Zn Metallothionein Research Articles

Sensitivity and reliability of zinc transporter and metallothionein gene expression in peripheral blood mononuclear cells as indicators of zinc status: responses toex vivozinc exposure and habitual zinc intake in humans

Zn is an essential nutrient for humans; however, a sensitive biomarker to assess Zn status has not been identified. The objective of this study was to determine the reliability and sensitivity of Zn transporter and metallothionein (MT) genes in peripheral blood mononuclear cells (PBMCs) to Zn exposure ex vivo and to habitual Zn intake in human subjects. In study 1, human PBMCs were cultured for 24 h with 0-50 µm ZnSO4 with or without 5 µm N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and mRNA expression of SLC30A1-10, SLC39A1-14, MT1 subtypes (A, B, E, F, G, H, L, M and X), MT2A, MT3 and MT4 mRNA was determined. In study 2, fifty-four healthy male and female volunteers (31·9 (sd 13·8) years, BMI 25·7 (sd 2·9) kg/m2) completed a FFQ, blood was collected, PBMCs were isolated and mRNA expression of selected Zn transporters and MT isoforms was determined. Study 1: MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, MT1X, MT2A and SLC30A1 increased with increasing concentrations of Zn and declined with the addition of TPEN. Study 2: Average daily Zn intake was 16·0 (sd 5·3) mg/d (range: 9-31 mg/d), and plasma Zn concentrations were 15·5 (SD 2·8) μmol/l (range 11-23 μmol/l). PBMC MT2A was positively correlated with dietary Zn intake (r 0·306, P = 0·03) and total Zn intake (r 0·382, P < 0·01), whereas plasma Zn was not (P > 0·05 for both). Findings suggest that MT2A mRNA in PBMCs reflects dietary Zn intake in healthy adults and may be a component in determining Zn status.

Evaluation of Zn Bioavailability and Metallothionein Expression by RT-PCR in Zinc Sulphate and Zinc Proteinate Fed Chickens

Evaluation of Zn Bioavailability and Metallothionein Expression by RT-PCR in Zinc Sulphate and Zinc Proteinate Fed Chickens

Quantitative distribution of Zn, Fe and Cu in the human lens and study of the Zn–metallothionein redox system in cultured lens epithelial cells by elemental MS

Study of metals and metallothioneins in the human lens by the multidisciplinary combination of MS and bio-analytical methodologies.

Relative bioavailability of tribasic zinc sulfate for broilers fed a conventional corn-soybean meal diet

An experiment was carried out to investigate the relative bioavailability of tribasic zinc (Zn) sulfate relative to Zn sulfate for broilers fed a conventional corn-soybean meal diet. A total of 504 1-d-old Arbor Acres commercial male chicks were randomly assigned by body weight of birds to one of seven treatments involving a 2×3 factorial arrangement with three levels of added Zn (30, 60, or 90 mg of Zn kg−1) and two Zn sources (tribasic Zn sulfate and Zn sulfate) plus a control with no added Zn for an experimental phase of 14 d. Plasma Zn, tibia ash Zn, pancreas Zn, and pancreas metallothionein (MT) messenger RNA (mRNA) were analyzed at 6 or 14 d of age post-hatching. The results showed that plasma Zn, tibia ash Zn, pancreas Zn, and pancreas MT mRNA increased linearly (P<0.002) as dietary Zn concentration increased at 6 or 14 d of age. The R2 for a linear model was greater on d 6 than on d 14 for the above 4 responsive criteria, and among these indices, the fitting of the tibia ash Zn concentration was the best (R2=0.99). Therefore, based on slope ratios from the multiple linear regressions of the above 4 indices of the birds at 6 d of age on daily intake of dietary Zn, the bioavailabilities of tribasic Zn sulfate relative to Zn sulfate (100%) were 95.6% (P=0.18), 83.5% (P=0.01), 87.9% (P=0.53), and 75.9% (P=0.38) for the tibia ash Zn, pancreas Zn, plasma Zn, and pancreas MT mRNA, respectively. The results indicated that generally, Zn from tribasic Zn sulfate was as available as Zn from Zn sulfate for broilers.

