Interaction of mercury(II) ions with immobilized apo-metallothioneins studied by scanning electrochemical microscopy combined with surface plasmon resonance

Scanning electrochemical microscopy (SECM) was combined with surface plasmon resonance (SPR) and applied for in-situ monitoring of the incorporation of Hg²⁺ by apo-metallothionein (apo-MT) immobilized on the SPR substrate. Hg²⁺ was anodically stripped from the Hg-coated SECM Pt tip and sequestered by apo-MT upon its diffusion to the SPR substrate. The high sensitivity of the SPR instrument enabled the detection of the change in the composition and structure of apo-MT molecules that was induced by the metal sequestration of Hg²⁺. The SPR response revealed that the saturation co-ordination number of Hg²⁺ binding to apo-MT was 18. Moreover, an unexpected collapse of the structure of MT was observed when the stoichiometric ratio of Hg²⁺/MT was ~70, and the structure cannot be further altered even by adding a large excess of Hg²⁺. This collapse was also confirmed by Raman spectroscopy. The results are potentially useful for a deeper understanding of the detoxification mechanism of MT to mercury ion.

一种新型结构的金属硫蛋白Pb-MT

金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎｓ,简称ＭＴｓ）是一大类富含巯基的小分子蛋白质（分子量＜９０００）,具有很强的金属结合能力,哺乳动物ＭＴ每分子可结合７个二价金属离子或１２个一价金属离子．ＭＴ的结构一直是科学家关注的热点,其中哺乳动物Ｃｄ７－Ｍ...     

过渡金属与新型硝基酚臂式氮杂18-冠-6配位性能的研究

合成并表征了1,10-二氧-4,7,13,16-四氮杂18-冠-6 (1)母体及其四取代硝基酚臂式衍生物 (2)。在H₂O-DMSO(V/V=1/4)混合溶剂中用UV-Vis光谱法对冠醚2与H⁺、Mn²⁺、Co²⁺、Ni²⁺、Cu²⁺、Zn²⁺、Cd²⁺和Hg^{2+相似文献}   

A highly selective ratiometric fluorescent chemosensor for Zn2+ ion based on a polyimine macrocycle

A new ratiometric fluorescent chemosensor based on a polyimine macrocycle ligand 1 has been synthesized. The chemosensor can exhibit a pronounced fluorescence response and high selectivity to Zn²⁺ ion over other 15 metal ions, including Cd²⁺. Sensor 1 appears an emission peak at 370 nm. Upon the addition of Zn²⁺ ion, the typical emission peak for 1 at 370 nm is obviously quenched, but a new emission peak at around 470 nm appears and shows a large enhancement due to the formation of a 1:1 Zn²⁺-1 complex. In addition, there is a good linear relationship between the fluorescence ratio I_470nm/I_370nm and the concentration of Zn²⁺, which makes a ratiometric assay of Zn²⁺ ion possible.     

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 ZnCl₂ 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^−0.01x, R² = 0.945 for 15 kDa peak area and y = 0.11 × e^−0.01x, R² = 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.     

Physico-chemical characterization of metal binding proteins using HPLC-ICP-MS, HPLC-MA-AAS and electrospray-MS

A size exclusion HPLC method was developed and interfaced with ICP-MS detection for determining the metal profiles of commercially available rabbit liver metallothioneins (MT) and metallothionein-like proteins (MLP) extracted from fresh water mussels and hemolyzed osprey blood. The redox state of the cysteine residues was indirectly evaluated via a cadmium saturation approach in the presence or absence of a reducing agent, followed by HPLC-microatomization (MA)-AAS and HPLC-ICP-MS analyses. An electrospray-MS protocol was also developed to accurately measure the molecular weight of rabbit MT isoform II. Nanogram quantities of Cd-MT/MLP were poorly chromatographed on silica based supports. A copolymeric styrene-divinylbenzene size exclusion support provided a symmetrical peak (rabbit MT standard) and linear HPLC-MA-AAS calibration curves [r=0.9988; from the LOD (27 ng, as protein) to about 300×LOD], indicating negligible losses of Cd during the chromatography of trace quantities. Co-injection of Cd²⁺ saturated samples with beta-mercaptoethanol (BMSH) was essential to repress Cd²⁺-support interactions which otherwise induced an undesirable metalaffinity retention mechanism. In the presence of added Cd²⁺, 22 mmol/L BMSH did not significantly compete for Cd²⁺ specifically bound to MT, while preventing non-specific binding to non-thiolic complexing sites. Crude mussel and osprey blood MLP extracts (in cold, deoxygenated Tris-HCl buffer) were obtained by ultracentrifugation (145,000 g) and thermocoagulation/centrifugation, respectively. Incubation with BMSH was prerequisite to obtain a maximum saturation of mussel and osprey blood MLP by Cd²⁺, even for samples conserved (–80° C) in the presence of BMSH (22 mmol/L). These observations indicated that a major proportion of the cysteine residues present in these MLP were oxidized. The assumption of a fully reduced MT/MLP pool binding metals in a definite stoichiometry has been the basis of several quantitative metal binding assays involving the saturation of the thiolic complexing sites with a metallic marker (Ag+, Cd²⁺, or Hg²⁺). Since thiolic agents may interfere, the metal saturation protocols do not include a reducing step to ensure that all cysteines in a MT/MLP extract are available for co-ordination. Given that variations in the redox state of crude MT/MLP extracts may compromise the accuracy of metal saturation assays, it is concluded that the preparation of reference samples certified for total metallothionein content would be desirable.     

