Metal stoichiometry of isolated and arsenic substituted metallothionein: PIXE and ESI-MS study
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- Garla, R., Mohanty, B.P., Ganger, R. et al. Biometals (2013) 26: 887. doi:10.1007/s10534-013-9665-8
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The stoichiometric analysis of the metal induced Metallothionein (MT) is pertinent for understanding the metal-MT interactions. Despite innumerable publications on MT, the literature addressing these aspects is limited. To bridge this gap, PIXE and ESI-MS analysis of the commercial rabbit liver MT1 (an isoform of MT), zinc induced isolated rat liver MT1, apo and Arsenic substituted rabbit liver MT1 have been carried out. These techniques in combination provide information about number and the signature of all the metal ions bound to MT. By using ESI-MS in the rabbit MT1, ions of ZnnMT1 (n = 0, 1, 4, 5, 6, 7) whereas, in rat MT1, the Zn1MT1 and Zn5MT1 ions are observed. PIXE analysis shows that some copper along with zinc is also present in the rabbit as well as rat MT1 which could not be assessed with ESI-MS. During As metallation reaction with rabbit MT1, with increase in arsenic concentration, the amount of arsenic bound to MT1 also increases, though not proportionally. The presence of both Zn and Cu in MT1 on Zn supplementation can be related to the role of MT in Zn and Cu homeostasis. Further, the presence of partially metallated MT1 suggests that MT1 may donate fractional amount of metal from it’s fully metallated form to other proteins where Zn acts as a cofactor.
Metallothioneins (MT) are a family of ubiquitous low molecular weight (6–7 kDa) proteins having different isoforms, with unusually high cysteine (30 %) content. MT binds to a number of heavy metals and plays a crucial role in the homeostasis of essential metals like Zinc and Copper (Bell and Vallee 2009). MT are known to be induced by a variety of agents like heavy metals, glucocorticoid, acute stress and various organic chemicals. Moreover, MT play a critical role in the protection against toxic non-essential metals such Cd, Hg and As etc. (Tandon et al. 2001; Klaassen et al. 2009). Three dimensional structure of MT reveals that it contains two independent domains: (i) M(II)3S9 in the N terminal, named the β domain (ii) M(II)4S11 in the C terminal, named the α domain (M(II) = divalent metal, S = sulphur of cysteine residue) (Arseniev et al. 1988; Robbins et al. 1991).
Arsenic is a well known carcinogen. The arsenic toxicity in the West Bengal, India is well known and has drawn the attention of international scientific community. It has been reported that inorganic arsenite (As (III)) induces MT in rat liver (Albores et al. 1992) and the induction of MT by arsenic is mediated by the metal-activated transcription factor 1 (Ma and He 2009). In the last decade, applying electrospray ionisation mass spectrometry (ESI-MS) and molecular modelling techniques, the group of Stillman has carried number of investigations to understand the reaction of As3+ with recombinant human MT1(rhMT1) and its α and β domains(Ngu and Stillman 2006; Ngu et al. 2010; Irvine et al. 2013). It has been reported that each individual domain fragment of the rhMT1 binds three As3+ ions in a ratio of one As3+ to three S of cysteines i.e. total six Arsenic ions are bound to MT1 (i.e. As6-βα-MT1) (Toyama et al. 2002; Ngu and Stillman 2006). Moreover, As3+ present in As6-βα-MT1 readily transfers to metal free apo-α-MT1 or apo-β-MT1 (Ngu et al. 2010). Recently, Irvine et al. (2013) has reported that during As3+ metallation, the accessibility of the cysteine thiols of the α and β domains of rhMT1 is different. More investigations are required to elucidate the mechanism of binding of As to MT and the role of MT in As detoxification.