Influence of formula versus sow milk feeding on trace element status and expression of zinc-related genes in the jejunum, liver and pancreas of neonatal piglets

Increasing litter sizes in modern swine production have raised an urgent need for artificial rearing strategies and formula feeding. The current experiment was conducted to study the influence of formula trace element concentration according to recommendations for weaned piglets on the mRNA concentration of zinc (Zn)-related genes in the jejunum, liver and pancreas of neonatal piglets. Eight artificially reared piglets were fed a cow-milk-based formula (Group FO) containing 100 mg Zn/kg dry matter. Eight of their sow-reared littermates (Group SM) were used as control. After 14 d, all 16 piglets were killed and the jejunum, liver and pancreas were evaluated for Zn, copper, manganese (Mn) and iron (Fe) concentration and mRNA concentration of metal and Zn-specific transporters, metallothioneins (MT) and interleukin 6 (IL-6). In Group FO the Zn concentration in liver tissue was increased (p < 0.05). Furthermore, Fe and Mn concentrations in liver and jejunal tissue were higher (p < 0.05) in Group FO, whereas neither Zn transporters nor MT in jejunal and pancreatic tissue showed differences between both groups. In the liver of Group FO, MT mRNA concentration was higher (p < 0.05), whereas Zn transporter protein 1 and divalent metal-ion transporter 1 (DMT1) mRNA concentration was lower (p < 0.05). Besides Zn-induced expression of transporters and MT, the significantly increased IL-6 expression in Group FO suggests the involvement of cytokine-mediated Mn and Fe sequestration in the liver and jejunum. The results revealed that dietary trace element concentration used in the study likely exceeded the requirements of neonatal pigs as reflected by homeostatic counter-regulation in different organs.

Relative bioavailability of zinc-methionine chelate for broilers fed a conventional corn-soybean meal diet.

An experiment was carried out to determine the bioavailability of the organic Zn-methionine chelate relative to inorganic Zn source (ZnSO4•7H2O) for broiler chicks fed a conventional corn-soybean meal diet. A total of 504 1-day-old Arbor Acres commercial male broiler chicks were randomly allotted to one of seven treatments in a completely randomized design involving a 2 × 3 factorial arrangement with three levels of added Zn (30, 60, or 90 mg of Zn/kg) and two Zn sources (Zn-methionine chelate and Zn sulfate) plus a Zn-unsupplemented control diet containing 29.2 mg of Zn/kg by analysis for an experimental phase of 21 days. Bone and pancreas were collected for testing Zn concentrations and pancreas metallothionein (MT) messenger RNA (mRNA) level at 7 or 21 days of age. The results showed that bone and pancreas Zn concentrations and MT mRNA level in pancreas increased linearly (P < 0.0001) at 7 or 21 days of age as added Zn level increased. Based on slope ratios from multiple linear regressions of the pancreas, MT mRNA level at 7 days and pancreas Zn concentration at 21 days on added Zn level and the bioavailability values of the Zn-methionine chelate relative to ZnSO4•7H2O (100%) were 120 and 115%, respectively (P > 0.22). The results indicated that the Zn from the Zn-methionine chelate was just as bioavailable as the Zn from Zn sulfate for broilers.

Metallothionein Zn2+- and Cu2+-clusters from first-principles calculations

Detailed electronic structures of Zn(II) and Cu(II) clusters from metallothioneins (MT) have been obtained using density functional theory (DFT), in order to investigate how oxidative stress-caused Cu(II) intermediates affect Zn-binding to MT and cooperatively lead to Cu(I)MT. The inferred accuracy is ∼0.02-0.03 Å for metal-thiolate bond lengths for the models that are the most realistic MT models so far studied by DFT. We find terminal Zn-S and Cu-S bond lengths of 2.35-2.38 Å and 2.30-2.34 Å, whereas bridging M-S bonds are 0.05-0.11 Å longer. This electronic effect is also reflected in changes in electron density on bridging sulfurs. Various imposed backbone constraints quantify the sensitivity of cluster electronic structure towards protein conformational changes. The large negative charge densities of the clusters are central to MT function, and the smaller β-clusters are more prone to modification. Oxidative stress-associated Cu(II) binding weakens the Zn-S bonds and is thus likely to impair the Zn(II) transfer function of MTs, providing a mechanism for cooperative Cu(II) binding leading to loss of Zn(II) and dysfunctional Cu(I)MT clusters.

