Dalton Transactions View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
PAPER
Cite this: DOI: 10.1039/c3dt52255e
View Journal
Mass spectrometry and potentiometry studies of Pb(II)–, Cd(II)– and Zn(II)–cystine complexes Emilia Furia, Donatella Aiello, Leonardo Di Donna, Fabio Mazzotti, Antonio Tagarelli, Hariprasad Thangavel, Anna Napoli* and Giovanni Sindona Cd(II)–, Pb(II)– and Zn(II)–cystine complexes were investigated by potentiometric and different mass spectrometric (MS) methodologies. Laser desorption mass spectrometry has provided both the composition and structure of metal–cystine complexes according to the speciation models proposed on the basis of the potentiometric data. Detection of neutral complexes was achieved by protonation or electrochemical reduction during mass spectrometric experiments. The redox activity of metal–cystine complexes
Received 18th August 2013, Accepted 17th October 2013
was confirmed by laser desorption and charge transfer matrix assisted laser assisted MS experiments, which allowed us to observe the formation of complexes with a reduction of cystine. The stoichiometry of Cd(II)–,
DOI: 10.1039/c3dt52255e
Pb(II)– and Zn(II)–cystine complexes was defined by observing the isotopic pattern of the investigated com-
www.rsc.org/dalton
pound. The results suggest that interaction occurs through the carboxylate group of the ligand.
Introduction Metal cations are widespread in the environment and in many dietary supplements present in the market of nutraceuticals.1 Their interaction with amino acids, proteins, nucleic acids or metabolites in biological systems may cause undesirable side effects due to the thermodynamics of the metal complexes thus formed in that particular setting. Cadmium ions, for example, can replace the native zinc ion from many proteins and enzymes to a degree that depends on their affinities. Lead ions also inhibit a variety of enzymes. The toxicities of Cd(II), Zn(II) and Pb(II) metals have been reviewed extensively in the literature. Interactions of some heavy metals with a restricted number of proteogenic amino acids have been investigated by different spectroscopic methods.2–7 Cystine (Cyss) is a nonproteogenic amino acid available as an individual supplement or as part of protein supplements. It participates in a variety of physiological functions, including antioxidant activity,8,9 and shows regulatory effects in the substrate-recognizing function of the eukaryotic proteasome.10,11 Furthermore, a significant fraction of heavy metals naturally present in blood plasma occurs in the form of amino acid complexes and a predominant fraction of these involves cystine. In the alkaline environment cystine displays four protonation sites due to (i) the two most basic amino groups and (ii) the two carboxylic groups. Both types of donor sites are likely to participate in metal
Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, P Bucci, cubo 12/d, I-87036Arcavacata di Rende, Italy. E-mail: [email protected]; Fax: +39 0984 493307
This journal is © The Royal Society of Chemistry 2013
coordination and the disulfide bond may also be involved in some specific cases. Studies on metal complex formation and even protonation equilibria of this amino acid have been hampered by very low solubility in neutral aqueous media.12 Recently, the solubility as well as the acidic constants of cystine in NaClO4 solutions at different ionic strengths have been determined.13 The solubility of cystine increases proportionally with the ionic medium concentration, thus the complexation equilibria of this ligand with the Cd(II), Pb(II) and Zn(II) ions have been studied by potentiometric titrations with a glass electrode at 25 °C and in 3 mol dm−3 NaClO4 ionic medium, where the variation of the reagent concentrations does not involve large changes in the activity coefficients.14 Therefore, the activities were replaced by the concentrations in the numerical evaluation. These systems should be formed by two, three and four monomeric coordination complexes with the same ligand. The structure of these species has not been fully characterized15 whilst the understanding of potential toxicological activity requires a deep knowledge of the sequestering ability of natural ligands in a given particular environment. Recent achievements in mass spectrometric methodologies provide a rapid and sensitive tool for the identification and quantitation16 of metabolites,17,18 amino acids and proteins19,20 and their post-translational modifications.21 Electrospray mass spectrometry (ESI MS)22–24 is a well-established method for the detection and analysis of metal ion complexes. Literature data include the study of gas-phase metal ion chemistry and the formation of metal ion adducts to assist in the detection and structural elucidation of organic compounds.25 Structural information on M(II)–Cyss compounds is quite
Dalton Trans.
View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Paper difficult, even under the softest ionization conditions because the labile involved non-covalent bonds are susceptible to protonolysis during the ionization process, and therefore the molecular adducts may be missing completely, or their relative abundances may be very low in the full-scan mass spectra. Charge transfer matrix-assisted laser desorption/ionization (CT-MALDI-MS) has successfully been used to ionize fragile complexes of polymetallic porphyrin derivatives.26 Thus, ESI MS, CT-MALDI-MS and laser desorption mass spectrometry (LD MS) can be exploited to determine either the composition and structure elucidation of Cd(II), Pb(II) and Zn(II) cations– Cyss complexes. The equilibrium behaviour and the speciation model in aqueous solution in a wide pH range, between Cd(II), Pb(II) and Zn(II) cations and Cyss and possible structures of the complexes formed will be discussed on the basis of potentiometric and mass spectrometric results.
Dalton Transactions Table 1 Best set of the overall stability constant, log βpqr (3σ), for the system Cyss–Pb(II) according to general equilibrium (1)
Species
Model 1
Model 2
Model 3
Model 4
PbL PbL22− Pb(HL)+ Pb(HL)2 Pb(HL)L− σ (mV) χ2 U
−5.87 ± 0.09 −5.45 ± 0.06 −5.62 ± 0.05 −5.79 ± 0.04 −17.0 ± 0.1 −17.18 ± 0.07 −17.38 ± 0.09 −17.43 ± 0.07 −8.64 ± 0.09 −8.8 ± 0.1 −8.85 ± 0.08 −10.48 ± 0.07 −9.56 ± 0.09 −9.67 ± 0.05 3.71 1.02 0.40 0.34 9.31 15.03 8.17 6.34 8.81 × 102 6.55 × 101 9.72 6.85
Results and discussion Characterization of Pb(II)–, Cd(II)– and Zn(II)–cystine complexes by potentiometric titration The potentiometric study was performed on the Pb(II)–, Cd(II)– and Zn(II)–Cyss systems in order to get the stoichiometry of the complexes and the magnitude of the related stability constants and to describe the speciation profile. A summary of the relevant data taken in all titrations is presented in Table 3 (Experimental section). By applying the reported value of the constants of the main hydrolytic products, i.e. PbOH+, Pb3(OH)42+, Pb4(OH)44+ and Pb6(OH)84+,27 the numerical treatment leads to the best set of log βpqr (3σ) for the considered system. The numerical evaluation initially was performed considering the formation of the complexes PbL and PbL22− (Model 1). Since the standard deviation observed was considerably higher than the experimental uncertainty (Model 1), other models were tested considering a set of n + 1 species for each one. The best agreement was obtained with Pb(HL)+ (Model 2) and a consequent diminution of the function U equal to 93%. The fit is significantly improved including in the previous model the species Pb(HL)L− (Model 3). An additional refinement was reached on adding the neutral complex Pb(HL)2 (Model 4). As no other species lowered the minimum, Model 4 was assumed to be the best in describing the data, also taking into consideration that the standard deviation (σ) is comparable with the experimental uncertainty. Using the values of the constants reported in Table 1 (Model 4), distribution diagrams were constructed (Fig. 1a–b). Speciation diagram (Fig. 1a) shows that PbL, PbL22− and Pb(HL)+ attain significance percentage when the analytical concentrations of the ligand are greater than those of the metal, e.g. CL = 5 mmol dm−3, CM = 0.5 mmol dm−3, while Pb(HL)2 is a minor species and reaches a percentage of only 10%. Otherwise, at comparable analytical concentrations of the ligand and the metal (Fig. 1b) the metal–ligand complexes
Dalton Trans.
Fig. 1 Speciation diagrams of M(II)–Cyss vs. −log[H3O+] at T = 25 °C. Free metal is shown by the dashed line: (a) Pb(II)–Cyss: CM = 0.5 mmol dm−3, CL = 5 mmol dm−3; (b) Pb(II)–Cyss: CM = 1 mmol dm−3, CL = 2 mmol dm−3; (c) Cd(II)–Cyss: CM = CL = 1 mmol dm−3; (d) Cd(II)–Cyss: CM = 1 mmol dm−3, CL = 2 mmol dm−3.