MT is usually isolated from the liver of the animals (rat, rabbit) after metal supplementation. The initial NMR and X-ray crystallographic studies to determine the structure of MT were carried out using isolated MT (Arseniev et al. 1988; Robbins et al. 1991). However, isolating milligram quantity of MT, required for spectroscopic studies, from animal tissues is a cumbersome task. This led to shift from the isolated MT to the recombinantly expressed MT for such studies (Blindauer and Leszczyszyn 2009). However, to see the MT response to metal supplementation, isolated MT from animal model is still needed.
With the advancement of techniques like particle induced X-ray emission (PIXE) and ESI-MS which requires only micrograms of sample, it is possible to characterize isolated MT. ESI-MS is an indirect method which determines the metal stoichiometry on the basis of total molecular weight of the protein-metal complex. With PIXE, simultaneous multi elemental analysis (from Al to U) can be done without any ambiguity as the characteristic X-ray of each element is unique and provides the direct signature of its presence in the protein without any sample consumption. The sulphur atoms present in MT (in the cysteine and methionine residues) can act as an internal standard in order to obtain the extent of metallation of MT. A number of metalloproteins have been studied with PIXE for their metal content (Garman and Grime 2005; Nagy et al. 2009; Warner et al. 2010; Solis et al. 2011), but no reports are available in literature regarding the PIXE analysis of MT.
The present study has been designed to understand the metal-MT interactions using complimentary features of PIXE and ESI-MS and, addresses two different problems. In the first part, molecular weight and metal analysis of the commercially available rabbit liver MT1 and the Zn induced isolated MT1 from rat liver have been carried out. It provides information about the presence of elements other than Zn and their stoichiometry in the isolated MT1. In the second part, both these techniques have been applied to follow the binding of As to rabbit apo-MT1 (in vitro), prepared by demetallation of commercial rabbit MT1. This provides information regarding the binding of As3+ to rabbit MT1 and substantiates the similar data available on rhMT1. Such information may be useful for understanding the role of MT in As toxicity.
Materials and methods
Isolation of MT
10 Male Wistar rats, weighing 200–250 g, were procured from the Central Animal House, Panjab University, Chandigarh, India, for the induction and isolation of MT. Sephadex G-75 and DEAE-Sephadex A-50 were obtained from Sigma Aldrich Co., USA. Analytical grade chemical reagents were used. The isolation of MT from the rat liver was carried out following the same procedure as used by Bremner and Davies (1975). Zinc (10 mg/kg body weight, as ZnSO4·7H2O per day) was injected subcutaneously for 2 days to rats to induce the synthesis and accumulation of MT. The livers were removed 18 h after the second injection. 20 % liver homogenates in 10 mM of Tris acetate buffer (pH 7.4) were prepared. Homogenates were centrifuged at 10,000×g for 30 min in the REMI C30BL centrifuge. Supernatants were again centrifuged at 100,000×g for 1 h in Brucker ultra centrifuge. The supernatant (cytosol) was fractionated on a Sephadex G-75 column (2.5 × 70 cm) equilibrated with 10 mM Tris acetate buffer (pH 7.4). The Zn containing fractions from low molecular weight region were applied to a DEAE-Sephadex A-50 column pre-equilibrated with 10 mM Tris acetate buffer (pH 7.4). The column was washed with same buffer and bound protein fractions were eluted with a linear gradient of 10–500 mM Tris acetate buffer (pH 7.4). High Zn containing fractions of protein (MT) were then pooled and concentrated using Centricons® (Millipore, 3 kDa MWCO). Zn quantitation of these fractions was carried by Atomic Absorption Spectrometer (Perkin Elmer 3100). Protein estimation of all the above fractions was done at 280 and 205 nm using UV–Vis spectrophotometer (Shimatzu Model UV-1800). The fractionation procedure was repeated multiple times to get required amount of protein for PIXE and ESI-MS studies. About 100–150 μg of MT1 was obtained from the liver of one animal. The experimental design and procedures were approved by the institutional Ethical Committee on Animal experiments.