Evaluation of metallothionein formation as a proxy for zinc absorption in an in vitro digestion/Caco-2 cell culture model

Caco-2 cell metallothionein (MT) formation was studied to determine if MT could be used as a proxy for zinc (Zn) absorption in a cell culture model. The MT intracellular concentration was determined using a cadmium/hemoglobin affinity assay. The cellular Zn uptake was determined by acid digests (5% HNO(3)) using inductively-coupled argon-plasma emission spectroscopy. The effect of phytic acid (PA) on cellular Zn and MT concentrations was also studied. Cells were treated with a media containing 0, 2, 5, 10, 25, 50, 75 μmol L(-1) Zn (ZnCl(2)). The effect of varying the Zn:PA molar ratios (1:0, 1:1, 1:5, 1:10, 1:20) on the Zn uptake and MT formation was determined. The results showed a positive linear correlation between Zn-media concentrations and cellular Zn uptake, and MT formation was observed. Zn and MT concentrations in the cells treated with increasing levels of Zn (>25 μmol L(-1) Zn) were elevated. The Zn and MT concentrations in the cells incubated with Zn (when <10 μmol L(-1)) were similar to the untreated cells. PA significantly lowered the cellular Zn and MT concentrations. When the Zn:PA molar ratios were >1:5, cellular MT concentrations were no different to untreated cells. When a combined in vitro digestion/cell model was used, the cellular MT concentrations in white or red beans and fish samples were no different to the cell baseline. This study suggests that measurements of cellular Zn and MT concentrations have some limitations (<10 μmol L(-1) Zn). PA was observed to be a potent inhibitor of Zn uptake. Under the conditions of this in vitro model, Caco-2 cell monolayers are not useful for evaluating the Zn availability from foods.

Effects of redox conditions and zinc(II) ions on metallothionein aggregation revealed by chip capillary electrophoresis

Metallothioneins (MTs) belong to cysteine-rich proteins with unique higher structure. One of the most known MT's functions is metals detoxification and maintaining their homeostasis in a cell. Structure of MT with naturally occurred zinc(II) ions can be affected by concentration of metal ions as well as redox milieu inside a cell, however the exact explanation and biochemical effects of the structural changes are still missing. In this study we used capillary electrophoresis on chip coupled with fluorescence detection to determine structural changes of MT with increasing concentration of zinc(II) ions and under various redox conditions. To investigate the structural-dependent effects, reduced and/or oxidized apo-MT (MT without natural occurred metal ion) was prepared. Zinc binding into reduced and/or oxidized apo-MT was compared. MT was incubated with 0, 5, 15, 25, 50 and 100 μM ZnCl 2 for 1 h in 37 °C. Formation of MT aggregates with increasing zinc concentration was observed by spectrophotometry, chip capillary electrophoresis, and SDS-PAGE. We found out that reduced MT forms aggregates more readily compared to oxidized MT. Using the chip capillary electrophoresis allowed us relative quantification of MT aggregation as a decrease in the area of the signal corresponding to the monomer form of MT (Mw 15 kDa, migration time 26.5 s) and its ratio to total signal (sum of all signals measured by the electrophoresis). The dependences had an exponential character with equation y = 2.4 × e −0.01 x , R 2 = 0.945 for 15 kDa peak area and y = 0.11 × e −0.01 x , R 2 = 0.938 for decrease of 15 kDa peak area ratio to the total signal. Zn–MT interaction was 30% faster during the first 15 min and 50% faster during the whole experiment for reduced MT. It can be concluded that formation of MT aggregates is dependent on redox state and Zn(II) concentration.