Pb(HL)L are the main species achieving a percentage of 50% at pH 7. Analogously, data treatment for Cd(II) was performed by considering the well-known hydrolysis product in 3 mol dm−3 perchlorate media (such as Cd(OH)+).28,29 Various models were tested by adding a single species but none of these lowered the minimum of the function. Then the only complex for the system Cyss–Cd(II) is CdL whose stability constant, log β121 according to the general equilibrium (1), is −5.5 ± 0.1 (the uncertainty represents 3σ). The refined equilibrium constant is used to construct the distribution diagrams of cadmium(II) in the different species (Fig. 1c–d). The speciation diagrams of the Cyss–Cd(II) system at different concentration ratios show that in the whole pH range, both at M : L = 1 : 1 (Fig. 1c) and at M : L = 1 : 2 (Fig. 1d), the most important species is CdL, which is predominant over the hydrolysis product, CdOH+, and the formation of which is over 90% when M : L = 1 : 2 (Fig. 1d). The numerical estimation of the equilibrium formation constants of the complexes between Cyss and Zn(II) was performed by considering the formation quotients of the hydrolytic species Zn(OH)3− and Zn(OH)42− (25 °C in 3 mol dm−3 NaClO4)27 and the various steps of the calculations are illustrated in Table 2. Model 1, which considers the presence of ZnL22−, is the best model with only one species. The introduction of ZnL
This journal is © The Royal Society of Chemistry 2013
View Article Online
Dalton Transactions
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Table 2
Paper
Best set of log βpqr (3σ) for the system CYSS–Zn(II)
Species
Model 1
Model 2
ZnL22−
−17.03 ± 0.09
ZnL σ (mV) χ2 U
0.51 14.88 21.92
−16.61 ± 0.08 −6.86 ± 0.06 0.32 5.13 7.12
(Model 2) lowered the minimum of the square error sum significantly (67%) and the standard deviation (σ) was comparable with the experimental uncertainty. No other species, introduced to improve the fit, were retained. All the proposed species reach a concentration level of at least 35% of the total metal; therefore significant concentrations were obtained for a correct definition of the equilibrium constants. Characterization of Pb(II)–, Cd(II)– and Zn(II)–cystine complexes by mass spectrometry Information regarding the structure, coordination site and metal oxidation state of the dissolved metal ion complexes cannot be obtained using electrochemical or spectroscopic techniques.30,31 “Soft” ionization techniques such as ESI and MALDI can ionize labile complexes with reduced fragmentation. ESI MS is well suited to study the dissolved-phase interaction between the metal ion and the organic ligand and has been successfully used to determine metal-binding stoichiometry, and for structural elucidation.22–24 Nevertheless, amino acids and peptides containing disulfide groups introduce some complications in the mass spectrum when collisioninduced dissociation (CID) is employed for protein identification. To overcome this drawback, metal ions are used as charge carriers in peptides. Metalated peptide-complexes with or without an auxiliary ligand could provide complementary ECD (electron-capture dissociation) fragments compared to their protonated species. Cyss represent the more simple system containing a disulfide bond, where the competition of other functional groups is reduced; thus the interaction between a metal ion and the disulfide bond could be increased. Unexpectedly, ESI MS of premixed solutions of M : L at different pHs did not show the complexed dication signals with sufficient intensity to explore the structure and coordination site by CID. Thus, an alternative ionization method should be desirable for these complexes, which are susceptible to protonolysis during the ionization process and their relative abundances are very low in the full-scan mass spectra. The first challenge was the identification of a suitable MALDI matrix for the M(II)–Cyss complex MS analysis. In fact, when the Pb–Cyss conjugated (1) was submitted to MALDI experiments using traditional matrixes (α-CHCA, DHB) no molecular ion peaks were detected. Thus, the interaction of M(II) cations with Cyss was exploited by CT MALDI MS and LD MS. The ionproviding information on M : L and M : L2 species (Scheme 1) was observed in the full-scan positive ion CT MALDI and LD mass spectra by deposition of a few μl of the reaction aqueous solution.
This journal is © The Royal Society of Chemistry 2013
Scheme 1
Formation of PbL, PbL2 radical cations by CT-MALDI and LD-MS.
MS/MS experiments were performed to elucidate the ionic structures and also to characterize fragmentation patterns. Fig. 2 shows the LD(+) mass spectrum obtained using 1 μl of the reaction aqueous solution of the conjugated 1. Pb(II) species are clearly identified by the characteristic distribution of lead isotopes, and the mass-to-charge (m/z) values reported refer solely to the most abundant (208Pb) isotope peaks. The spectrum shows the formation of reduced1 and reduced2 forms of ML2 and ML and ion fragments due to the partial decomposition of the complexes. On the other hand, the protonation of the neutral two-coordinate species [Cyss,Pb(II)] would result in the formation of the pseudomolecular ions [Cyss,Pb(II) + H]+, [(2Cyss,Pb(II) + H]+ (m/z 446.99 and m/z 687.02, respectively). The absence of complexes containing chloride and free Cyss suggests that the complex is formed quantitatively and that the perchlorate anion does not compete towards cations under the adopted experimental conditions. The comparison of the measured experimental isotopic distribution with the theoretically calculated distribution of the expected summary formula suggests that the ion clusters of m/z 688.02–686.00 and m/z 447.99–445.98 result from the overlap of the reduced1/reduced2 form of [ML2]•+ ([C12H20N4O8S4Pb]•+; [C12H24N4O8S4 Pb]•+, Δm = −5 ppm) and [ML]•+ ([C6H12N2O4S2 Pb]•+, [C6H10N2O4S2Pb]•+; Δm = −5 ppm), respectively (Fig. 2). The expected oxidizing nature of higher valence metals such as Pb(IV), together with the known ease of reduction of Cyss, suggests that it is the ligand rather than the metal ion which is reduced. Therefore, the ionization process of this system is represented by a redox process, for which a laser induced reduction initiates the chemical process. Initially, the reduction of the complex, ML2, ML, generates a formal positive charge localized on the coordinated Cyss ligand, [ML2]•+ and [ML]•+; a subsequent intra-cluster H transfer drives the formation of reduced species [ML2 + 2H]•+ and [ML + 2H]•+ at m/z 688.02 and 447.99, respectively. Such ionic complexes are,
Fig. 2
LD MS of the conjugated 1.
Dalton Trans.
View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Paper
Dalton Transactions
therefore, best represented as radical cations of the ligand oxidized and reduced forms, i.e. [ML2]•+, [ML]•+ and [ML2 + 2H]•+, [ML + 2H]•+. The peak of m/z 686.00 (Fig. 2) corresponds to the ion [(Cyss)2,Pb(II)]•+ ([ML2]•+), in which Cyss is deprotonated (i.e. Cyss2−) and presumably bound covalently to Pb(II) via oxygen. Furthermore, the ion peak of m/z 688.02 represents [(Cyss)2,Pb(II)]•+ ([ML2 + 2H]•+), in which the reduced ligand is likewise bound covalently via oxygen to the metal(II). The same considerations can be extended to the observed [ML]•+ species (Scheme 1). CT MALDI MS was exploited as an alternative ionization process for these systems, which are susceptible to ease reduction because the ionization process is initiated by photoabstraction of an electron from matrix molecules. This step generates a radical cation, which in turn abstracts an electron from the analyte. Therefore, it was hypothesized that 2,7dimethoxynaphthalene (DMN) could promote intracluster single electron transfer between the matrix and analytes allowing one to exploit the redox chemistry of these complexes. A few μl of the reaction mixture 1 was loaded on to the sample plate with either a sandwich layer method and a premixing sample/matrix solution (MeOH–CH3Cl (1 : 1)) at different ratios. The CT MALDI MS spectrum of 1 (Fig. 3) exhibits prominent signals for the oxidized/reduced form of the complexes [ML]•+, [ML2]•+ (m/z 446–448, 686–688), electron bound clusters [M2L2]•+ (m/z 892.98), [M2L2 − 32]•+ (m/z 861.98), [M2L2 − 64]•+ (m/z 828.01), [M2L2 − 64]•2+ (m/z 412.99), [M2L3]•+ (m/z 1135.01), [M2L4–Cys–H2O]•+ (m/z 1238.04), and [M2L4–Cys– H2O]•2+ (m/z 619.05). In all cases, the isotopic pattern corresponds to that simulated for the intact molecular cation, providing unequivocal confirmation of the molecular composition. However, the formation of mono and doubly charged electron bound dimers suggested that the matrix improves the desorption process favoring the intra-cluster single electron/H transfer and thus the re-reduction of the ligand. The LD in-source fragmentation of oxidized and reduced forms of [ML2]•+ and [ML]•+ showed fragments arising from the consecutive mass loss of 16 Da. This mass loss can be explained by the release of a NH2• radical suggesting the
Fig. 3
CT MALDI of the conjugated 1.
Dalton Trans.
formation of O → Pb(II) oxidized and reduced forms of [ML2]•+ [ML]•+. MS information on the most prevailing peaks combined with that obtained by MS/MS at different collision energies are used to assign the most probable structure of the complexes [ML2]•+and [ML]•+. LD MS experiments clearly indicate the formation of the species [C12H24N4O6S4Pb]•+ (m/z 656.03, Δm: −6 ppm), [C12H24N4O6S4Pb]•+ (m/z 654.01, Δm: −5 ppm), [C6H10N2O3S2Pb]•+ (m/z 429.99 Δm: −5 ppm), [C6H10N2O2S2Pb]•+ (m/z 413.99 Δm: −6 ppm), and [C6H12N2O3S2Pb]•+ (m/z 432.00 Δm: −5 ppm), [C6H12N2O2S2Pb]•+ (m/z 416.01 Δm: −5 ppm), [C6H12N2O2S2]•+ (m/z 208.00), [C5H11NOS2]•+ (m/z 165.00), probably due to gasphase rearrangements of the molecular cations with the consecutive loss of a OH•, thus excluding the presence of N → Pb coordination. MS/MS of the precursor [ML2 + 2H]•+ (m/z 688.02, 1) yields a fragment rich product mass spectrum (Fig. 4a). The spectrum revealed the formation of the cations [ML]•+ (m/z 448.0) [C6H12N2O3S2Pb]•+ (m/z 432.0), [C6H12N2O2S2Pb]•+ (m/z 416.0), [C5H11NOS2]•+ (m/z 165.0), resulting from the fragmentation of the Cyss, and [Pb]•+ (m/z 208.0), [PbO2]•+ (m/z 240.0). The CID mass spectrum (Fig. 4b) obtained from the precursor ion [ML + 2H]•+ (m/z 447.99) showed product ions arising from the loss of Cyss followed by the consecutive release of OH and the formation of [Pb]•+ (m/z 208.0), [PbO2]•+ (m/z 240.0). The same fragmentation pattern was observed for the oxidized form [ML2]•+, [ML]•+. The observed fragmentation pattern of the oxidized and reduced species confirmed that Cyss is bound covalently to Pb(II) via oxygen. The gas-phase ion chemistry of the system involving Cd(II) and Zn(II) is quite different from the corresponding [Pb(II), Cyss]. According to the speciation diagrams of Cd(II) and Zn(II), which showed that the main complex species in solution is [ML] (Fig. 1c–d), the formation of species of higher coordination number was not observed. In both cases, the metal was bound exclusively by oxidized Cyss, giving [CdL]+ and [ZnL]+. The stability of these complexes was confirmed by the absence of free ligand ions in the LD mass spectra. According to the oxidation potential of the couple Cd(II)/Cd, Zn(II)/Zn, no laser
Fig. 4 LD MS/MS spectra of the reduced form of the conjugated 1: (a) [ML2 + 2H]+ (m/z 688.02) and (b) [ML + 2H]+ (m/z 447.99).