Electrochemical methods for quantification and characterization of metallothioneins induced in Mytilus galloprovincialis

The quantification of the purified metallothionein (MT) component, isolated from the digestive gland of cadmium-exposed Mytilus galloprovincialis, is described based on the analysis of Cd(II) and SH-groups content, applying electrochemical methods. Advantages and disadvantages of the Brdika procedure for the determination of the MT content is discussed. The saturation of binding positions of purified MT with Cd²⁺ ions can be directly followed voltammetrically. Irrespective of the MT concentration, the saturation with Cd²⁺ of in vivo induced mussel MT is achieved at a molar ratio of 5. Cd²⁺ ions are rapidly displaced from the Cd-Th complex after the addition of Pb²⁺ ions, which indicates the kinetically labile type of the complex.     

Simple imine linked colorimetric and fluorescent receptor for sensing Zn ions in aqueous medium based on inhibition of ESIPT mechanism

Herein, we report the highly selective binding of Zn²⁺ ion by the salicylaldimine based Schiff base chromogenic receptor 1 [(N,N′-bis (salicylidine)-o-phenylenediamine]. Receptor 1 senses Zn²⁺ ion in aqueous medium by colorimetric and fluorescent response in the presence of other metal ions like Pb²⁺, Hg²⁺, Sn²⁺, Cd²⁺. Receptor 1 on binding with Zn²⁺ ion exhibits fluorescence enhancement which is due to the inhibition of the (ESIPT) mechanism.     

Photo- and ionochromic properties of new spirobenzochromene-pyranoquinoline

A compound of the new spirobipyran-quinoline series possesses weakly pronounced photochromic properties at room temperature. Its interaction with Co²⁺, Ni²⁺, Zn²⁺ and Cd²⁺ ions leads to intensely colored complexes absorbing in the λ> 600 nm region, which is important for the study of biological objects. Up to three merocyanine molecules are capable of binding one metal ion, which is not typical for the previously studied indoline analogs.     

Cadmium-113 NMR and Theoretical Studies of Complexation of Cadmium Ion with 15-Crown-5 and Benzo-15-Crown-5 in Acetonitrile and Its Binary Mixtures with Water and Nitromethane

Cadmium-113 nuclear magnetic resonance was used as a sensitive probe to study the interaction of Cd²⁺ ion with 15-crown-5 and benzo-15-crown-5 in acetonitrile and its binary mixtures with water and nitromethane. The observed ¹¹³Cd-NMR chemical shift changes at a constant Cd²⁺ ion concentration and varying crown concentrations were found to be consistent with a fast exchange model. The formation constants of the resulting 1:1 complexes were evaluated from computer fitting of the chemical shift-mole ration data to an equation which relates the observed chemical shifts to the formation constant. The geometries of the macrocyclic ligands and their cadmium complexes were optimized by an ab initio method, and the calculated binding energies of the resulting complexes were compared. Both the ¹¹³Cd-NMR and ab initio studies revealed that, in all cases, 15-crown-5 forms a more stable Cd²⁺ complex than benzo-15-crown-5. In the case of the both crown ethers, there is an inverse relationship between the stability of complexes and solvating ability of the solvent systems used.     