Preparation and functional analysis of recombinant protein transduction domain-metallothionein fusion proteins

In order for proteins to be used as pharmaceuticals, delivery technologies need to be developed to overcome biochemical and anatomical barriers to protein drug transport, to protect proteins from systemic degradation, and to target the drug action to specific sites. Protein transduction domains (PTDs) are used for the non-specific transduction of bio-active cargo, such as proteins, genes, and particles, through cellular membranes to overcome biological barriers. Metallothionein (MT) is a low molecular weight intra-cellular protein that consists of 61 amino acids, including 20 cysteine residues, and is over-expressed under stressful conditions. Although MT has the potential to improve the viability of islet cells and cardiomyocytes by inhibiting diabetic-induced apoptosis and by removing reactive oxygen species (ROS), and thereby prevent or reduce diabetes and diabetic complications, all MT applications have been made for gene therapy or under induced over-expression of endogenous MT. To overcome the drawbacks of ineffective intra-cellular MT protein uptake, a human MT gene was cloned and fused with protein transduction domains (PTDs), such as HIV-1 Tat and undeca-arginine, in a bacterial expression vector to produce PTD–MT fusion proteins. The expression and purification of three types of proteins were optimized by adding Zn ions to maintain their stability and functionality mimicking intra-cellular stable conformation of MT as a Zn–MT cluster. The Zn–MT cluster showed better stability than MT in vitro. PTD–MT fusion proteins strongly protected Ins-1 beta cells against oxidative stress and apoptosis induced by glucolipotoxicity with or without hypoxia, and also protected H9c2 cardiomyocytes against hyperglycemia-induced apoptosis with or without hypoxia. PTD–MT recombinant fusion proteins may be useful protein therapeutics for the treatment or prevention of diabetes and diabetes-related complications.

Interaction of dietary zinc and intracellular binding protein metallothionein in postnatal bone growth

Zinc and its binding protein, metallothionein (MT), are important in regulating growth and development, and yet it is unclear how dietary Zn and MT interact in regulating bone growth. Here, 3.5-week female MT-I&II knockout (MT −/−) and wild type (MT +/+) mice were fed diets containing 2.5 (limiting, Zn-L), 15 or 50 mg Zn/kg (Zn adequate) for 5 or 9 weeks, and effects were analysed on structure and function of growth plate and metaphysis, two structures important for bone growth. Zn limitation did not affect bone growth in MT +/+ mice. However, MT −/− mice, having lower Zn concentrations in plasma and long bone, showed growth retardation as demonstrated by lower body length gain, shorter and smaller tibia/femur, lower chondrocyte proliferation, reduced metaphysis heights, but increased osteoclast densities on trabecular bone, particularly in mice fed Zn-L diet. Interestingly, mRNA expression of MT-I&II was induced in the growth plate of MT +/+ mice fed the Zn-L diet possibly compensating for Zn limitation. Growth plate MT-III expression increased in MT −/− mice fed the adequate Zn diet, whereas metaphyseal MT-III was significantly upregulated in MT −/− mice fed Zn-L diet, possibly as a compensatory mechanism or exacerbating effects of Zn limitation. Consistent with the increased osteoclast numbers, a higher ratio of RANKL/OPG gene expression was found in bone of mutant mice fed lower Zn diets. These results indicate that interaction between dietary Zn and endogenous MT is important for maximal bone growth, and MT is particularly important in the regulation of Zn pool for bone growth during moderate Zn limitation.