This journal is © The Royal Society of Chemistry 2013
View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Dalton Transactions
Scheme 2
MS/MS fragmentation pattern of [ML + H]+ (m/z 352.92).
induced reduction of the complexes was observed for these systems. The LD(+) mass spectrum, obtained using 1 μL of the aqueous solution of the sample 2, showed the ion peak of 352.92 corresponding to the species [ML + H]+ ([C6H11N2O4S2Cd]+, Δm = 5.5 ppm) in which Cyss is deprotonated and therefore presumably bound covalently to Cd(II) via oxygen. In particular, the detection of species [C6H11N2O4S2Cd]+ of m/z 352.92 (Δm = 5.5 ppm) suggested the formation of cyclic structures in which Cyss could act as a bidentate ligand. The MS/MS spectrum of [ML − H]+ (m/z 352.92), acquired with a precursor mass window of ±0.5 Da, showed an ion fragment with a mass loss of 16 Da; this mass loss can be explained by the loss of OH• or NH2•, and both possibilities can be postulated. Moreover, the comparison of the measured experimental isotopic distribution with the theoretically calculated distribution of the expected summary formula for each fragment suggested that the ions of m/z 337.9 ([C6H10NO4S2Cd]+) and 320.9 ([C6H7O4S2Cd]+) arose from an intramolecular rearrangement followed by the release of the amino group excluding the presence of N → Cd coordination. Furthermore, the formation of [CdCys–H]+ (m/z 233.9; Cys: cysteine), [(CdCys-H2S)H]+ (m/z 199.9), [(CdCys–CdS)H]+ (m/z 74.0) and [(M-Cd(OH)2)-H]+ (m/z 207.0) indicates that O → Cd bonds are involved in the formation of [ML − H]+ species. The observed fragmentation pattern could be explained by an intramolecular proton transfer to the uncoordinated sulphur leading to a formal negative charge localized on thiol function. The arrangement of the thiolate group promotes the formation of thiiranes with the release of the amino group (m/z 337.9 and 320.9), and the formation of [CdCysH]+ (m/z 233.9) followed by the loss of H2S (m/z 199.9) and CdS (m/z 74.0) (Scheme 2).
Experimental section Materials and methods A perchloric acid stock solution was prepared and standardized as described previously.32 A sodium perchlorate stock
This journal is © The Royal Society of Chemistry 2013
Paper solution was prepared and standardized according to Biedermann.33 Sodium hydroxide titrant solutions were obtained by dilution of a saturated solution filtered on a Gooch crucible (G4) under a nitrogen atmosphere. The hydroxide concentration was determined by titration with standardized HClO4 using methyl-red as a visual indicator. The results agreed to within 0.1%. Purissimum grade (≥99.0% Aldrich) Cyss product was used without further purification. It was kept in a desiccator over silica gel. Cadmium(II) perchlorate stock solutions were prepared by dissolving an exactly weighed amount of pure metal (Aldrich) in a small excess of 57% HI (Merck) which was then added a known amount of HClO4 standardized to ensure an excess of 10%. Most hydroiodic acid was transformed into iodine by the addition of HIO3 (Baker) and the released iodine was removed by boiling. Finally, the last traces of iodine were eliminated by bubbling oxygen into the solution. The Cd(II) concentration was controlled by electrogravimetry using a Pt anode, with an intensity current of 0.5 A for 12 hours; after this time the cadmium deposit was washed with water, dried with ethyl alcohol and then conditioned in an oven at 102 °C to constant weight. The hydrogen ion concentration was determined potentiometrically with a glass electrode using Gran’s method.34 The perchlorate ion concentration was obtained from the sum: [H+] + 2[Cd(II)]. Lead(II) perchlorate stock solutions were prepared starting from a stock solution of Cu(ClO4)2. Into this solution, placed in a closed funnel and purged with nitrogen gas, was poured a lead amalgam (1%) and the solution was stirred under inert atmosphere for a week. After this time 0.02 cm3 of this solution was analyzed by differential pulse polarography in the range between −0.900 V and +0.100 V to detect the disappearance of Cu(II). The Pb(II) concentration was determined gravimetrically according to Winkler.35 The results agreed to within 0.06%. The hydrogen ion concentration was determined potentiometrically with a glass electrode using Gran’s method. The perchlorate ion concentration was obtained from the sum: [H+] + 2[Pb(II)]. Zinc(II) perchlorate stock solutions was prepared and standardized as reported by Iuliano.36 All solutions were prepared by using ultrapure water with a resistivity of 18.2 MΩ cm, obtained from a Milli-Q plus system (Millipore, Bedford, MA, USA). Potentiometric measurements The complexation between Pb(II), Cd(II) and Zn(II) was evaluated assuming for all four systems that the reagents act according to the following equilibrium. pMðiiÞ þ rH2 L ⇄ Mp Hq ðH2 LÞr ð2pqÞ þ qHþ ; βpqr
ð1Þ
The most probable p, q, r values and the corresponding constants βpqr were computed by a numerical approach based on the least squares procedure using the program SUPERQUAD.37 The minimum of the function U = Σwi (Eexp − Ecalc) was sought, where wi represents a statistical weight assigned to each point. Calculations of the chi-square statistic have been considered to test the fit between a theoretical frequency
Dalton Trans.
View Article Online
Paper
Dalton Transactions
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
distribution and a frequency distribution of observed data. The numerical treatment concerning the species (eqn (2)–(5)) takes into account the equilibrium constants determined in a previous study.13 L2 þ Hþ ⇄ HL
log K 1 ¼ 9:50
ð2Þ
HL þ Hþ ⇄ H2 L
log K 2 ¼ 8:50
ð3Þ
H2 L þ Hþ ⇄ H3 Lþ
log K 3 ¼ 2:352
ð4Þ
H3 Lþ þ Hþ ⇄ H4 L2þ
log K 4 ¼ 1:960
ð5Þ
Potentiometric titrations were carried out (at 25.0 ± 0.1 °C) with a programmable computer controlled data acquisition switch unit 34 970 A supplied by Hewlett Packard. Glass electrodes, manufactured by Metrohm, were of the 6.0133.100 type. They acquired a constant potential within 10 min after the addition of the reagents and remained unchanged to within ±0.1 mV for several hours. The EMF values were measured with a precision of ±10–5 V using an OPA 111 lownoise precision DIFET operational amplifier. The cell arrangement was similar to the one described by Forsling et al.38 Ag/AgCl electrodes were prepared according to Brown.39 A slow stream of nitrogen gas was passed through four bottles (a–d) containing: (a) 1 mol dm−3 NaOH, (b) 1 mol dm−3 H2SO4, (c) twice distilled water, and (d) 3 mol dm−3 NaClO4, and then into the test solutions, stirred during titrations, through the gas inlet tube. The complexation equilibria have been studied, at 25 °C and in 3 mol dm−3 NaClO4, by measuring with a glass electrode the competition of the Cyss, H2L, for the metal and H+ ions. The potentiometric measurements were performed with cell (G) RE=Test Solution=Glass Electrode
ðGÞ
where RE, reference electrode = Ag/AgCl/0.01 mol dm−3 AgClO4, 2.99 mol dm−3 NaClO4/3 mol dm−3 NaClO4 and Test Solution = CM mol dm−3 M(ClO4)2, CL mol dm−3 H2L, CA mol dm−3 HClO4, CB mol dm−3 NaOH, (3–2CM–CA–CB) mol dm−3 NaClO4. The metal and the ligand concentrations, CM and CL respectively, were in the range (0.5 × 10−3 to 5 × 10−3) mol dm−3, the upper limit being imposed by the limited solubility of the ligand, while the ligand-to-metal ratio varied between 1 and 10 (1 ≤ CL/CM ≤ 10). The hydrogen ion concentration was decreased stepwise from 10−4 mol dm−3 to incipient precipitation of a basic salt of each metal, whose formation depends on the particular metal ion and the specific ligand-to-metal ratio. Since the effects of composition changes on activity coefficients can be considered negligible, the EMF of cell (G) can be written, in mV, at the temperature of 25 °C, as (6) E ¼ E° þ 59:16 log ½Hþ þ Ej
ð6Þ
where E° is constant in each series of measurements, Ej is the liquid junction potential which is a function of [H+] only.14 In
Dalton Trans.
a previous study we have found27 that Ej = −17 [H+] in 3 mol dm−3 NaClO4. Each titration was divided into two parts. In the first part, E° was determined in the absence of M(II) ions and the acidity was decreased stepwise by coulometric generation of OH− ions with the circuit (C): –Pt=Test Solution=AEþ
ðCÞ
where AE, auxiliary electrode = 3 M NaClO4/0.1 M NaCl, 2.9 M NaClO4/Hg2Cl2/Hg. Assuming that the only reactions occurring at the cathode are H+ + e− → 1/2 H2 and H2O + e− → 1/2 H2 + OH−, then, in the Test Solution of a given volume V dm3, CB = μF 10−6/V mol dm−3, where μF stands for the microfaradays passed through the cell. In the second part of the investigation of metal ion complexes, alkalification was achieved by adding NaOH since the coulometric generation did not proceed with 100% current efficiency. The primary CM, CL, CA, CB and [H+] data represent the basis of the treatment to obtain the equilibrium constants. Table 3 lists a summary of relevant data taken in all titrations. The metal–Cyss conjugated. All the complexes (1, [(Cyss), Pb(II)]; 2, [(Cyss),Cd(II)]; 3, [(Cyss),Zn(II)]) were prepared starting from 1 mmol of metal perchlorate dissolved in a water–ethanol 1/1 (v/v) mixture. These solutions were heated until the solvent refluxed and then the solution of 2 equivalents ligand was added slowly while stirring. Compounds (1–3) were not isolated, and 1 μL of the resulting reaction mixture was directly loaded on the probe and analyzed by LD MS and MS/MS. (+)LD MS of 3: m/z 320.96, [ML + H]+ ([C6H13N2O5S2Zn]+, Δm = 5.3 ppm).
