Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis

We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60–80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.     

Determination of metallothionein-1/metallothionein-2 ratios in the mouse liver and pancreas by capillary zone electrophoresis using a polyacrylamide-coated capillary at neutral pH

Metallothionein (MT) isoforms, MT-1 and MT-2, in biological specimens are clearly separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated capillary. The effectiveness of CZE analysis in the study of MT isoforms in biological specimens is discussed. We did two experiments to determine the MT-1/MT-2 ratio in biological specimens. The ratio of MT-1/MT-2 can be determined by CZE under a neutral pH without any detergents. One of these studies is time-dependent changes of the MT-1/MT-2 ratio in the cytosol of the pancreas and liver in mice after Zn or Cd injection. In the pancreas, both isoforms were detected in the control mice and the ratio of MT-1/MT-2 was below 1.0. When Zn was injected, the maximum peak areas of both isoforms were obtained at 24 h, and the ratios increased over a value of 1.0 at 3 h and peaked at 10 h. However, in the Cd-injected mice, the peak areas of both isoforms increased up to 72 h, and the ratios were below 1.0 up to 72 h. On the contrary, neither isoform was detected in the livers of control mice. The ratios of Zn-injected mice liver were near the value 1.0 between 6 and 72 h, although the areas of both isoforms showed peaks at 48 h. The ratios of Cd-injected mice livers were detected to be over 1.0 from 10 h, but there were no significant difference between 10 and 72 h, and the areas of both isoforms showed peaks at 24 h. The other experiment investigated the ratio in each fraction of cell fractionation. Cell fractionation was done in the livers of Zn-treated mice. Twenty-four hours after the injection, the ratio of MT-1/MT-2 was 0.80+/-0.12 and 1.19+/-0.21 (mean+/-SD) in nuclear and cytosol fractions, respectively. Neither isoform was detected in mitochondrial or microsomal fraction. From the present results, CZE analysis is a suitable method for observation of the ratio of MT-1/MT-2 in biological specimens, and dynamic changes in both isoforms can be detected.     

Identification of metallothionein isoforms with capillary zone electrophoresis using a polyacrylamide-coated tube

Metallothionein (MT) isoforms from various liver tissues were separated with capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH. The electrophoresis was performed on MT-1 and MT-2 purified from mouse, rat, rabbit and human livers. The retention times of mouse and rat MT-1 coincided, while the retention times of rabbit and human MT-1 were longer. The retention times of MT-2 purified from the four sources were the same. MT-1 and MT-2 separated more definitely with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-Tris buffer (25 mM, pH 7.4) than with N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS)-Tris buffer (25 mM, pH 7.7) or with N-(2-acetamido)iminodiacetic acid (ADA)-Tris buffer (25 mM, pH 7.4). In addition, liver MT isoforms prepared from Zn- or Cd-administered mice could be separated.     

Identification of metallothionein isoforms on capillary zone electrophoresis by adding anti-metallothionein antibody

The aim of this study was to identify metallothionein (MT) isoforms in mouse liver by using capillary zone electrophoresis (CZE). Purified MT-1 and MT-2 isoforms were completely separated by CZE using a polyacrylamide-coated tube at physiologic pH. There were two peaks in the cytosol fraction prepared from zinc-injected mouse liver, in which the migration times corresponded with those of purified MT-1 and MT-2 isoforms. When anti-MT monoclonal antibody was added with the purified MT-1 or MT-2 solution, the peaks decreased. Furthermore, the two peaks in the cytosol prepared from Zn-injected mouse liver decreased in a time-dependent manner from the electropherogram after the addition of the antibody. Therefore, those peaks were identified as MT-1 and MT-2 isoforms, respectively. In conclusion, the addition of anti-MT monoclonal antibody to the cytosol fraction of tissues is an effective method for identification of MT isoforms after separation using CZE.     

White-sided dolphin metallothioneins: purification, characterisation and potential role

Metallothioneins (MTs) were characterised in the kidneys of a white-sided dolphin Lagenorhynchus acutus stranded along the Belgian coast, displaying high levels of cadmium (Cd) and mercury (Hg) in liver and kidney. The protein has two isoforms: MT-1 and MT-2. MT-1 binds Cu, Zn, Hg and Cd, while MT-2 only binds Zn, Hg and Cd. This suggests different metabolic functions for the two isoforms: MT-1 is mainly involved in Cu homeostasis; MT-2, which was four-fold more abundant than MT-1, detoxifies most of the accumulated cadmium.     

Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1–100 ng for MT-2 and 10–1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our anti-serum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2. Therefore, the MT-2 specific antibodies may be useful for the study of the functions of MT isoforms. Levels of total MT in tissues and biological fluids of rats injected with CdCl₂ (0.3 mg Cd/kg) and Cd-MT (0.3 mg Cd/kg) were estimated by ELISA. The results suggest urinary MT levels may be related to kidney damage.     

Metal binding to brain-specific metallothionein-3 studied by electrospray ionization mass spectrometry.

Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.     

Separation and quantitation of metallothionein isoforms from liver of untreated rats by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry

A sensitive method for determination of metallothionein (MT) isoform levels in rat liver by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry was developed. Critical steps in sample preparation, like MT extraction, MT saturation with Cd and protein separation, were optimized. This method is capable of measuring levels of 2.0 μg/g liver for metallothionein-1 (MT-1) and 1.3 μg/g liver for metallothionein-2 (MT-2), respectively, with a high recovery of 103% on average. The method described, thus, proved suitable for analyzing metallothionein isoform concentrations even in untreated animals. The ratio of MT-1 to MT-2 was found to be 1:1 on average. MT decomposition during storage was very high in whole livers, but could be reduced by about 80% when extracted liver samples were used.     

Zinc and copper accumulation and isometallothionein induction in mouse ascites sarcoma S180A cells.

To investigate Zn and Cu accumulation and isometallothionein (iso-MT) induction in ascites-sarcoma S180A cells, 5 micrograms of Zn2+ or Cu2+/g body weight was administered to tumour-bearing mice intraperitoneally. In the tumour cells the Zn or Cu concentration increased more than in the host liver, which is the target organ for those metals; the maximum Zn or Cu level was about 2-3 times that in the host liver. The amounts of Zn-MT or Cu-MT accumulated in the tumour cells and host liver were proportional to such dose accumulation levels in the each cytosol; the maximum level of Zn-MT or Cu-MT was 4 or 2 times higher than in the host liver. MT accumulated in the tumour cells showed two subfractions (MT-1 and MT-2); the ratio of Zn (or Cu) bound to MT-1 to that bound to MT-2 in the host liver and tumour cells was 1.0 (or 1.0) and 0.7 (or 0.25) respectively, suggesting that the induction level of MT-2 in the tumour cells is more than that of MT-1. The h.p.l.c. profiles (using an anion-exchange column) of the isolated MT-1 and MT-2 subfractions from Zn-treated normal-mouse liver showed a single peak (MT-1-1) and two peaks (MT-2-1 and MT-2-2) respectively; mouse MTs were separated into three isoforms. In the ascites cells, the MT fraction obtained by a gel filtration was also separated into three isoforms; however, the amount of MT-2-1 isoform was 3 times that in the Zn-treated normal-mouse liver.     

Chemistry and biology of mammalian metallothioneins

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal–thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discussed.     

Purification of recombinant human apometallothionein-3 and reconstitution with zinc

Metallothioneins (MT) are small cysteine-rich proteins, expressed in many life forms. They are involved primarily in the metabolism of zinc and copper, and in metal detoxification processes. Metallothionein-3 is a mammalian brain-specific MT, which is down-regulated in Alzheimer's disease brains. In this report, we describe a new procedure for purification of recombinant human apo-MT-3 by three steps, size exclusion at neutral pH, followed by cation-exchange and reverse-phase HPLC, both at low pH. Purified apo-MT-3 was reconstituted with seven Zn(2+) ions and reconstitution products were analyzed with electrospray ionization mass spectrometry. The mass spectrum of reconstituted ZnMT-3 was identical with that of native ZnMT-3 isolated by size exclusion chromatography proving the efficiency of the reconstitution process. It showed that ZnMT-3 exists in solution as a dynamic mixture of several metalloforms, where the main metalloform is Zn(7)MT-3 and minor forms are Zn(6)MT-3 and Zn(8)MT-3.     