Cloning, expression and characterization of metallothionein from the Antarctic clam Laternula elliptica

The genes for two apparent subtypes of metallothionein (MT) isoform were isolated from the Antarctic clam Laternula elliptica. Determination of the nucleotide sequence showed that the gene consists of 222 bp that code a 73-amino acid protein. The comparison between MT cDNA sequences of L. elliptica and other bivalves showed strong homologies on positions of cysteine residues, which are important for their metal binding abilities. The gene for the MT was inserted into a pET vector and overexpressed as a carboxyl terminal extension of glutathionein- S-transferase (GST) in Escherichia coli. After the GST fusion proteins had been purified by glutathione–Sepharose affinity chromatography column and digested with enterokinase, the MT was purified with gel filtration and analyzed for its biochemical properties. Recombinant MTs were reconstituted with Cd, Cu, and Zn, and kinetic studies of the reactions with electrophilic disulphide, DTNB, were investigated to explore their metal binding ability. It is revealed that the Cd–MT and Zn–MT react with DTNB biphasically, and that Zn–MT reacts with DTNB more rapidly, and with a significantly greater pseudo-first-order rate constant. Cu–MT reacts monophasically and releases metal slowly from MT.

Constant Current Stripping Chronopotentiometry for the Study of Adsorbing Inert and Electrochemically Nonreversible Metal Complexes at Low Concentrations: Application to Cd and Zn Metallothioneins

Constant current chronopotentiometric stripping analysis using adsorptive accumulation and negative stripping current (AdSCP) was applied for the study of behavior of rabbit liver Cd-Zn and Zn metallothionein (Cd-Zn-MT, ZnMT) on hanging mercury drop electrode. Electrochemically inert or labile behavior of complexes can be distinguished with the application of high (1000 nA) or low (100 to 20 nA) current. Using high current, no influence of added Cd2+ or Zn2+ ions on the reduction of Cd(II) or Zn(II) complexed within MT molecule was observed, except of additions of Cd2+ to ZnMT, where bound Zn(II) was substituted by cadmium ions. With lowering of stripping current and increasing concentration of added Cd2+ or Zn2+ ions in solution progressive formation of reorganized complex with labile behavior is observed. Parallel measurement using DC voltammetry with different rates of polarization or differential pulse voltammetry were in agreement with AdSCP measurement. However, only chronopotentiometric method combines good sensitivity and signal separation at μM concentrations, inevitable in MT studies.

Dietary Pharmacological or Excess Zinc and Phytase Effects on Tissue Mineral Concentrations, Metallothionein, and Apparent Mineral Retention in the Newly Weaned Pig

Feeding pharmacological zinc (Zn) to weaned pigs improves growth, and dietary phytase improves P and Zn availability. Metallothionein (MT) increases in the duodenum, kidney, and liver of pigs fed 1000 mg Zn/kg with phytase or 2000 mg Zn/kg with or without phytase when fed for 14 d postweaning. The goal of this study was to determine the effects of feeding pharmacological Zn and phytase on tissue minerals, MT, mineral excretion, and apparent retention. Twenty-four newly weaned pigs (20 d; 7.2 kg) were individually fed twice daily, a basal diet supplemented with 0, 1000, or 4000 mg Zn/kg as Zn oxide, without or with phytase (500 phytase units [FTU]/kg) for 14 d, followed by a basal diet (100 mg Zn/kg) without phytase for 7 d. Pigs fed 4000 mg Zn/kg without phytase had higher (p=0.01) plasma, hepatic, renal Zn, renal Cu, and hepatic, renal, and jejunal MT than pigs fed the basal diet or 1000 mg Zn/kg. Duodenal MT was higher (p=0.0001) in pigs fed 1000 and 4000 mg Zn/kg than in pigs fed the basal diet. In pigs fed 1000 and 4000 mg Zn/kg, Zn loading occurred during the first 11 d of supplementation; by d 14, excess Zn was being excreted in the feces.

