Metallothionein isoforms from horse, rabbit and rat separated by capillary zone electrophoresis at low pH

A comparative study of MT isoforms in rat liver and in commercial Sigma MT preparations from rabbit liver and horse kidney was performed using capillary zone electrophoresis (CZE). Electropherograms revealed the co-migration of MT forms from these species. A special form, the a-form (not binding Cd), occurred in various MT samples in different amounts, depending on the method used for MT purification. In the rabbit liver electropherogram a main form appeared (the b-form), which might be a modified MT form. A band of unknown composition, running ahead of the rat liver MT-I and -II forms on polyacrylamide gels, not having Cd binding affinity, probably had its counterpart in a yet unidentified CZE peak. CZE electropherograms of purified MT samples may contain main peaks that do not represent genuine and functional MT isoforms. Results are also presented which indicate that at low pH the MT-II form is more unstable than MT-I.     

Improved separation of metallothionein isoforms by the presence of cyclodextrin in capillary zone electrophoresis

Capillary zone electrophoresis (CZE) with cyclodextrin (CD) in the polyacrylamide-coated capillary was used to study metallothionein (MT) forms in the horse kidney preparation produced commercially by Sigma. It is known that CDs form complexes with hydrophobic amino acids. The results show that the presence of CD improves the separability of the various MT forms, including the MT-IA and the MT-IB forms, metallothionein aggregates, as well as the so far unidentified a and b forms. This was true both below and above the isoelectric points (pIs), although the migration times were somewhat longer at increasing CD concentrations for runs at constant voltage than with constant current.     

Anion-exchange high performance liquid chromatography hyphenated to inductively coupled plasma-isotope dilution-time-of-flight mass spectrometry for speciation analysis of metal complexes with metallothionein isoforms in gibel carp (Carassius auratus gibelio) exposed to environmental metal pollution

The capability of post-column isotope dilution (ID) combined with anion-exchange HPLC-ICP-time-of-flight (TOF)-MS was for the first time investigated for environmental quality assessment through metal speciation analysis of metallothionein (MT) isoforms in cytosols of gibel carp (Carassius auratus gibelio), used as biomarkers for environmental metal exposure. A full spectral scanning of the biological sample (with 50 microl injection volume) using ICP-TOF-MS in transient mode allowed fast multi-isotope screening of cytosolic metal-containing fractions and to investigate the presence of matrix-induced interferences. The MT cytosolic fraction of liver and kidney of the carp, sampled at three different sampling sites in Belgium, was partially purified using size-exclusion (SE) HPLC. Quantification of the elements Cd (toxic) and Zn and Cu (essential) associated with MT isoforms in this fraction was addressed using an hybrid approach based on post-column addition of the enriched isotopes 65Cu, 67Zn, 106Cd and monitoring on-line the isotope ratios 63Cu/65Cu, 64Zn/67Zn and 114Cd/106Cd by ICP-MS with a time of flight instrument, which was coupled to anion-exchange HPLC. With this separation method, baseline separation of up to five MT isoforms, which is required for quantitative metal speciation by HPLC-ICP-IDMS, was achieved within a run of 15 min. The MT fraction of the cytosols was also analysed for the total metal content using IDMS with size-exclusion HPLC-ICP-MS and species-unspecific calibration. Results showed significant differences between speciation results and total MT concentrations of control fish and fish from the most contaminated sampling sites, revealing the potential of gibel carp MT for sequestering excess intracellular free-ions (essential and toxic elements) and for its protection against metal toxicity. Preferences for metal sequestration of metal complexes with MT isoforms were also found to be tissue-specific: excess of Cd was found preferably bound to a major MT isoform (tR = 8.0 min) in kidney, whereas excess intracellular Zn appeared to be mostly sequestered by four MT isoforms (tR=7.3, 8.0, 12.2 and 14.4 min) in liver, the MT form with tR = 8.0 min being the main Zn scavenger form. Such kind of quantitative speciation information on the preferences of MT isoforms in different fish organs for sequestering heavy metals, reported here for the first time, is important to elucidate the role of isoform-specific induction of vertebrate fish MT in metal detoxification and the use of MT as biomarker.     