Preparation of apo-MT1
Rabbit MT1 (95 % pure, procured from United Bo Tai Biotech Co., Ltd., China) was demetallated by adopting the procedure of Ngu and Stillman (2006) with only difference of using buffer of pH 3 instead of 2.7. This pH change was implemented because of the stability range of pH 3–8 for rabbit MT1, as quoted by the supplier. Elution was carried through a Sephadex G-25 column using 25 mM ammonium formate at pH 3 (pH adjusted with HCl) and the fractions were monitored by observing the absorption at 205 nm. Fractions that contained apo-MT1 were pooled and concentrated using centrifugal filter devices (Centricons®, Millipore, 3 kDa MWCO).
Preparation of As-MT1
The apo-MT1 (7.7 μmol) was reacted with increasing stoichiometric ratios (1, 2, 3) of As3+ (100 μM sodium arsenite in 25 mM ammonium formate, pH 3). Each sample was incubated for 2 h at 4 °C. To remove the unbound metal, the samples were loaded on Sephadex G-25 column (equilibrated with 25 mM ammonium formate at pH 3) and eluted with the same buffer. Fractions containing the protein–metal complex were collected by monitoring absorption at 205 nm. These fractions were pooled and concentrated using centrifugal filter devices (Centricons®, Millipore, 3 kDa MWCO).
Particle induced X-ray emission (PIXE)
ESI-MS experiments were performed with Waters® micromass® Q-TOF Mass Spectrometer at Regional Sophisticated Instrumentation Centre (RSIC), Panjab University, Chandigarh, India. The experimental conditions were as follows: capillary voltage 3973 V, sample cone voltage 21 V and extraction cone voltage 2 V. The spectrometer was calibrated using Leucine enkephalin. The m/z range chosen for the recording of the spectra of MT1 was 500 to 2000 amu. All samples were analyzed thrice and prepared under the environment of argon gas to avoid the oxidation of MT.
Results and discussion
Rabbit MT1 and Rat liver MT1
Ratio of different elements found in the PIXE spectrum of the rabbit 1 and the Zn induced isolated rat MT1
Major species observed in the ESI-MS spectrum of the Rabbit MT1
Peak intensity (%)
Molecular ion ([ion]charge)
[Zn7MT1 + 5H]5+
[Zn6MT1 + 5H]5+
[Zn7MT1–CO2 + 5H]5+
[Zn6MT1–CO2 + 5H]5+
[Zn5MT1 + 5H]5+
[Zn7MT1 + Na + 4H]5+
[Zn7MT1 + Na + K + 3H]5+
[Zn6MT1 + 4H]4+
[Zn7MT1–CO2 + 4H]4+
[Zn7MT1 + 4H]4+
[Zn7MT1 + Na + 3H]4+
[Zn6MT1–CO2 + 4H]4+
[Zn5MT1 + 4H]4+
[Zn6MT1 + 6H]6+
[Zn4MT1 + Na + 6H]7+
[Zn1MT1–H2O + 8H]8+
[MT1 + 9H]9+
Major species observed in the ESI-MS spectrum of the isolated Zn induced MT1 from rat liver
Peak intensity (%)
Molecular ion ([ion]charge)
[Zn5MT–NH3 + 9H]9+
[Zn1MT + K + 8H]9+
The ratio of Cu to Zn in the commercial rabbit MT1 (0.009) and the rat MT1 (0.35) are found to be differing by a factor of 35. It is interesting to note that on Zn supplementation both Cu and Zn bind to MT1 instead of only Zn as expected. PIXE being a multi-elemental analysis technique also shows that besides Cu and Zn, no other metal binds with MT1 in these conditions. To the best of the authors knowledge, no reports in literature are available on the application of multielemental analysis techniques on isolated MT. Bremner and Davies (1975) observed the presence of Cu along with Zn in the MT fraction of gel filtration chromatography using atomic absorption spectrometry (AAS). The AAS being a single element detection technique can’t rule out the presence of other metals in MT. Moreover, their objective to use Cu and Zn estimation was for the MT isolation and not the metal stoichiometry because the absorbance measured at 280 nm was not suitable for the MT estimation, as it lacks aromatic amino acids. Similarly, while preparing samples for the NMR investigations of Cd induced rat MT1, Nicholson et al. (1983), found the Cd:Zn:Cu proportions (normalized to 7.0 metal ions) to be 4.46:2.35:0.19. Zn and Cu are both essential metals and both have high affinity for MT. Zn and Cu work in synergy for the coactivation of proteins like superoxide dismutase and phospholipase C. Out of various functions proposed for MT, one function is to act as a storage molecule for essential metals in order to maintain the balance between concentration of free Zn and Cu inside the cell. The presence of both Zn and Cu in rat liver MT1 on Zn supplementation can be related to the role of MT1 in Zn and Cu homeostasis.