ESI-, CT-, LD-MS and MS/MS High-resolution ESI experiments were carried out using a hybrid Q-Star Pulsar-i (MDS Sciex Applied Biosystems, Toronto, Canada) mass spectrometer equipped with an ionspray ionization source. Samples were introduced by direct infusion (5 mL min−1) at the optimum ion spray voltage of 4500 V. The nitrogen gas pressure was set at 137.89 kPa and the declustering was set at 50 V.
Table 3
Summary of the relevant experimental data for the systems investigated
M(II)
CM (mol dm−3)
CL (mol dm−3)
pH range
Pb(II)
0.5 × 10−3 1 × 10−3 1 × 10−3 1 × 10−3 5 × 10−3 1 × 10−3 1 × 10−3 0.5 × 10−3 5 × 10−3 0.5 × 10−3 1 × 10−3 1 × 10−3 5 × 10−3
5 × 10−3 1 × 10−3 2 × 10−3 3 × 10−3 5 × 10−3 1 × 10−3 2 × 10−3 5 × 10−3 5 × 10−3 5 × 10−3 1 × 10−3 2 × 10−3 5 × 10−3
6.2–8.0 6.0–8.0 6.1–7.8 6.2–7.4 5.4–7.5 4.3–7.0 4.5–6.7 5.5–6.3 4.0–6.0 6.5–7.9 6.3–7.6 6.0–8.0 6.2–7.5
Cd(II)
Zn(II)
This journal is © The Royal Society of Chemistry 2013
View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Dalton Transactions CT- and LD MS and MS/MS analyses were performed using a 5800 MALDI TOF–TOF Analyzer (AB SCIEX) equipped with a neodymium–yttrium–aluminum–garnet laser (laser wavelength 349 nm), in reflection positive-ion mode with a mass accuracy of 5 ppm. At least 4000 laser shots were typically accumulated with a laser pulse rate of 400 Hz in the MS mode, whereas in the MS/MS mode spectra up to 5000 laser shots were acquired and averaged with a pulse rate of 1000 Hz. MS/MS experiments were performed at a collision energy of 1–2 kV, and ambient air was used as the collision gas with a medium pressure of 10−6 Torr. After acquisition, spectra were handled using Data Explorer version 4.0. CT MALDI MS and MS/MS were performed using 2,7-DMN as the matrix. For these experiments the sample loading was performed by the sandwich layer method and by direct spotting of the sample/matrix premixed solution (1 : 5–1 : 25 v/v ratio). A 0.5 μL aliquot of a premixed solution of the sample and DMN dissolved in MeOH–CH3Cl (1 : 1) was spotted on the sample plate, dried at room temperature, and analyzed into the mass spectrometer. CT, LD MS and MS/MS experiments were performed to elucidate the ionic structure and also to characterize the fragmentation patterns. Cd(II), Pb(II) and Zn(II) species are clearly identified by the characteristic distribution of metal isotopes, and the mass-tocharge (m/z) values reported refer solely to the most abundant (114Cd, 208Pb, 64Zn) isotope peaks.
Conclusions The speciation models and the formation constants proposed on the basis of potentiometric results were in agreement with M(II)–Cyss systems MS data. The latter provided information regarding the structure, the stoichiometry and the metal oxidation state of metal ion complexes. CT MALDI MS and LD MS allow an accurate determination of the metal–cystine conjugated giving a quantitative ionization of neutral species by protonation or laser induced ligand reduction. Thus, several different fragmentation pathways are observed. The Pb(II)– cystine complexes ionize through the reduction of the ligand producing a formal positive charge on the coordinated Cyss. The most abundant MS/MS fragments arise from the cleavage of the disulfide bond, suggesting that Cyss has the canonical structure with the charge and the radical located on the sulfur atoms. For Zn(II) and Cd(II) complexes the proton transfer is the only significant reaction.
Notes and references 1 O. Yamauchi and A. Odani, Pure Appl. Chem., 1996, 68(2), 469. 2 J. S. Magyar, T. C. Weng, C. M. Stern, D. F. Dye, B. W. Rous, J. C. Payne, B. M. Bridgewater, A. Mijovilovich, G. Parkin, J. M. Zaleski, J. E. Penner-Hahn and H. A. Godwin, J. Am. Chem. Soc., 2005, 127, 9495.
This journal is © The Royal Society of Chemistry 2013
Paper 3 C. D. L. Saunders, L. E. Longobardi, N. Burford, M. D. Lumsden, U. Werner-Zwanziger, B. Chen and R. McDonalds, Inorg. Chem., 2011, 50, 2799. 4 N. Burford, D. M. Eelman, G. W. LeBlank, T. S. Cameron and N. K. Robertson, Chem. Commun., 2004, 332. 5 N. Burford, D. M. Eelman and G. W. LeBlanc, Can. J. Chem., 2004, 1254. 6 R. G. Parr and R. G. Pearson, J. Am. Chem. Soc., 1983, 105, 7512. 7 F. Mazzotti, H. Benabdelkamel, L. Di Donna, C. M. Athanassopoulos, A. Napoli and G. Sindona, J. Mass Spectrom., 2012, 47, 932. 8 W. Dröge, Physiol. Rev., 2002, 82, 47. 9 D. Bohrer, V. Gabbi Polli, P. Cícero do Nascimento, J. K. A. Mendonça, L. Machado de Carvalho and S. Garcia Pomblum, J. Biol. Inorg. Chem., 2006, 11, 991. 10 M. Kaiser, M. Groll, C. Siciliano, I. Assfalg-Machleidt, E. Weyher, J. Kohno, A. G. Milbradt, C. Renner, R. Huber and L. Moroder, ChemBioChem., 2004, 5, 1256. 11 M. Kaiser, A. G. Milbradt, C. Siciliano, I. Assfalg-Machleidt, W. Machleidt, M. Groll, C. Renner and L. Moroder, Chem. Biodiversity, 2004, 1, 161. 12 E. Königsberger, Z. Wang and L. C. Königsberger, Monatsch. Chem., 2000, 131, 39. 13 E. Furia, M. Falvo and R. Porto, J. Chem. Eng. Data, 2009, 54, 3037. 14 G. Biedermann and L. G. Sillén, Ark. Kemi., 1953, 5, 425. 15 A. D. Kulkarni, S. A. Patil and P. S. Badami, Int. J. Electrochem. Sci., 2009, 4, 717. 16 A. Napoli, C. M. Athanassopoulos, P. Moschidis, D. Aiello, L. Di Donna, F. Mazzotti and G. Sindona, Anal. Chem., 2010, 82, 5552. 17 L. Di Donna, D. Taverna, F. Mazzotti, H. Benabdelkamel, M. Attya, A. Napoli and G. Sindona, Food Chem., 2013, 141, 2328. 18 F. Mazzotti, H. Benabdelkamel, L. Di Donna, L. Maiuolo, A. Napoli and G. Sindona, Food Chem., 2012, 135(3), 1006. 19 A. Napoli, D. Aiello, L. Di Donna, A. Sajjad, E. Perri and G. Sindona, Anal. Chem., 2006, 78, 3434. 20 A. Napoli, D. Aiello, L. Di Donna, H. Prendushi and G. Sindona, Anal. Chem., 2007, 79, 5941. 21 A. Napoli, D. Aiello, L. Di Donna, P. Moschidis and G. Sindona, J. Proteome Res., 2008, 7(7), 2723. 22 P. Cardiano, O. Giuffre, A. Napoli and S. Sammartano, New J. Chem., 2009, 33, 2286. 23 G. Falcone, C. Foti, A. Gianguzza, O. Giuffrè, A. Napoli, A. Pettignano and D. Piazzese, Anal. Bioanal. Chem., 2013, 405, 881. 24 P. Cardiano, G. Falcone, C. Foti, O. Giuffrè and A. Napoli, J. Inorg. Biochem., 2013, 129, 84. 25 L. Di Donna, F. Mazzotti, A. Napoli, R. Salerno, A. Sajjad and G. Sindona, Rapid Commun. Mass Spectrom., 2007, 21, 273. 26 I. Aiello, L. Di Donna, M. Ghedini, M. La Deda, A. Napoli and G. Sindona, Anal. Chem., 2004, 76, 5985. 27 A. Olin, Acta Chem. Scand., 1960, 14, 126.
Dalton Trans.
View Article Online
Paper
34 G. Gran, Analyst, 1952, 77, 661. 35 L. W. Winkler, Ausgewählte Untersuchungsverfahren für das chemische Laboratorium, Enke Verlag, Stuttgart, 1936. 36 M. Iuliano, Ann. Chim., 1994, 84, 187. 37 P. Gans, A. Sabatini and A. Vacca, J. Chem. Soc., Dalton Trans., 1985, 1195. 38 W. Forsling and S. Hietanen, Acta Chem. Scand., 1952, 6, 901. 39 A. S. Brown, J. Am. Chem. Soc., 1934, 56, 646.
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
28 C. F. Baes and R. E. Mesmer, The hydrolysis of cations. A Wiley–Interscience Publication, New York, 1976. 29 D. Dyrssen and P. Lumme, Acta Chem. Scand., 1962, 16, 1785. 30 P. Hu, Q. Z. Ye and J. A. Loo, Anal. Chem., 1994, 66, 4190. 31 R. Feng, A. L. Castelhano, R. Billedeau and Z. Yuan, J. Am. Soc. Mass Spectrom., 1995, 6, 1105. 32 E. Furia and R. Porto, Ann. Chim., 2002, 92, 521. 33 G. Biedermann, Svensk Kem Tidsk, 1964, 76, 362.
Dalton Transactions
Dalton Trans.