Effects of Chronic Cadmium Poisoning on Zn, Cu, Fe, Ca, and Metallothionein in Liver and Kidney of Rats

An experiment was conducted to invest effects of chronic cadmium poisoning on Zn, Cu, Fe, Ca, and metallothionein gene expression and protein synthesis in liver and kidney in rats. Forty rats, 6?weeks old, were randomly allocated into two groups. A group was given CdCl(2) (1?mg/KgCd(2+)) by intraperitoneal injection once a day. The other group was treated with normal saline in the same way. Liver and kidney were collected for analysis at the end of the third week. Results showed that Cd exposure increased Cd (P?相似文献   

Investigation of metal complexes with metallothionein in rat tissues by hyphenated techniques.

The potential of hyphenated techniques based on a combination of microbore reversed-phase (RP) HPLC or capillary zone electrophoresis (CZE) with inductively coupled plasma (ICP) or electrospray (ES) mass spectrometry (MS) was demonstrated for the characterization of metal complexes with metallothionein in rat liver and kidney. The mixture of MT complexes was isolated from the tissues by size-exclusion LC and further characterized in neutral pH conditions (pH 6.8-7.2) by RP-HPLC or CZE. The metal stoichiometry and the molar mass of the eluted complexes was measured by ICP-MS and ES-MS, respectively. An additional dimension to the analysis was achieved by post-column acidification of the chromatographic eluent that allowed the determination of the molecular weight of the demetallated complexes with 10-fold higher sensitivity. The approach allowed the detection of two major metallothionein (MT) isoforms (MT-1 and MT-2) in liver and one MT isoform in kidney. The actual number of peaks in chromatograms and electropherograms was bigger because of the formation of mixed Cd-Cu complexes of the same MT isoform that showed different hydrophobicities.     

The Properties of the Metal–Thiolate Clusters in Recombinant Mouse Metallothionein-4

Metallothioneins (MTs) are metal-binding proteins with low molecular weight and conservative cysteine residues. Metallothionein-4 (MT-4), one of MT isoforms, is first reported to be distributed in a tissue-specific manner, mainly in stratified squamous epithelia. Here, we compare the properties of metal–thiolate clusters in MT-4 to those in MT-1 and MT-3, including the stabilities toward both pH change and EDTA, as well as the exposure of thiolates to solvent. The metal–thiolate clusters in MT-3 show different property and activity to the reactions compared with MT-4 and MT-1. The structure of metal–thiolate clusters in MT-4 is similar to that of MT-1 from the UV and CD spectra. During pH titration and DTNB reaction, MT-4 and MT-1 exhibit comparable behavior. But while reacting with EDTA, the metal–thiolate clusters in MT-4 are more stable than those of MT-1. We suppose the negative charge of the β-domain of MT-4 prevents the EDTA attack to MT-4.     

Metalloprotein analysis by capillary isoelectric focusing

Capillary isoelectric focusing (cIEF) was used to analyze three metalloproteins: conalbumin, transferrin and metallothionein (MT). Two different ampholyte mixtures were employed that generated linear pH gradients of 3–10 and 5–8. Several different proteins and one peptide known isoelectric points (pIs) were used to establish linear relationships between peak migration time and pI. These standards were also used as internal markers to estimate peak pI values of the metalloproteins subjected to cIEF. Conalbumin (iron-free) subjected to cIEF with a pH gradient of 3–10 yielded a single major component (pI 7.17). When the protein was saturated with iron (2 Fe³⁺/mol protein), a shift to lower pI was observed with a major peak (pI 6.24) and a lesser peak (pI 6.09). Mixing iron-free with iron-saturated conalbumin or adding iron to iron-free conalbumin prior to cIEF produced an additional peak (pI 6.68) that was presumed to be conalbumin containing a single iron atom (monoferric form). Human transferrin subjected to cIEF with a pH range of 3–10 gave a similar separation pattern to conalbumin with four major peaks at pI values of 6.25 (apotransferrin), 5.96 (monoferric form), 5.48 and 5.34 (differic forms). Additional resolution of the molecular forms of both conalbumin and transferrin was achieved using a narrower pH gradient (5–8). Rabbit liver MT subjected to cIEF with a pH gradient of 3–10 gave a complex separation pattern with two prominent peaks (pI values of 3.73 and 3.56) that were presumed to be the fully metal-saturated MT-1 and MT-2 isoforms. When individual MT isoforms (MT-1 and MT-2) were separately subjected to cIEF with a pH gradient of 3–10, heterogeneous peaks with higher pI values (4.12–4.74) were observed. In contrast, horse kidney MT gave a single predominant peak with a pI of 4.09. MT samples could be separated using pH gradient of 5–8 despite the fact that their apparent pI values were below the limits of the pH gradient established. In general, the heterogeneity observed for conalbumin, transferrin and MT proteins subjected to cIEF reflects the presence or absence of bound metal. Thus, cIEF represents a potentially useful analytical method which can provide information concerning the metal-binding characteristics of these and perhaps other metalloproteins.     