Zn selective luminescent ‘off-on’ probes derived from diaryl oxadiazole and aza-15-crown-5

New photo-induced electron transfer (PET) probes OMOX and OBOX, carrying an additional binding site in the form of ‘oxadiazole nitrogen’ have been designed to evaluate binding interactions with biologically significant Li⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, and Zn²⁺ including environmentally toxic Ba²⁺ and Cd²⁺ using optical spectral techniques. While Li⁺, Na⁺, and K⁺ did not appreciably perturb either the absorption or emission spectra, Ba²⁺, Ca²⁺, Mg²⁺, Zn²⁺, and Cd²⁺ induced slight red shifts (2-8 nm) in the UV-visible spectra as well as pronounced chelation induced enhanced fluorescence (CHEF). Both OMOX and OBOX exhibited the highest CHEF in contact with the zinc ion, whereas Ba²⁺, Ca²⁺, Mg²⁺, and Cd²⁺ induced relatively less emission enhancements. OBOX, which is a poorer emitter (Φ_f=0.0062) than OMOX (Φ_f=0.015), showed highly promising 160-fold emission enhancement in the presence of Zn²⁺. Potential, therefore is available in OBOX to function as a selective luminescent ‘off-on’ sensor for Zn²⁺ in the presence of coordinatively competing Ba²⁺, Ca²⁺, Mg²⁺, and Cd²⁺ ions.     

Cd2+掺杂ZnWO4纳米棒的合成和光致发光性能研究

采用简单的水热法合成了Cd2+掺杂ZnWO4纳米棒.通过SEM、TEM、EDX和XRD等手段对产物进行了表征.实验结果表明,产物为直径约20 nm.长300～500 nm的Cd2+掺杂ZnWO4纳米棒.研究了Cd2+掺杂量对ZnWO4纳米棒的光致发光性能的影响,随着Cd2+掺杂量的增加,ZnWO4纳米棒的光致发光强度随之增强.     

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

Cisplatin 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–Pt_n–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 Cd_x–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 & Sons, Ltd.     

Structural Adaptation of a Protein to Increased Metal Stress: NMR Structure of a Marine Snail Metallothionein with an Additional Domain

In this study, we present an NMR structure of the metallothionein (MT) from the snail Littorina littorea (LlMT) in complex with Cd²⁺. LlMT is capable of binding 9 Zn²⁺ or 9 Cd²⁺ ions. Sequence alignments with other snail MTs revealed that the protein is likely composed of three domains. The study revealed that the protein is divided into three individual domains, each of which folds into a single well‐defined three‐metal cluster. The central α2 and C‐terminal β domains are positioned with a unique relative orientation. Two variants with longer and shorter linkers were investigated, which revealed that specific interdomain contacts only occurred with the wild‐type linker. Moreover, a domain‐swap mutant in which the highly similar α1 and α2 domains were exchanged was structurally almost identical. It is suggested that the expression of a three‐domain MT confers an evolutionary advantage on Littorina littorea in terms of coping with Cd²⁺ stress and adverse environmental conditions.     

Poly(2-acrylamido glycolic acid-co-acryloyl morpholine) and poly(2-acrylamido glycolic acid-co-acrylamide): Synthesis, characterization, and retention properties for environmentally impacting metal ions

Poly(2-acrylamido glycolic acid-co-acrylamide), P(AGA-co-AAm), and poly(2-acrylamido glycolic acid-co-4-acryloylmorpholine), P(AGA-co-AMo), were synthesized by radical polymerization. The water-soluble polymers containing tertiary amine, amide, hydroxyl, and carboxylic acid groups were investigated as polychelatogen, in view of their metal ion binding properties by using the liquid-phase polymer-based retention technique under different experimental conditions. The retention properties for the following metal ions were investigated: Ag⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Al²⁺, Cr³⁺ and Fe³⁺. P(AGA-co-AMo) showed a selective retention for tri-valent cation Al³⁺ at pH 3, but no retention at higher pH. P(AGA-co-AAm) showed the highest metal ion retention capability, specially at pH 5 and pH 7 with values close to 100% to di-valent cations.     

Multiphoton dissociation/ionisation of dimethyl sulphide (CH3SCH3) at 355 and 532 nm

Multiphoton dissociation/ionization has been studied for CH₃SCH₃ at 355 and 532 nm using a time-of-flight mass spectrometer. The major ion signals observed at 355 nm are C⁺, CH₃ ⁺, HCS⁺, CH₂S⁺, CH₃S⁺ and CH₃SCH₃ ⁺. Power dependence studies at 355 nm show a (2+1) REMPI process for the formation of parent ion. Peaks atm/e = 46, 47 and 61 show two-photon laser power dependence whereasm/e = 15 and 45 peaks show four-photon dependence. However, in 532 nm photo-ionisation, no parent ion signal is observed. A peak atm/e = 35 corresponding to SH₃ ⁺ has been observed. SH₃ ⁺ has been suggested to originate from CH₃SCH₂ ⁺ via a cyclic transition state. Photoionisation results of CH₃SCH₃ have been compared with those of CH₃SSCH₃, at these two wavelengths.     