This journal is © The Royal Society of Chemistry 2013
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
PAPER
Cite this: DOI: 10.1039/c3dt52255e
View Journal
Mass spectrometry and potentiometry studies of Pb(II)–, Cd(II)– and Zn(II)–cystine complexes Emilia Furia, Donatella Aiello, Leonardo Di Donna, Fabio Mazzotti, Antonio Tagarelli, Hariprasad Thangavel, Anna Napoli* and Giovanni Sindona Cd(II)–, Pb(II)– and Zn(II)–cystine complexes were investigated by potentiometric and different mass spectrometric (MS) methodologies. Laser desorption mass spectrometry has provided both the composition and structure of metal–cystine complexes according to the speciation models proposed on the basis of the potentiometric data. Detection of neutral complexes was achieved by protonation or electrochemical reduction during mass spectrometric experiments. The redox activity of metal–cystine complexes
Received 18th August 2013, Accepted 17th October 2013
was confirmed by laser desorption and charge transfer matrix assisted laser assisted MS experiments, which allowed us to observe the formation of complexes with a reduction of cystine. The stoichiometry of Cd(II)–,
DOI: 10.1039/c3dt52255e
Pb(II)– and Zn(II)–cystine complexes was defined by observing the isotopic pattern of the investigated com-
www.rsc.org/dalton
pound. The results suggest that interaction occurs through the carboxylate group of the ligand.
Introduction Metal cations are widespread in the environment and in many dietary supplements present in the market of nutraceuticals.1 Their interaction with amino acids, proteins, nucleic acids or metabolites in biological systems may cause undesirable side effects due to the thermodynamics of the metal complexes thus formed in that particular setting. Cadmium ions, for example, can replace the native zinc ion from many proteins and enzymes to a degree that depends on their affinities. Lead ions also inhibit a variety of enzymes. The toxicities of Cd(II), Zn(II) and Pb(II) metals have been reviewed extensively in the literature. Interactions of some heavy metals with a restricted number of proteogenic amino acids have been investigated by different spectroscopic methods.2–7 Cystine (Cyss) is a nonproteogenic amino acid available as an individual supplement or as part of protein supplements. It participates in a variety of physiological functions, including antioxidant activity,8,9 and shows regulatory effects in the substrate-recognizing function of the eukaryotic proteasome.10,11 Furthermore, a significant fraction of heavy metals naturally present in blood plasma occurs in the form of amino acid complexes and a predominant fraction of these involves cystine. In the alkaline environment cystine displays four protonation sites due to (i) the two most basic amino groups and (ii) the two carboxylic groups. Both types of donor sites are likely to participate in metal
Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, P Bucci, cubo 12/d, I-87036Arcavacata di Rende, Italy. E-mail: [email protected]; Fax: +39 0984 493307
This journal is © The Royal Society of Chemistry 2013
coordination and the disulfide bond may also be involved in some specific cases. Studies on metal complex formation and even protonation equilibria of this amino acid have been hampered by very low solubility in neutral aqueous media.12 Recently, the solubility as well as the acidic constants of cystine in NaClO4 solutions at different ionic strengths have been determined.13 The solubility of cystine increases proportionally with the ionic medium concentration, thus the complexation equilibria of this ligand with the Cd(II), Pb(II) and Zn(II) ions have been studied by potentiometric titrations with a glass electrode at 25 °C and in 3 mol dm−3 NaClO4 ionic medium, where the variation of the reagent concentrations does not involve large changes in the activity coefficients.14 Therefore, the activities were replaced by the concentrations in the numerical evaluation. These systems should be formed by two, three and four monomeric coordination complexes with the same ligand. The structure of these species has not been fully characterized15 whilst the understanding of potential toxicological activity requires a deep knowledge of the sequestering ability of natural ligands in a given particular environment. Recent achievements in mass spectrometric methodologies provide a rapid and sensitive tool for the identification and quantitation16 of metabolites,17,18 amino acids and proteins19,20 and their post-translational modifications.21 Electrospray mass spectrometry (ESI MS)22–24 is a well-established method for the detection and analysis of metal ion complexes. Literature data include the study of gas-phase metal ion chemistry and the formation of metal ion adducts to assist in the detection and structural elucidation of organic compounds.25 Structural information on M(II)–Cyss compounds is quite
Dalton Trans.
View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Paper difficult, even under the softest ionization conditions because the labile involved non-covalent bonds are susceptible to protonolysis during the ionization process, and therefore the molecular adducts may be missing completely, or their relative abundances may be very low in the full-scan mass spectra. Charge transfer matrix-assisted laser desorption/ionization (CT-MALDI-MS) has successfully been used to ionize fragile complexes of polymetallic porphyrin derivatives.26 Thus, ESI MS, CT-MALDI-MS and laser desorption mass spectrometry (LD MS) can be exploited to determine either the composition and structure elucidation of Cd(II), Pb(II) and Zn(II) cations– Cyss complexes. The equilibrium behaviour and the speciation model in aqueous solution in a wide pH range, between Cd(II), Pb(II) and Zn(II) cations and Cyss and possible structures of the complexes formed will be discussed on the basis of potentiometric and mass spectrometric results.
Dalton Transactions Table 1 Best set of the overall stability constant, log βpqr (3σ), for the system Cyss–Pb(II) according to general equilibrium (1)
Species
Model 1
Model 2
Model 3
Model 4
PbL PbL22− Pb(HL)+ Pb(HL)2 Pb(HL)L− σ (mV) χ2 U
−5.87 ± 0.09 −5.45 ± 0.06 −5.62 ± 0.05 −5.79 ± 0.04 −17.0 ± 0.1 −17.18 ± 0.07 −17.38 ± 0.09 −17.43 ± 0.07 −8.64 ± 0.09 −8.8 ± 0.1 −8.85 ± 0.08 −10.48 ± 0.07 −9.56 ± 0.09 −9.67 ± 0.05 3.71 1.02 0.40 0.34 9.31 15.03 8.17 6.34 8.81 × 102 6.55 × 101 9.72 6.85
Results and discussion Characterization of Pb(II)–, Cd(II)– and Zn(II)–cystine complexes by potentiometric titration The potentiometric study was performed on the Pb(II)–, Cd(II)– and Zn(II)–Cyss systems in order to get the stoichiometry of the complexes and the magnitude of the related stability constants and to describe the speciation profile. A summary of the relevant data taken in all titrations is presented in Table 3 (Experimental section). By applying the reported value of the constants of the main hydrolytic products, i.e. PbOH+, Pb3(OH)42+, Pb4(OH)44+ and Pb6(OH)84+,27 the numerical treatment leads to the best set of log βpqr (3σ) for the considered system. The numerical evaluation initially was performed considering the formation of the complexes PbL and PbL22− (Model 1). Since the standard deviation observed was considerably higher than the experimental uncertainty (Model 1), other models were tested considering a set of n + 1 species for each one. The best agreement was obtained with Pb(HL)+ (Model 2) and a consequent diminution of the function U equal to 93%. The fit is significantly improved including in the previous model the species Pb(HL)L− (Model 3). An additional refinement was reached on adding the neutral complex Pb(HL)2 (Model 4). As no other species lowered the minimum, Model 4 was assumed to be the best in describing the data, also taking into consideration that the standard deviation (σ) is comparable with the experimental uncertainty. Using the values of the constants reported in Table 1 (Model 4), distribution diagrams were constructed (Fig. 1a–b). Speciation diagram (Fig. 1a) shows that PbL, PbL22− and Pb(HL)+ attain significance percentage when the analytical concentrations of the ligand are greater than those of the metal, e.g. CL = 5 mmol dm−3, CM = 0.5 mmol dm−3, while Pb(HL)2 is a minor species and reaches a percentage of only 10%. Otherwise, at comparable analytical concentrations of the ligand and the metal (Fig. 1b) the metal–ligand complexes
Dalton Trans.
Fig. 1 Speciation diagrams of M(II)–Cyss vs. −log[H3O+] at T = 25 °C. Free metal is shown by the dashed line: (a) Pb(II)–Cyss: CM = 0.5 mmol dm−3, CL = 5 mmol dm−3; (b) Pb(II)–Cyss: CM = 1 mmol dm−3, CL = 2 mmol dm−3; (c) Cd(II)–Cyss: CM = CL = 1 mmol dm−3; (d) Cd(II)–Cyss: CM = 1 mmol dm−3, CL = 2 mmol dm−3.