Application of a polyamine-coated capillary to the separation of metallothionein isoforms by capillary zone electrophoresis

In this study a fused-silica capillary treated internally with a polyamine coating which reverses electroosmotic flow in the direction of the anode was evaluated for its ability to resolve metallothionein (MT) isoforms. Analysis of different MTs purified from liver and kidney tissue revealed the following numbers of putative isoform peaks resolved: rabbit (3–6); horse (3–5); rat (2–3), chicken (1); human MT-1 (5–6); sheep (4–5) and pig (4–5). The greater degree of MT isoform heterogeneity detected in this study using the polyamine-coated capillary suggested a higher resolving capacity for capillary zone electrophoresis conducted with this capillary compared to an uncoated one. Using the single isoform of chicken MT (cMT) as a reference standard, relative standard deviations of 2.53, 1.85 and 2.21% for peak migration time, area and height, respectively, were observed for eight consecutive runs. A standard curve for cMT established linearity (r² = 0.99) for integrated peak area over three log units of cMT concentration with a lower limit of detection estimated to be 5 μg/ml. Acetonitrile extracts of chick liver tissue homogenates were successfully analyzed for the presence of MT isoforms from both control and zinc-injected animals. Based on our initial evaluation, capillary zone electrophoresis using the polyamine-coated capillary appears to be a very useful analytical method for the separation and quantification of individual MT isoforms.     

Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic and/or necrotic processes and on MT isoform gene expression

It has been shown in various systems that zinc is able to antagonize the catalytic properties of the redox-active transition metals iron and copper, although the process is still unclear. Probably, the protective effect of Zn against oxidative stress is mainly due to the induction of a scavenger metal binding protein such as metallothionein (MT), rather than a direct action. To support this hypothesis, in this study, the effects of Zn, Cu, Fe, Zn + Cu and Zn + Fe treatments were investigated in a fibroblast cell line corresponding to an SV40-transformed MT-1/-2 mutant (MT-/-), and in wild type (MT+/+), by valuing metal concentrations and apoptotic and/or necrotic processes. We also investigated the synthesis of MT and the levels of both MT-1 and MT-2 mRNAs. In MT+/+ cells, co-treatment with Zn + Fe caused a decrease in Fe content compared to treatment with Fe alone. After Zn and Zn + Cu exposure the expression of MT-1 and MT-2 isoforms increased with a concomitant increase in MT synthesis. Annexin V-FITC and propidium iodide staining revealed necrotic or apoptotic cells in terminal stages, especially after Fe treatments. Immunofluorescent staining with an anti-ssDNA Mab and annexin detected a lower signal in co-treated cells compared to the single treatments in both cell lines. The intensity and quantity of fluorescence resulting from anti-ssDNA and Annexin V staining of MT null cells was higher compared to wild type cells. These results suggest that Zn alone does not completely exert an anti-oxidant effect against Cu and Fe toxicity, but that induction of MT is necessary.     

Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis

Structure and function of molluscan metallothioneins (MTs) are still poorly understood. The sea mussel Mytilus galloprovincialis displays two MT isoforms which differ in both primary sequences and physiological functions. MT-10 is the constitutive isoform, whereas MT-20 is mainly induced by cadmium (Cd). Both MTs were produced as recombinant proteins and showed identical Cd content and similar Cd-binding properties. Conversely, circular dichroism disclosed marked differences in the secondary conformations of the two Cd(7)-MTs. The possible relapses of these structural differences on protein stability and function were assessed. MT-10 presented a higher thermal stability and a more compact structure than MT-20, as it was inferred by absorption and emission spectroscopy studies. Moreover, the kinetics of Cd-release clearly indicated that MT-10 is much more sensitive to oxidation than is MT-20. The observed differences between MT-10 and MT-20 are discussed in terms of the different physiological roles exerted by the two isoforms in mussel.