A Highly Selective Two‐Photon Fluorescent Probe for Detection of Cadmium(II) Based on Intramolecular Electron Transfer and its Imaging in Living Cells

A new quinoline‐based probe was designed that shows one‐photon ratiometric and two‐photon off–on changes upon detecting Cd²⁺. It exhibits fluorescence emission at 407 nm originating from quinoline groups in Tris‐HCl (25 mM , pH 7.40), H₂O/EtOH (8:2, v/v). Coordination with Cd²⁺ causes quenching of the emission at 407 nm and simultaneously yields a remarkable redshift of the emission maximum to 500 nm with an isoemissive point at 439 nm owing to an intramolecular charge‐transfer mechanism. Thus, dual‐emission ratiometric measurement with a large redshift (Δλ=93 nm) and significant changes in the ratio (F₅₀₀/F₄₃₉) of the emission intensity (R/R₀ up to 27) is established. Moreover, the sensor H₂L displays excellent selectivity response, high sensitive fluorescence enhancement, and strong binding ability to Cd²⁺. Coordination properties of H₂L towards Cd²⁺ were fully investigated by absorption/fluorescence spectroscopy, which indicated the formation of a 2:1 H₂L/Cd²⁺ complex. All complexes were characterized by X‐ray crystallography, and TD‐DFT calculations were performed to understand the origin of optical selectivity shown by H₂L. Two‐photon fluorescence microscopy experiments have demonstrated that H₂L could be used in live cells for the detection of Cd²⁺.     

Investigation of the uranil(II)—3-Hydroxyflavone complex

Summary By the application of suitable spectrophotometric methods andpH-metric measurements it has been established that uranyl ion and 3-hydroxyflavone form a [UO₂(C₁₅H₉O₃)]⁺ complex. The concentration stability constant of the complex was determined by Bent-French's (₁=8.59 atpH=3.5) and Bjerrum's (from 8.68 atpH=4 to 6.14 atpH=7) methods. Conditions are given for the spectrophotometric determination of 3-hydroxyflavone by means of the complex. The regression equation is calculated and the accuracy of the method is determined. All investigations were carried out with 80% ethanolic solutions at room temperature (20°), the spectrophotometric measurements being performed at a constantpH (3.5) and a constant ionic strength (0.01M).

---

Untersuchung des Uranil(II)—3-Hydroxyflavon-Komplexes

Zusammenfassung Unter der Anwendung von geeigneten spektrophotometrischen Methoden sowiepH-metrischen Messungen wurde gefunden, daß das UO ₂ ²⁺ -Ion und 3-Hydroxyflavon den Komplex [UO₂(C₁₅H₉O₃)]⁺ bilden. Die Stabilitätskonstante des Komplexes wurde nach Bent und French (₁=8.59 beipH=3.5) und nach Bjerrum (von 8.68 beipH=4 bis 6.14 beipH=7) bestimmt. Es werden die Bedingungen für die spektrophotometrische Bestimmung des 3-Hydroxyflavons mit Hilfe des Komplexes angegeben, die Regreßgleichung berechnet und die Genauigkeit der Methode ermittelt. Alle Untersuchungen wurden in 80proz. Ethanol bei Raumtemperatur (20°), die spektrophotometrischen Untersuchungen bei konstantempH-Wert und Ionenstärke (0.01M) durchgeführt.

     

Application of semidifferential electroanalysis to anodic stripping voltammetry

A new voltammetric technique, Semidifferential electroanalysis, in which the semiderivative, e, of the current, i, is measured as a function of electrode potential, has been applied for detection in anodic stripping voltammetry. The semiderivative of the current is defined by [fx131-1.tif] Cd²⁺, Pb²⁺, and Tl⁺ in 0.1 M KNO₃ at different pH values were tested as samples on a hanging mercury drop working electrode. Symmetrical sharp peaks were observed for the re-dissolution processes of metal amalgams formed during pre-electrolysis at -1.0 V vs. SCE. The peak potentials of e vs. E curves for the above three amalgams agreed well with the literature values for d.c. polarographic half-wave potentials. The peak heights were proportional to the pre-electrolysis time up to about 5 min, to the potential scan rate in the range 60–160 mV s^-1, and to the concentrations of Cd²⁺, Pb²⁺, and Tl⁺ in the original solution in the range 10^-6–10^-9 M. The relative standard deviation for the determination of Cd²⁺ was about ±4% at the 2 × 10^-5 M level.