The calculated masses of metal bound MT1 using the molecular masses of the amino acids and metal are slightly different (up to 10.7 Da) from the experimentally observed masses of ions in the ESI-MS spectra. This can be attributed to the presence of Cu ions in lieu of Zn ions as is evident from PIXE. ESI-MS can’t discriminate between Zn and Cu ions when bound to protein because the atomic mass of Zn and Cu are very close (i.e. 65 and 63 respectively) and a broad isotopic variation in the mass of Zn is also present. The number of Zn and Cu ions bound to MT1 is difficult to evaluate at this stage and require further investigations.
Rabbit apo-MT1 and arsenic substituted MT1
Major species observed in the ESI-MS spectrum of rabbit MT1 at pH 3
Peak intensity (%)
Molecular ion ([ion]charge)
[MT1 + 9H]9+
[Zn3MT1 + Na + 8H]9+
[Zn1MT1 + Na + 7H]8+
[MT1 + Na + 6H]7+
[Zn3MT1 + Na + 6H]7+
Major species observed in the ESI-MS spectrum of As substituted rabbit MT1 at pH 3
Peak intensity (%)
Molecular ion ([ion]charge)
[As2 MT1 + K + 6H]7+
[As6MT1 + Na + 5H]6+
[As5MT1 + 5H]5+
[As4MT1 + 8H]8+
The present work shows that PIXE and ESI-MS can be clubbed together in order to extract complimentary information regarding the identity of the metal and its stoichiometry when bound to MT. ESI-MS analysis of isolated rat MT1 shows the presence of only partially metallated MT1 is further substantiated by the high S/Zn ratio (i.e. 3.23, instead of 1.468 for Zn7MT1) observed with PIXE analysis. PIXE results also show that isolated rat MT1 contains significant number of Cu ions along with Zn ions despite being induced by Zn supplementation, suggesting the role of MT in metal homeostasis. By lowering the pH to 3 for rabbit MT1, few Zn ions still remain bound. On As3+ metallation reaction with MT1 at this pH forms AsxMT1 (x = 2, 4, 5 and 6) complexes. With an increase in the As3+ ion concentration, the As bound to MT1 increases. To further understand the role of MT in Arsenic toxicity more studies are needed like the isolation of MT induced by As which can then be analyzed by PIXE, ESI-MS and other spectroscopic techniques.
The authors are thankful to the Pelletron crew of IOP, Bhubneswar and the staff of Panjab University Cyclotron, Chandigarh for the support rendered during PIXE experiment. Department of Science and Technology, New Delhi and Sophisticated Analytical Instrumentation Facility, Panjab University, Chandigarh are gratefully acknowledged for providing ESI-MS facility. This work is funded by University Grant Commission (UGC), New Delhi and UGC-DAE Consortium for Scientific Research, Kolkatta. Roobee Garla and Renuka Ganger are thankful to UGC, New Delhi for providing financial assistance in the form of Junior Research Fellowship.