金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAE Sephadex A-25离子交换柱,结合离线电感耦合等离子体质谱(ICP-MS)对锌诱导金属硫蛋白的两个异构体MT-1和MT-2进行分离和检测;利用Sephadex G-25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。     

Characterization of the metal composition of metallothionein isoforms using reversed-phase high-performance liquid chromatography with atomic absorption spectrophotometric detection

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to separate metallothionein (MT) isoforms and on-line atomic absorption spectrophotometric (AAS) detection was used to quantitatively determine their metal content. With this coupled system (HPLC-AAS), it was possible to determine the zinc, cadmium and copper content of individual horse kidney MT isoforms. When rabbit liver MT and the purified isoforms (MT-1 and MT-2) were subjected to RP-HPLC and the zinc-containing peaks of the MT sample to MT-1 or MT-2. HPLC-AAS was used to identify zinc-induced MT in heat-treated cytosol from turkey hen liver, thereby demonstrating its application to the analysis of crude tissue extracts. A standard curve was established using turkey liver MT for the quantitative determination of the zinc content of MT isoforms. There was excellent linear correlation between the micrograms of zinc bound to MT injected onto the column (ranging from 0.34 to 3.43 micrograms of MT-bond zinc) and the integrated peak area of the atomic absorbance for zinc. Using this standard curve, it was possible to quantitate the amount of MT-bound zinc in cytosol extracts of cultured turkey embryo hepatocytes exposed to varying levels of supplemental zinc in the culture medium.     

Differential ESI-MS behaviour of highly similar metallothioneins

ESI-MS can only be accepted as a quantification method when using standards with a high resemblance to the analyte(s). Unfortunately, this is usually not applicable to metallothioneins (MTs), a superfamily of singular metal-binding cysteine-rich proteins, present in all living organisms, since the absence of suitable reference material due to the high diversity among metal-MT species precludes their quantification by molecular mass spectrometry. Even thus, it is widely assumed that the intensities of the ESI-MS peaks of similar species are directly correlated with their relative concentration in the sample, and this has been extended to the determination of different MT proteins coexisting in a sample.Practically all organisms contain several MT isoforms, some of them exhibiting highly similar sequences, with conserved coordinating Cys residues. For the current analysis, we used as a model system the MT isoforms of two terrestrial snails (Helix pomatia and Cornu aspersum). Hence, distinct samples were prepared by mixing, at different molar ratios, the recombinant HpCuMT and HpCdMT isoforms from H. pomatia, or the recombinant CaCuMT, CaCdMT and CaCdCuMT isoforms from C. aspersum, and they were analyzed by ESI-MS both at neutral pH (for Zn-loaded MT forms) and at acidic pH (for the corresponding apo-forms). The results here presented reveal that the ESI-MS peak intensity of a single MT species strongly depends on its sensitivity to be ionized, and thus, on the presence or absence of metal ions bound. Furthermore, our data demonstrate that very similar MT isoforms of the same organism with similar pI (ranging from 7.9 to 8.3) can show a clear different sensitivity to ES ionization, something that cannot be readily predicted only by consideration of their amino acid content. In conclusion, even in this optimum case, deductions about quantity features of MT samples drawn from ESI-MS measurements should be carefully considered.     

Structural changes in metallothionein isoforms revealed by capillary electrophoresis and Brdicka reaction

Metallothionein (MT) as a potential cancer marker is at the center of interest and its properties, functions and behavior under various conditions is intensively studied. In the present study, two major mammalian MT isoforms (MT‐1 and MT‐2) were separated using capillary electrophoresis (CE) coupled with UV detector in order to describe their basic behavior. Under the optimized conditions, the separation of both isoforms was enabled as well as estimation of detection limits as subunits and units of ng per μL for MT‐2 and MT‐1, respectively. Further, the effects of thermal treatment and the presence of denaturing agent such as urea on MT‐1 and MT‐2 isoforms were studied by CE‐UV. Thermal treatment caused an increase in the signals of both isoforms. A new parameter called precipitation rate has been defined based on this finding. This parameter can be expressed as a slope of the linear regression of the time dependency curve recalculated on the MT concentration. The thermal precipitation rate for MT‐1 and MT‐2 was determined as 1.1 and 0.9 ng of MT/min, respectively. The chemical precipitation rate calculated from the linear regression for both isoforms provided the same value of 0.25 ng of MT/min. The results were confirmed by manual spectrometric measurements and by differential pulse voltammetry Brdicka reaction. Based on these results, a model of MT behavior under the conditions studied was suggested.     

