, Pt and Ru concentrations in samples prepared as described above for MS analysis was performed in triplicate by a Varian 720-ES Inductively Coupled Plasma Atomic Emission Spectrometer (ICP AES). Before analysis, samples were diluted to 1 : 10 in 5 mL of 0.1% high purity nitric acid obtained by sub-boiling distillation. Samples were spiked with 400 ppb of Ge, used as internal standard, and calibration standards were prepared by gravimetric serial dilution from mono standards at 1000 mg L⁻¹.

Acknowledgements

A. C. thanks the Swiss National Science Foundation for financial support (AMBIZIONE project N° PZ00P2_121933).

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Footnote

† Electronic supplementary information (ESI) available: SEC details and Figures S1 and S2. See DOI: 10.1039/b909185h

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