Pb(HL)L are the main species achieving a percentage of 50% at pH 7. Analogously, data treatment for Cd(II) was performed by considering the well-known hydrolysis product in 3 mol dm−3 perchlorate media (such as Cd(OH)+).28,29 Various models were tested by adding a single species but none of these lowered the minimum of the function. Then the only complex for the system Cyss–Cd(II) is CdL whose stability constant, log β121 according to the general equilibrium (1), is −5.5 ± 0.1 (the uncertainty represents 3σ). The refined equilibrium constant is used to construct the distribution diagrams of cadmium(II) in the different species (Fig. 1c–d). The speciation diagrams of the Cyss–Cd(II) system at different concentration ratios show that in the whole pH range, both at M : L = 1 : 1 (Fig. 1c) and at M : L = 1 : 2 (Fig. 1d), the most important species is CdL, which is predominant over the hydrolysis product, CdOH+, and the formation of which is over 90% when M : L = 1 : 2 (Fig. 1d). The numerical estimation of the equilibrium formation constants of the complexes between Cyss and Zn(II) was performed by considering the formation quotients of the hydrolytic species Zn(OH)3− and Zn(OH)42− (25 °C in 3 mol dm−3 NaClO4)27 and the various steps of the calculations are illustrated in Table 2. Model 1, which considers the presence of ZnL22−, is the best model with only one species. The introduction of ZnL
This journal is © The Royal Society of Chemistry 2013
View Article Online
Dalton Transactions
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Table 2
Paper
Best set of log βpqr (3σ) for the system CYSS–Zn(II)
Species
Model 1
Model 2
ZnL22−
−17.03 ± 0.09
ZnL σ (mV) χ2 U
0.51 14.88 21.92
−16.61 ± 0.08 −6.86 ± 0.06 0.32 5.13 7.12
(Model 2) lowered the minimum of the square error sum significantly (67%) and the standard deviation (σ) was comparable with the experimental uncertainty. No other species, introduced to improve the fit, were retained. All the proposed species reach a concentration level of at least 35% of the total metal; therefore significant concentrations were obtained for a correct definition of the equilibrium constants. Characterization of Pb(II)–, Cd(II)– and Zn(II)–cystine complexes by mass spectrometry Information regarding the structure, coordination site and metal oxidation state of the dissolved metal ion complexes cannot be obtained using electrochemical or spectroscopic techniques.30,31 “Soft” ionization techniques such as ESI and MALDI can ionize labile complexes with reduced fragmentation. ESI MS is well suited to study the dissolved-phase interaction between the metal ion and the organic ligand and has been successfully used to determine metal-binding stoichiometry, and for structural elucidation.22–24 Nevertheless, amino acids and peptides containing disulfide groups introduce some complications in the mass spectrum when collisioninduced dissociation (CID) is employed for protein identification. To overcome this drawback, metal ions are used as charge carriers in peptides. Metalated peptide-complexes with or without an auxiliary ligand could provide complementary ECD (electron-capture dissociation) fragments compared to their protonated species. Cyss represent the more simple system containing a disulfide bond, where the competition of other functional groups is reduced; thus the interaction between a metal ion and the disulfide bond could be increased. Unexpectedly, ESI MS of premixed solutions of M : L at different pHs did not show the complexed dication signals with sufficient intensity to explore the structure and coordination site by CID. Thus, an alternative ionization method should be desirable for these complexes, which are susceptible to protonolysis during the ionization process and their relative abundances are very low in the full-scan mass spectra. The first challenge was the identification of a suitable MALDI matrix for the M(II)–Cyss complex MS analysis. In fact, when the Pb–Cyss conjugated (1) was submitted to MALDI experiments using traditional matrixes (α-CHCA, DHB) no molecular ion peaks were detected. Thus, the interaction of M(II) cations with Cyss was exploited by CT MALDI MS and LD MS. The ionproviding information on M : L and M : L2 species (Scheme 1) was observed in the full-scan positive ion CT MALDI and LD mass spectra by deposition of a few μl of the reaction aqueous solution.
This journal is © The Royal Society of Chemistry 2013
Scheme 1
Formation of PbL, PbL2 radical cations by CT-MALDI and LD-MS.
MS/MS experiments were performed to elucidate the ionic structures and also to characterize fragmentation patterns. Fig. 2 shows the LD(+) mass spectrum obtained using 1 μl of the reaction aqueous solution of the conjugated 1. Pb(II) species are clearly identified by the characteristic distribution of lead isotopes, and the mass-to-charge (m/z) values reported refer solely to the most abundant (208Pb) isotope peaks. The spectrum shows the formation of reduced1 and reduced2 forms of ML2 and ML and ion fragments due to the partial decomposition of the complexes. On the other hand, the protonation of the neutral two-coordinate species [Cyss,Pb(II)] would result in the formation of the pseudomolecular ions [Cyss,Pb(II) + H]+, [(2Cyss,Pb(II) + H]+ (m/z 446.99 and m/z 687.02, respectively). The absence of complexes containing chloride and free Cyss suggests that the complex is formed quantitatively and that the perchlorate anion does not compete towards cations under the adopted experimental conditions. The comparison of the measured experimental isotopic distribution with the theoretically calculated distribution of the expected summary formula suggests that the ion clusters of m/z 688.02–686.00 and m/z 447.99–445.98 result from the overlap of the reduced1/reduced2 form of [ML2]•+ ([C12H20N4O8S4Pb]•+; [C12H24N4O8S4 Pb]•+, Δm = −5 ppm) and [ML]•+ ([C6H12N2O4S2 Pb]•+, [C6H10N2O4S2Pb]•+; Δm = −5 ppm), respectively (Fig. 2). The expected oxidizing nature of higher valence metals such as Pb(IV), together with the known ease of reduction of Cyss, suggests that it is the ligand rather than the metal ion which is reduced. Therefore, the ionization process of this system is represented by a redox process, for which a laser induced reduction initiates the chemical process. Initially, the reduction of the complex, ML2, ML, generates a formal positive charge localized on the coordinated Cyss ligand, [ML2]•+ and [ML]•+; a subsequent intra-cluster H transfer drives the formation of reduced species [ML2 + 2H]•+ and [ML + 2H]•+ at m/z 688.02 and 447.99, respectively. Such ionic complexes are,
Fig. 2
LD MS of the conjugated 1.
Dalton Trans.
View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Paper
Dalton Transactions
therefore, best represented as radical cations of the ligand oxidized and reduced forms, i.e. [ML2]•+, [ML]•+ and [ML2 + 2H]•+, [ML + 2H]•+. The peak of m/z 686.00 (Fig. 2) corresponds to the ion [(Cyss)2,Pb(II)]•+ ([ML2]•+), in which Cyss is deprotonated (i.e. Cyss2−) and presumably bound covalently to Pb(II) via oxygen. Furthermore, the ion peak of m/z 688.02 represents [(Cyss)2,Pb(II)]•+ ([ML2 + 2H]•+), in which the reduced ligand is likewise bound covalently via oxygen to the metal(II). The same considerations can be extended to the observed [ML]•+ species (Scheme 1). CT MALDI MS was exploited as an alternative ionization process for these systems, which are susceptible to ease reduction because the ionization process is initiated by photoabstraction of an electron from matrix molecules. This step generates a radical cation, which in turn abstracts an electron from the analyte. Therefore, it was hypothesized that 2,7dimethoxynaphthalene (DMN) could promote intracluster single electron transfer between the matrix and analytes allowing one to exploit the redox chemistry of these complexes. A few μl of the reaction mixture 1 was loaded on to the sample plate with either a sandwich layer method and a premixing sample/matrix solution (MeOH–CH3Cl (1 : 1)) at different ratios. The CT MALDI MS spectrum of 1 (Fig. 3) exhibits prominent signals for the oxidized/reduced form of the complexes [ML]•+, [ML2]•+ (m/z 446–448, 686–688), electron bound clusters [M2L2]•+ (m/z 892.98), [M2L2 − 32]•+ (m/z 861.98), [M2L2 − 64]•+ (m/z 828.01), [M2L2 − 64]•2+ (m/z 412.99), [M2L3]•+ (m/z 1135.01), [M2L4–Cys–H2O]•+ (m/z 1238.04), and [M2L4–Cys– H2O]•2+ (m/z 619.05). In all cases, the isotopic pattern corresponds to that simulated for the intact molecular cation, providing unequivocal confirmation of the molecular composition. However, the formation of mono and doubly charged electron bound dimers suggested that the matrix improves the desorption process favoring the intra-cluster single electron/H transfer and thus the re-reduction of the ligand. The LD in-source fragmentation of oxidized and reduced forms of [ML2]•+ and [ML]•+ showed fragments arising from the consecutive mass loss of 16 Da. This mass loss can be explained by the release of a NH2• radical suggesting the
Fig. 3
CT MALDI of the conjugated 1.
Dalton Trans.
formation of O → Pb(II) oxidized and reduced forms of [ML2]•+ [ML]•+. MS information on the most prevailing peaks combined with that obtained by MS/MS at different collision energies are used to assign the most probable structure of the complexes [ML2]•+and [ML]•+. LD MS experiments clearly indicate the formation of the species [C12H24N4O6S4Pb]•+ (m/z 656.03, Δm: −6 ppm), [C12H24N4O6S4Pb]•+ (m/z 654.01, Δm: −5 ppm), [C6H10N2O3S2Pb]•+ (m/z 429.99 Δm: −5 ppm), [C6H10N2O2S2Pb]•+ (m/z 413.99 Δm: −6 ppm), and [C6H12N2O3S2Pb]•+ (m/z 432.00 Δm: −5 ppm), [C6H12N2O2S2Pb]•+ (m/z 416.01 Δm: −5 ppm), [C6H12N2O2S2]•+ (m/z 208.00), [C5H11NOS2]•+ (m/z 165.00), probably due to gasphase rearrangements of the molecular cations with the consecutive loss of a OH•, thus excluding the presence of N → Pb coordination. MS/MS of the precursor [ML2 + 2H]•+ (m/z 688.02, 1) yields a fragment rich product mass spectrum (Fig. 4a). The spectrum revealed the formation of the cations [ML]•+ (m/z 448.0) [C6H12N2O3S2Pb]•+ (m/z 432.0), [C6H12N2O2S2Pb]•+ (m/z 416.0), [C5H11NOS2]•+ (m/z 165.0), resulting from the fragmentation of the Cyss, and [Pb]•+ (m/z 208.0), [PbO2]•+ (m/z 240.0). The CID mass spectrum (Fig. 4b) obtained from the precursor ion [ML + 2H]•+ (m/z 447.99) showed product ions arising from the loss of Cyss followed by the consecutive release of OH and the formation of [Pb]•+ (m/z 208.0), [PbO2]•+ (m/z 240.0). The same fragmentation pattern was observed for the oxidized form [ML2]•+, [ML]•+. The observed fragmentation pattern of the oxidized and reduced species confirmed that Cyss is bound covalently to Pb(II) via oxygen. The gas-phase ion chemistry of the system involving Cd(II) and Zn(II) is quite different from the corresponding [Pb(II), Cyss]. According to the speciation diagrams of Cd(II) and Zn(II), which showed that the main complex species in solution is [ML] (Fig. 1c–d), the formation of species of higher coordination number was not observed. In both cases, the metal was bound exclusively by oxidized Cyss, giving [CdL]+ and [ZnL]+. The stability of these complexes was confirmed by the absence of free ligand ions in the LD mass spectra. According to the oxidation potential of the couple Cd(II)/Cd, Zn(II)/Zn, no laser
Fig. 4 LD MS/MS spectra of the reduced form of the conjugated 1: (a) [ML2 + 2H]+ (m/z 688.02) and (b) [ML + 2H]+ (m/z 447.99).