Metal speciation of metallothionein in white sea catfish, Netuma barba, and pearl cichlid, Geophagus brasiliensis, by orthogonal liquid chromatography coupled to ICP-MS detection

Metal speciation analysis in MTs was carried out in two tropical fish species of Brazil, the freshwater fish pearl cichlid (Geophagus brasiliensis) and the marine fish white sea catfish (Netuma barba), that are presently used to monitor the effects of heavy metal pollution in aquatic ecosystems in Brazil. In order to obtain the MT fraction, liver cytosols from both fish species where subjected to size exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. That MT fraction was then separated by anion-exchange (AE)-FPLC, whose optimal chromatographic conditions were optimized for the separation of the different hepatic MT isoforms existing in both fish species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with the ICP-MS instrument. The analytical results showed that MTs of these fish species, unknown so far, exhibited unique characteristics in comparison with standard MTs and other fish liver MTs. In fact, MT isoforms of N. barba turned out to be very anionic, as indicated by their high retention in the Mono Q column and the strong ionic strength required to separate them. As for G. brasiliensis, cadmium was exclusively present in only one of the peaks of the MT isoforms showing a unique metal-binding behavior for MT in this fish species. The differences between the MTs among these species and the different association of metals in particular MT isoforms display the importance of the metal speciation analysis in these proteins prior to its use as bioindicators.     

Analysis of metallothionein isoforms by capillary electrophoresis: optimisation of protein separation conditions using micellar electrokinetic capillary chromatography

Capillary electrophoresis (CE) techniques have been successfully applied to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diameter capillaries we conclude that 25 μm I.D. columns give better separations than 50 or 75 μm I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C₁ or C₁₈ hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress. Analysis of sheep liver extracts using optimised conditions showed the expression of at least 4 MT isoforms in response to Zn injection and 3 of these forms were evident in extracts from untreated sheep. We therefore conclude that MECC is a suitable method for MT isoform analysis.     

金属硫蛋白的色谱/电喷雾电离质谱联用表征方法

通过反相液相色谱(RPLC)与电喷雾电离质谱(ESI-MS)的联用技术,对镉诱导金属硫蛋白标准物质MT-1和MT-2的结构进行表征分析。采用Vydac C8 反相色谱柱(250 mm×2.1 mm i.d., 5 μm, 30 nm),流动相A为pH 6.0的5 mmol/L乙酸铵水溶液,流动相B为pH 6.0的5 mmol/L乙酸铵的甲醇-水(体积比为1∶1)溶液,流动相流速为0.20 mL/min,在40 min内流动相B的体积分数从10%增加到37.5%进行梯度洗脱。分别用紫外(UV)和ESI-M     

An alternative interface for CE-ICP-MS cadmium speciation in metallothioneins based on volatile species generation

An alternative CE-ICP-MS interface based on volatile species generation (VSG) is here developed, evaluated and compared to the conventional sample introduction systems via nebulisation. For this purpose, the speciation of Cd-metallothioneins (MTs) in rabbit liver is taken as a model. Cd, bound to the different MT isoforms previously separated by CE, is transformed into volatile species at the exit of the capillary and on-line detected by ICP-MS. Optimum conditions for Cd VSG have been investigated in a flow injection device, using NaBH₄ as hydrogenation reagent in a HCl medium containing cobalt and thiourea as catalysts. Sample volume injected, CE separation voltage and reagents flows have been optimised. Analytical performance characteristics of the CE-VSG-ICP-(Q)MS coupling developed were evaluated, in terms of repeatability and linearity of response, using standard rabbit liver metallothionein isoforms (MT1 and MT2). Detection limits for Cd-MTs turned out to be almost one order of magnitude better than those derived from using a conventional Babington nebuliser-based interface. Compared to a MicroMist-based interface detection limits resulted to be similar, but the observed peak height was eight times higher using the VSG interface, indicating the enhanced analyte transport efficiency derived from VSG sample introduction systems.     

Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries

Human serum transferrin is a mixture of isoforms (isoproteins) having different amounts of carbohydrates. Each isoform may exist in iron-free and iron-complexed molecular form. The genetic variations in different populations increase the number of combinations of the different forms of transferrin. To resolve the many components in transferrin preparations, the new high performance capillary technique was employed for isoelectric focusing. Iron-free transferrin and transferrin samples of known iron content were examined. The above method gives an exceptionally rapid analysis (within 15-25 min) of small amounts of samples (less than 1 microgram protein) and as good as or better resolution than other isoelectric focusing techniques previously used for transferrin analysis. By monitoring the focused protein zones at both 280 and 460 nm the molecular forms of transferrin (iron-free, monoferric and differic complexes) can easily be identified. Both steps of isoelectric focusing in capillaries (i.e., prefocusing and mobilization) can be used for analysis. We observed that chelating agents (e.g., carrier ampholytes, nitrilotriacetate) may release iron from microsyringes having metal pistons causing the formation of iron-transferrin complexes.     

A novel extractant-impregnated resin containing carminic acid for selective separation and pre-concentration of uranium(VI) and thorium(IV)

The present work proposes the use of a novel extractant-impregnated resin (EIR) as an adsorbent in trace separation and pre-concentration of U(VI) and Th(IV) ions. The new EIR was prepared by impregnating carminic acid onto Amberlite XAD-16 resin beads. The morphology of new EIR was studied by BET surface area measurements and SEM micrographs. A column packed with CA/XAD-16 was used for selective separation and pre-concentration of the metal ions. Maximum adsorption of Th(IV) and U(VI) ions occurred at pHs of 3.50–5.75 and 3.75–6.50, respectively. The adsorbed metals could be eluted sequentially using 0.55?mol?L^?1 HCl for U(VI) and 2.25?mol?L^?1 HCl for Th(IV). The dynamic capacity of EIR was found to be 0.832 and 0.814?mmol?g^?1 for Th(IV) and U(VI), respectively. The tolerance limit of some foreign ions was also studied. The proposed method showed a good performance in analyzing geological reference materials and a synthetic seawater sample. Furthermore, the above procedure was successfully employed for the analysis of natural water samples.     

Speciation analysis of trace elements in the brains of individuals with Alzheimer's disease with special emphasis on metallothioneins

A speciation analysis of protein-bound elements in the cytosol of human brain was achieved by size exclusion chromatographical separation of the biomolecules and on-line detection of the metal profiles in the eluate by hyphenated inductively coupled plasma-mass spectrometry. Post-mortem samples from Alzheimer's disease brains and from brains of a control group were investigated to elucidate changes in the trace element distribution during the pathological process. Special attention was paid to the metallothioneins (MT) - cysteine-rich, metal-binding proteins of low molecular weight, existing in several isoforms. The isoform MT-3 is found especially in the brain and has a growth inhibition function on neurons. The MT peaks were identified in the element profiles. For this purpose, the metal binding capability and the heat stability of MT were taken into consideration. For verification, a comparison with pure MT-3 was carried out and further biochemical and analytical methods were applied to the fractions of the chromatographical run. A comparison between Alzheimer's disease and control brains showed a significant difference concerning the MT-1/-2 and MT-3 metal levels, leading to the assumption that there were oxidative processes having taken place in the Alzheimer's brain samples.     