The role of metallothionein and zinc in hepatic copper accumulation in cattle

The role of metallothionein (MT) and zinc (Zn) in hepatic copper (Cu) accumulation in calves from a region in North–West Spain has been investigated. In this region there is intensive pig farming, and animals with liver Cu concentrations above normal are not uncommon. Concentrations of hepatic MT were not related to Cu accumulation but were strongly dependent on the Zn status of the animal. When analysing the metal content bound to MT it was observed that Cu–MT values, in the same way as Zn–MT, were directly correlated with MT concentrations in the liver, indicating that although Cu is a poor inducer of MT synthesis, it can compete with Zn for MT binding sites. The ability of Cu to displace Zn from MT is highly dependent on the Cu:Zn ratio in the cell, Cu being the main metal in MT at the higher levels of Cu exposure in Galician cattle. In spite of this, the percentage of the total hepatic Cu bound to MT is very low in these animals, indicating that cattle have a very limited capacity to accumulate Cu–MT in the liver, and may therefore have a greater susceptibility to Cu toxicosis.

Direct evidence for co‐binding of cisplatin and cadmium to a native zinc‐ and cadmium‐containing metallothionein

AbstractCisplatin is widely used to treat a number of cancers, and its covalent binding to DNA is believed to cause cell death; however, the roles of cisplatin–protein interactions in the mechanisms of action, toxicity, and resistance of the drug largely remain to be elucidated. Here, we investigate the interactions of cisplatin and a native rabbit metallothionein (MT), containing 1.4% zinc and 7.9% cadmium, using nanospray tandem quadrupole time‐of‐flight mass spectrometry (MS) and size‐exclusion high‐performance liquid chromatography with inductively coupled plasma MS. At near‐neutral pH conditions, reactions between cisplatin and MT resulted in the formation of complexes that contained Cd4–Ptn–MT (n = 1–7). While zinc was displaced by cisplatin, both platinum and cadmium were bound to the same MT molecule. This is the first report to provide direct evidence for the co‐binding of cadmium and platinum to MT, which suggests that the mechanism of the binding of cisplatin to the native MT may not be through the displacement of cadmium as previously proposed. A tandem MS investigation into the binding sites of the platinum and cadmium to MT showed platinum‐ and cadmium‐related fragments, such as (PtS2C2H7N)+ and (CdS3C5H17N2)+, demonstrating the platinum–cysteine and cadmium–cysteine binding. In addition, detection of Cd4–Pt7–MT demonstrated more than ten metals bound to a single MT molecule. This finding was extended to the binding of MT with a five‐fold excess of CdCl2. As many as 14 metal atoms (13 cadmium and one zinc) were detected bound to a single MT molecule, the complexes being Cdx–Zn–MT (x = 5–13). The high binding capacity of MT for cadmium and platinum is consistent with the role of MT in reduction of metal toxicity and its involvement in drug resistance. Copyright © 2003 John Wiley &amp; Sons, Ltd.

Roles of metallothionein in copper homeostasis: responses to Cu-deficient diets in mice

Metallothionein (MT) protects the body from both harmful non-essential and excessive essential metals. Copper (Cu) is an essential metal, and its concentration in the body is regulated at a constant level between excess and deficient ones. Cu accumulating in the livers of Wilson disease patients and its animal model, Long-Evans rats with a cinnamon-like coat color (LEC) rats, is in the form of Cu,Zn–MT, MT being an antioxidant. Contrary to the efficient production of MT in response to excessive accumulation of Cu in LEC rats, Cu-binding to MT only occurs marginally under normal conditions. However, the present study revealed that Cu binds to MT more with a severe Cu-deficiency. Namely, male C57BL/6J mice were fed a Cu-deficient diet (0.037 mg Cu/g) and deionized water containing trientine, and then the concentration and distribution of Cu were determined. It was suggested that the cessation of biliary excretion and limitation of the Cu supply to ceruloplasmin are the first responses on feeding of a Cu-deficient diet, followed by an increase in Cu–MT with maintenance of the Cu concentration in the liver. These results suggest that MT causes the recruitment of Cu in a Cu-deficient environment by sequestering Cu from degraded Cu-enzymes and delivering it to Cu chaperones.