This journal is © The Royal Society of Chemistry 2013
View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Dalton Transactions
Scheme 2
MS/MS fragmentation pattern of [ML + H]+ (m/z 352.92).
induced reduction of the complexes was observed for these systems. The LD(+) mass spectrum, obtained using 1 μL of the aqueous solution of the sample 2, showed the ion peak of 352.92 corresponding to the species [ML + H]+ ([C6H11N2O4S2Cd]+, Δm = 5.5 ppm) in which Cyss is deprotonated and therefore presumably bound covalently to Cd(II) via oxygen. In particular, the detection of species [C6H11N2O4S2Cd]+ of m/z 352.92 (Δm = 5.5 ppm) suggested the formation of cyclic structures in which Cyss could act as a bidentate ligand. The MS/MS spectrum of [ML − H]+ (m/z 352.92), acquired with a precursor mass window of ±0.5 Da, showed an ion fragment with a mass loss of 16 Da; this mass loss can be explained by the loss of OH• or NH2•, and both possibilities can be postulated. Moreover, the comparison of the measured experimental isotopic distribution with the theoretically calculated distribution of the expected summary formula for each fragment suggested that the ions of m/z 337.9 ([C6H10NO4S2Cd]+) and 320.9 ([C6H7O4S2Cd]+) arose from an intramolecular rearrangement followed by the release of the amino group excluding the presence of N → Cd coordination. Furthermore, the formation of [CdCys–H]+ (m/z 233.9; Cys: cysteine), [(CdCys-H2S)H]+ (m/z 199.9), [(CdCys–CdS)H]+ (m/z 74.0) and [(M-Cd(OH)2)-H]+ (m/z 207.0) indicates that O → Cd bonds are involved in the formation of [ML − H]+ species. The observed fragmentation pattern could be explained by an intramolecular proton transfer to the uncoordinated sulphur leading to a formal negative charge localized on thiol function. The arrangement of the thiolate group promotes the formation of thiiranes with the release of the amino group (m/z 337.9 and 320.9), and the formation of [CdCysH]+ (m/z 233.9) followed by the loss of H2S (m/z 199.9) and CdS (m/z 74.0) (Scheme 2).
Experimental section Materials and methods A perchloric acid stock solution was prepared and standardized as described previously.32 A sodium perchlorate stock
This journal is © The Royal Society of Chemistry 2013
Paper solution was prepared and standardized according to Biedermann.33 Sodium hydroxide titrant solutions were obtained by dilution of a saturated solution filtered on a Gooch crucible (G4) under a nitrogen atmosphere. The hydroxide concentration was determined by titration with standardized HClO4 using methyl-red as a visual indicator. The results agreed to within 0.1%. Purissimum grade (≥99.0% Aldrich) Cyss product was used without further purification. It was kept in a desiccator over silica gel. Cadmium(II) perchlorate stock solutions were prepared by dissolving an exactly weighed amount of pure metal (Aldrich) in a small excess of 57% HI (Merck) which was then added a known amount of HClO4 standardized to ensure an excess of 10%. Most hydroiodic acid was transformed into iodine by the addition of HIO3 (Baker) and the released iodine was removed by boiling. Finally, the last traces of iodine were eliminated by bubbling oxygen into the solution. The Cd(II) concentration was controlled by electrogravimetry using a Pt anode, with an intensity current of 0.5 A for 12 hours; after this time the cadmium deposit was washed with water, dried with ethyl alcohol and then conditioned in an oven at 102 °C to constant weight. The hydrogen ion concentration was determined potentiometrically with a glass electrode using Gran’s method.34 The perchlorate ion concentration was obtained from the sum: [H+] + 2[Cd(II)]. Lead(II) perchlorate stock solutions were prepared starting from a stock solution of Cu(ClO4)2. Into this solution, placed in a closed funnel and purged with nitrogen gas, was poured a lead amalgam (1%) and the solution was stirred under inert atmosphere for a week. After this time 0.02 cm3 of this solution was analyzed by differential pulse polarography in the range between −0.900 V and +0.100 V to detect the disappearance of Cu(II). The Pb(II) concentration was determined gravimetrically according to Winkler.35 The results agreed to within 0.06%. The hydrogen ion concentration was determined potentiometrically with a glass electrode using Gran’s method. The perchlorate ion concentration was obtained from the sum: [H+] + 2[Pb(II)]. Zinc(II) perchlorate stock solutions was prepared and standardized as reported by Iuliano.36 All solutions were prepared by using ultrapure water with a resistivity of 18.2 MΩ cm, obtained from a Milli-Q plus system (Millipore, Bedford, MA, USA). Potentiometric measurements The complexation between Pb(II), Cd(II) and Zn(II) was evaluated assuming for all four systems that the reagents act according to the following equilibrium. pMðiiÞ þ rH2 L ⇄ Mp Hq ðH2 LÞr ð2pqÞ þ qHþ ; βpqr
ð1Þ
The most probable p, q, r values and the corresponding constants βpqr were computed by a numerical approach based on the least squares procedure using the program SUPERQUAD.37 The minimum of the function U = Σwi (Eexp − Ecalc) was sought, where wi represents a statistical weight assigned to each point. Calculations of the chi-square statistic have been considered to test the fit between a theoretical frequency
Dalton Trans.
View Article Online
Paper
Dalton Transactions
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
distribution and a frequency distribution of observed data. The numerical treatment concerning the species (eqn (2)–(5)) takes into account the equilibrium constants determined in a previous study.13 L2 þ Hþ ⇄ HL
log K 1 ¼ 9:50
ð2Þ
HL þ Hþ ⇄ H2 L
log K 2 ¼ 8:50
ð3Þ
H2 L þ Hþ ⇄ H3 Lþ
log K 3 ¼ 2:352
ð4Þ
H3 Lþ þ Hþ ⇄ H4 L2þ
log K 4 ¼ 1:960
ð5Þ
Potentiometric titrations were carried out (at 25.0 ± 0.1 °C) with a programmable computer controlled data acquisition switch unit 34 970 A supplied by Hewlett Packard. Glass electrodes, manufactured by Metrohm, were of the 6.0133.100 type. They acquired a constant potential within 10 min after the addition of the reagents and remained unchanged to within ±0.1 mV for several hours. The EMF values were measured with a precision of ±10–5 V using an OPA 111 lownoise precision DIFET operational amplifier. The cell arrangement was similar to the one described by Forsling et al.38 Ag/AgCl electrodes were prepared according to Brown.39 A slow stream of nitrogen gas was passed through four bottles (a–d) containing: (a) 1 mol dm−3 NaOH, (b) 1 mol dm−3 H2SO4, (c) twice distilled water, and (d) 3 mol dm−3 NaClO4, and then into the test solutions, stirred during titrations, through the gas inlet tube. The complexation equilibria have been studied, at 25 °C and in 3 mol dm−3 NaClO4, by measuring with a glass electrode the competition of the Cyss, H2L, for the metal and H+ ions. The potentiometric measurements were performed with cell (G) RE=Test Solution=Glass Electrode
ðGÞ
where RE, reference electrode = Ag/AgCl/0.01 mol dm−3 AgClO4, 2.99 mol dm−3 NaClO4/3 mol dm−3 NaClO4 and Test Solution = CM mol dm−3 M(ClO4)2, CL mol dm−3 H2L, CA mol dm−3 HClO4, CB mol dm−3 NaOH, (3–2CM–CA–CB) mol dm−3 NaClO4. The metal and the ligand concentrations, CM and CL respectively, were in the range (0.5 × 10−3 to 5 × 10−3) mol dm−3, the upper limit being imposed by the limited solubility of the ligand, while the ligand-to-metal ratio varied between 1 and 10 (1 ≤ CL/CM ≤ 10). The hydrogen ion concentration was decreased stepwise from 10−4 mol dm−3 to incipient precipitation of a basic salt of each metal, whose formation depends on the particular metal ion and the specific ligand-to-metal ratio. Since the effects of composition changes on activity coefficients can be considered negligible, the EMF of cell (G) can be written, in mV, at the temperature of 25 °C, as (6) E ¼ E° þ 59:16 log ½Hþ þ Ej
ð6Þ
where E° is constant in each series of measurements, Ej is the liquid junction potential which is a function of [H+] only.14 In
Dalton Trans.
a previous study we have found27 that Ej = −17 [H+] in 3 mol dm−3 NaClO4. Each titration was divided into two parts. In the first part, E° was determined in the absence of M(II) ions and the acidity was decreased stepwise by coulometric generation of OH− ions with the circuit (C): –Pt=Test Solution=AEþ
ðCÞ
where AE, auxiliary electrode = 3 M NaClO4/0.1 M NaCl, 2.9 M NaClO4/Hg2Cl2/Hg. Assuming that the only reactions occurring at the cathode are H+ + e− → 1/2 H2 and H2O + e− → 1/2 H2 + OH−, then, in the Test Solution of a given volume V dm3, CB = μF 10−6/V mol dm−3, where μF stands for the microfaradays passed through the cell. In the second part of the investigation of metal ion complexes, alkalification was achieved by adding NaOH since the coulometric generation did not proceed with 100% current efficiency. The primary CM, CL, CA, CB and [H+] data represent the basis of the treatment to obtain the equilibrium constants. Table 3 lists a summary of relevant data taken in all titrations. The metal–Cyss conjugated. All the complexes (1, [(Cyss), Pb(II)]; 2, [(Cyss),Cd(II)]; 3, [(Cyss),Zn(II)]) were prepared starting from 1 mmol of metal perchlorate dissolved in a water–ethanol 1/1 (v/v) mixture. These solutions were heated until the solvent refluxed and then the solution of 2 equivalents ligand was added slowly while stirring. Compounds (1–3) were not isolated, and 1 μL of the resulting reaction mixture was directly loaded on the probe and analyzed by LD MS and MS/MS. (+)LD MS of 3: m/z 320.96, [ML + H]+ ([C6H13N2O5S2Zn]+, Δm = 5.3 ppm).