兔肝金属硫蛋白亚型异构体的高效液相色谱分离与ESI-MS,MALDI-TOF-MS鉴定

由于金属硫蛋白(MT)基因的多态性,决定不同亚型的MT异构体的存在,MT亚型异构体的结构是MT功能研究的基础.通过离子交换柱可将MT分成MT-1和MT-2两个异构体,用不同条件的反相高效液相色谱(RP-HPLC)可将MT-1和MT-2分成不同的亚型异构体,并利用MALDI-TOFMS和LC-ESI-MS对比确定了它们的分子量.结果表明,兔肝MT在不同的pH条件下分离得到不同分子量的亚型异构体.在酸性条件下,MT-1可分为2个主要亚型异构体,分子量分别为6149.0和6244.5,而MT-2主要分为3个亚型异构体,分子量分别为6149.0,6244.0和6127.0.MT-1和MT-2有2个亚型异构体分子量相同的异构体存在.在酸性条件下,MT-1的2个异构体及MT-2分子量为6127的亚型异构体可稳定存在.     

类水滑石/蒙脱土体系的特殊粘弹性和触变性研究

本文对由类水滑石（HTlc） /钠质蒙脱土（MT）组成的复杂体系的粘弹性和触变性进行了详细的研究,我们共研究了两种Fe-Mg-Al- HTlc样品,从样品1到样品2,Al 对 Fe的比例保持不变,而Mg2+的含量逐渐增加。研究发现,随着类水滑石中Mg2+的含量增加,HTlc/MT分散体系的结构强度逐渐增加;触变环的实验结果也表明,随着类水滑石中Mg2+的含量增加,HTlc/MT分散体系的触变程度逐渐增加。由此我们可以得到这样的结论,通过调节类水滑石金属的配比我们就可以对类水滑石/蒙脱土体系的流变性进行宏观的调节。     

Electrochemistry of and Redox‐Induced Metal Release from Metallothioneins at a Nafion‐Coated Bismuth Film Electrode

Nafion‐coated bismuth film electrodes (NCBiFEs) were used to investigate the redox behavior of and metal release from rabbit liver metallothioneins (MTs) in an acetate buffer. Owing to the permselective exchange between positively charged MT molecules and cations in Nafion and the absence of detectable MT adsorption at bismuth surface, a diffusion‐controlled Nernstian redox wave of MTs (E_pc=?0.869 V) was observed for the first time. The Nernstian behavior of the diffusing MTs is in contrast to the voltammetric responses of MTs at thin mercury films or Nafion‐coated mercury film electrodes, which either result in the replacement of the metals originally present in MTs by mercury or lead to a noticeable MT adsorption. By avoiding these undesirable features, the NCBiFE provides an excellent milieu for voltammetric studies of different types of MTs and related isoforms, paving the way for studying the redox‐modulated metal transfer of MTs and understanding the biological role of MTs in heavy metal detoxification and essential metal regulations.     

Narrow-bore HPLC–ICP–MS for speciation of copper in mutant mouse neonates bearing a defect in Cu metabolism

Minute amounts of tissue supernatants from mouse neonates bearing a mutation in the copper (Cu)-transporter gene, Atp7a, were injected into narrow-bore HPLC coupled with an inductively coupled plasma–mass spectrometer (ICP–MS) to examine Cu metabolism. In the 14-day-old mutant neonates, Cu accumulated in the intestine in the metallothionein (MT)-bound form, and mRNA expression of the two MT isoforms was increased. Meanwhile, Cu in the MT-bound form (Cu-MT) was depleted in the liver and mRNA expression decreased in comparison with wild-type mice. These results suggest that Cu is not secreted by intestinal microvillus cells into bloodstream due to the defect of Atp7a, and systemic depletion of Cu occurred. On the other hand, in the kidneys of mutant mice, Cu accumulated in the MT-bound form despite the fact that mRNA expression of the two MT isoforms was low. Part of Cu-MT in microvillus cells may be released into bloodstream at turnover and be preferably taken up by the kidneys. Consequently, the mRNA expression of MT isoforms was not always coincident with the amounts of MT proteins binding Cu, and narrow bore HPLC–ICP–MS used for MT protein determination is a complementary technique to real-time RT-PCR used for MT mRNA determination in Cu speciation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.