Characterisation of zinc metallothioneins by electroanalytical techniques

The electrochemical behaviour of zinc metallothionein (MT) from commercially available rabbit liver, with respect to solution pH as well as the influence of the addition of cadmium and/or zinc was studied using differential pulse polarography and square wave voltammetry. This Zn–MT has an extremely low content of cadmium in the molecule. The complexation equilibrium of dissociation and the formation from their depleted form was investigated, going from basic to acid solution and vice versa. Results obtained allow one to conclude that the equilibrium of complexation is reversible in the pH range studied. Three different responses due to the Zn(II) reduction were distinguished, with a better resolution over the basic pH value range indicating different forms of complexation of zinc with the thionein. Additions of zinc to the MT do not provoke changes in the features of voltammograms indicating that the zinc, if it is incorporated in the molecule, does not modify its structure. On the contrary, the additions of cadmium, which is quickly incorporated into the molecule, provoke a clear reorganisation of its structure affecting not only peaks of the Cd(II) reduction attributed to two forms of complexation of cadmium–thionein, but also changes in the co-ordination of zinc complexes. The pH plays an important role in the stability of the different sorts of cadmium and zinc complexes. Voltammetric peaks attributed to different species are interrelated.

Complexation Properties of the Metallothionein Fragment Lys-Cys-Thr-Cys-Cys-Ala [56-61] MT I with Zinc and Cadmium Using Square Wave Voltammetry

An electrochemical study using square wave voltammetry of the complexing properties of the peptide Lys-Cys-Thr-Cys-Cys-Ala [56–61] MT 1 (FT), a part of the metallothionein structure, with cadmium and zinc was performed. The applied technique allows one to obtain well-defined peaks corresponding to the free metal ions as well as providing evidence that different forms of coordination of Cd(II) and Zn(II) with the peptide exist. FT is capable of complexing Cd2+and Zn2+ separately in a concentration equal to FT, obtaining complexes with a ratio of 1:1. However, the total metal ion concentration bound to the FT is higher when both cations are simultaneously present. The maximum total concentration that can be complexed with FT is a ratio of [Zn]:[Cd]:[FT] like 0.6:1.0:1.0 which leads to the ratio of Zn4Cd7S21, comparable to that of the metallothionein structure. The coordination of Cd(II) with FT not only depends on the Cd:FT ratio, but also on the proportion in which Zn(II) is added. However, the Zn(II) complexes are not affected by the presence of Cd-FT complexes in solution. The complexation equilibrium was investigated going from acid to basic solutions and viceversa. From the slopes of the variation Ep=f (pH), the number of thiol groups at which the cations are bound were evaluated. This study allows us to enable comparisons between the behavior of [Zn]:[Cd]:[FT] with that of Cd,Zn metallothioneins.

Binding properties and stoichiometries of a palladium(II) complex to metallothioneins in vivo and in vitro

This paper will be the first to discuss the in vivo and in vitro properties of a Pd(II) complex, K 2PdCl 4, interacting with metallothioneins (MTs). In vivo experiments revealed that intraperitoneal injections of K 2PdCl 4 into rabbits led to the simultaneous synthesis of Pd–MT in the kidney and Zn 7MT in the liver. The renal Pd–MT complex contains 3.6 ± 0.3 Pd, 2.1 ± 0.2 Zn, and 1.0 ± 0.1 Cu per mole protein. It was found that pre-treatment with Zn(NO 3) 2 before K 2PdCl 4 injections significantly enhanced renal Pd–MT level. The same pre-treatment also increases hepatic Zn–MT levels. These results strongly suggest that Pd(II) ions can be bound in vivo by MT existing in the rabbit kidneys to form Pd–MT. Gel-filtration chromatographic studies after the incubation of either native Cd 5Zn 2MT2 or Zn 7MT2 with K 2PdCl 4 in vitro demonstrate that Pd(II) ions promote the non-oxidative oligomerization of native MTs. Increasing the level of Pd(II) relative to MT led to a concomitant increase in the apparent yield of MT oligomers. At relatively low Pd–MT ratio, Pd(II) is found predominantly in the oligomers while the monomeric products are chiefly composed of the reactants, Cd 5Zn 2MT2 or Zn 7MT2. Based on our experimental data, the mechanisms of the reactions between Pd(II) and MTs in vivo and in vitro are discussed.