ESI-, CT-, LD-MS and MS/MS High-resolution ESI experiments were carried out using a hybrid Q-Star Pulsar-i (MDS Sciex Applied Biosystems, Toronto, Canada) mass spectrometer equipped with an ionspray ionization source. Samples were introduced by direct infusion (5 mL min−1) at the optimum ion spray voltage of 4500 V. The nitrogen gas pressure was set at 137.89 kPa and the declustering was set at 50 V.
Table 3
Summary of the relevant experimental data for the systems investigated
M(II)
CM (mol dm−3)
CL (mol dm−3)
pH range
Pb(II)
0.5 × 10−3 1 × 10−3 1 × 10−3 1 × 10−3 5 × 10−3 1 × 10−3 1 × 10−3 0.5 × 10−3 5 × 10−3 0.5 × 10−3 1 × 10−3 1 × 10−3 5 × 10−3
5 × 10−3 1 × 10−3 2 × 10−3 3 × 10−3 5 × 10−3 1 × 10−3 2 × 10−3 5 × 10−3 5 × 10−3 5 × 10−3 1 × 10−3 2 × 10−3 5 × 10−3
6.2–8.0 6.0–8.0 6.1–7.8 6.2–7.4 5.4–7.5 4.3–7.0 4.5–6.7 5.5–6.3 4.0–6.0 6.5–7.9 6.3–7.6 6.0–8.0 6.2–7.5
Cd(II)
Zn(II)
This journal is © The Royal Society of Chemistry 2013
View Article Online
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
Dalton Transactions CT- and LD MS and MS/MS analyses were performed using a 5800 MALDI TOF–TOF Analyzer (AB SCIEX) equipped with a neodymium–yttrium–aluminum–garnet laser (laser wavelength 349 nm), in reflection positive-ion mode with a mass accuracy of 5 ppm. At least 4000 laser shots were typically accumulated with a laser pulse rate of 400 Hz in the MS mode, whereas in the MS/MS mode spectra up to 5000 laser shots were acquired and averaged with a pulse rate of 1000 Hz. MS/MS experiments were performed at a collision energy of 1–2 kV, and ambient air was used as the collision gas with a medium pressure of 10−6 Torr. After acquisition, spectra were handled using Data Explorer version 4.0. CT MALDI MS and MS/MS were performed using 2,7-DMN as the matrix. For these experiments the sample loading was performed by the sandwich layer method and by direct spotting of the sample/matrix premixed solution (1 : 5–1 : 25 v/v ratio). A 0.5 μL aliquot of a premixed solution of the sample and DMN dissolved in MeOH–CH3Cl (1 : 1) was spotted on the sample plate, dried at room temperature, and analyzed into the mass spectrometer. CT, LD MS and MS/MS experiments were performed to elucidate the ionic structure and also to characterize the fragmentation patterns. Cd(II), Pb(II) and Zn(II) species are clearly identified by the characteristic distribution of metal isotopes, and the mass-tocharge (m/z) values reported refer solely to the most abundant (114Cd, 208Pb, 64Zn) isotope peaks.
Conclusions The speciation models and the formation constants proposed on the basis of potentiometric results were in agreement with M(II)–Cyss systems MS data. The latter provided information regarding the structure, the stoichiometry and the metal oxidation state of metal ion complexes. CT MALDI MS and LD MS allow an accurate determination of the metal–cystine conjugated giving a quantitative ionization of neutral species by protonation or laser induced ligand reduction. Thus, several different fragmentation pathways are observed. The Pb(II)– cystine complexes ionize through the reduction of the ligand producing a formal positive charge on the coordinated Cyss. The most abundant MS/MS fragments arise from the cleavage of the disulfide bond, suggesting that Cyss has the canonical structure with the charge and the radical located on the sulfur atoms. For Zn(II) and Cd(II) complexes the proton transfer is the only significant reaction.
Notes and references 1 O. Yamauchi and A. Odani, Pure Appl. Chem., 1996, 68(2), 469. 2 J. S. Magyar, T. C. Weng, C. M. Stern, D. F. Dye, B. W. Rous, J. C. Payne, B. M. Bridgewater, A. Mijovilovich, G. Parkin, J. M. Zaleski, J. E. Penner-Hahn and H. A. Godwin, J. Am. Chem. Soc., 2005, 127, 9495.
This journal is © The Royal Society of Chemistry 2013
Paper 3 C. D. L. Saunders, L. E. Longobardi, N. Burford, M. D. Lumsden, U. Werner-Zwanziger, B. Chen and R. McDonalds, Inorg. Chem., 2011, 50, 2799. 4 N. Burford, D. M. Eelman, G. W. LeBlank, T. S. Cameron and N. K. Robertson, Chem. Commun., 2004, 332. 5 N. Burford, D. M. Eelman and G. W. LeBlanc, Can. J. Chem., 2004, 1254. 6 R. G. Parr and R. G. Pearson, J. Am. Chem. Soc., 1983, 105, 7512. 7 F. Mazzotti, H. Benabdelkamel, L. Di Donna, C. M. Athanassopoulos, A. Napoli and G. Sindona, J. Mass Spectrom., 2012, 47, 932. 8 W. Dröge, Physiol. Rev., 2002, 82, 47. 9 D. Bohrer, V. Gabbi Polli, P. Cícero do Nascimento, J. K. A. Mendonça, L. Machado de Carvalho and S. Garcia Pomblum, J. Biol. Inorg. Chem., 2006, 11, 991. 10 M. Kaiser, M. Groll, C. Siciliano, I. Assfalg-Machleidt, E. Weyher, J. Kohno, A. G. Milbradt, C. Renner, R. Huber and L. Moroder, ChemBioChem., 2004, 5, 1256. 11 M. Kaiser, A. G. Milbradt, C. Siciliano, I. Assfalg-Machleidt, W. Machleidt, M. Groll, C. Renner and L. Moroder, Chem. Biodiversity, 2004, 1, 161. 12 E. Königsberger, Z. Wang and L. C. Königsberger, Monatsch. Chem., 2000, 131, 39. 13 E. Furia, M. Falvo and R. Porto, J. Chem. Eng. Data, 2009, 54, 3037. 14 G. Biedermann and L. G. Sillén, Ark. Kemi., 1953, 5, 425. 15 A. D. Kulkarni, S. A. Patil and P. S. Badami, Int. J. Electrochem. Sci., 2009, 4, 717. 16 A. Napoli, C. M. Athanassopoulos, P. Moschidis, D. Aiello, L. Di Donna, F. Mazzotti and G. Sindona, Anal. Chem., 2010, 82, 5552. 17 L. Di Donna, D. Taverna, F. Mazzotti, H. Benabdelkamel, M. Attya, A. Napoli and G. Sindona, Food Chem., 2013, 141, 2328. 18 F. Mazzotti, H. Benabdelkamel, L. Di Donna, L. Maiuolo, A. Napoli and G. Sindona, Food Chem., 2012, 135(3), 1006. 19 A. Napoli, D. Aiello, L. Di Donna, A. Sajjad, E. Perri and G. Sindona, Anal. Chem., 2006, 78, 3434. 20 A. Napoli, D. Aiello, L. Di Donna, H. Prendushi and G. Sindona, Anal. Chem., 2007, 79, 5941. 21 A. Napoli, D. Aiello, L. Di Donna, P. Moschidis and G. Sindona, J. Proteome Res., 2008, 7(7), 2723. 22 P. Cardiano, O. Giuffre, A. Napoli and S. Sammartano, New J. Chem., 2009, 33, 2286. 23 G. Falcone, C. Foti, A. Gianguzza, O. Giuffrè, A. Napoli, A. Pettignano and D. Piazzese, Anal. Bioanal. Chem., 2013, 405, 881. 24 P. Cardiano, G. Falcone, C. Foti, O. Giuffrè and A. Napoli, J. Inorg. Biochem., 2013, 129, 84. 25 L. Di Donna, F. Mazzotti, A. Napoli, R. Salerno, A. Sajjad and G. Sindona, Rapid Commun. Mass Spectrom., 2007, 21, 273. 26 I. Aiello, L. Di Donna, M. Ghedini, M. La Deda, A. Napoli and G. Sindona, Anal. Chem., 2004, 76, 5985. 27 A. Olin, Acta Chem. Scand., 1960, 14, 126.
Dalton Trans.
View Article Online
Paper
34 G. Gran, Analyst, 1952, 77, 661. 35 L. W. Winkler, Ausgewählte Untersuchungsverfahren für das chemische Laboratorium, Enke Verlag, Stuttgart, 1936. 36 M. Iuliano, Ann. Chim., 1994, 84, 187. 37 P. Gans, A. Sabatini and A. Vacca, J. Chem. Soc., Dalton Trans., 1985, 1195. 38 W. Forsling and S. Hietanen, Acta Chem. Scand., 1952, 6, 901. 39 A. S. Brown, J. Am. Chem. Soc., 1934, 56, 646.
Published on 18 October 2013. Downloaded by Laurentian University on 28/10/2013 18:00:57.
28 C. F. Baes and R. E. Mesmer, The hydrolysis of cations. A Wiley–Interscience Publication, New York, 1976. 29 D. Dyrssen and P. Lumme, Acta Chem. Scand., 1962, 16, 1785. 30 P. Hu, Q. Z. Ye and J. A. Loo, Anal. Chem., 1994, 66, 4190. 31 R. Feng, A. L. Castelhano, R. Billedeau and Z. Yuan, J. Am. Soc. Mass Spectrom., 1995, 6, 1105. 32 E. Furia and R. Porto, Ann. Chim., 2002, 92, 521. 33 G. Biedermann, Svensk Kem Tidsk, 1964, 76, 362.
Dalton Transactions
Dalton Trans.
This journal is © The Royal Society of Chemistry 2013
