Electrochemical measurements with glucose oxidase immobilized in polyacrylamide gel: Constant current voltametry

Constant current voltametry was shown to be a suitable method for evaluation of electrodes containing immobilized oxidative enzymes as catalysts. The half-cell potential for the oxdidation of D -glucose at constant pH was measured at a series of constant dc currents, using glucose oxidase immobilized in a polyacrylamide gel-platinum gauze matrix as catalyst. Over the range studied the potential versus current data were linear and could be extrapolated to give the open-circuit half-cell potential at zero current. The difference between the measured potential and the open-circuit potential at a constant current was indicative of the loss in useful voltage due to concentration gradients within or adjacent to the enzyme electrode assembly. The relationship between the open-circuit potential and the pH of the reaction mixture was linear with a slope of ?0.056, in good agreement with the predicted slope of ?0.061.     

Electrochemical evaluation of glucose oxidase immobilized by different methods

Glucose oxidase electrodes were constructed on a platinum screen using polyacrylamide gel, glutaraldehyde crosslinking, and glutaraldehyde crosslinking with +0.04 volts dc on the platinum screen as the methods of enzyme immobilization. The electrodes were evaluated in an electrochemical cell for the oxidation of glucose at the enzyme electrode and the reduction of oxygen at a platinum auxiliary electrode, using constant current voltametry or under external load operation. The method of immobilization affected the extrapolated opencircuit potential as well as the half-cell potential and the steady current under external load operation. The charged glutaraldehyde electrode gave the best current performance; however, the small output (microamps) indicated that major problems in electron transfer from an enzyme catalyst to an external circuit must be resolved before such electrodes can be used in practical application.     

Review on Methods for Determination of Metallothioneins in Aquatic Organisms

One aspect of environmental degradation in coastal areas is pollution from toxic metals, which are persistent and are bioaccumulated by marine organisms, with serious public health implications. A conventional monitoring system of environmental metal pollution includes measuring the level of selected metals in the whole organism or in respective organs. However, measuring only the metal content in particular organs does not give information about its effect at the subcellular level. Therefore, the evaluation of biochemical biomarker metallothionein may be useful in assessing metal exposure and the prediction of potential detrimental effects induced by metal contamination. There are some methods for the determination of metallothioneins including spectrophotometric method, electrochemical methods, chromatography, saturation-based methods, immunological methods, electrophoresis, and RT-PCR. In this paper, different methods are discussed briefly and the comparison between them will be presented.     

An electrophoretic profiling method for thiol-rich phytochelatins and metallothioneins

Thiol-rich peptides such as phytochelatins (PCs) and metallothioneins (MTs) are important cellular chelating agents which function in metal detoxification and/or homeostasis. The variations in molecular sizes and lack of chromophores of these peptides make their analysis difficult. This paper reports an electrophoresis-based method for a broad screen of thiol-rich peptides and proteins. The method uses the thiol-selective fluorescent tag, monobromobimane, coupled with Tricine--sodium dodecyl sulphate--urea polyacrylamide gel electrophoresis for a sensitive determination of both PCs and MTs. Results for PCs were confirmed by two-dimensional NMR and HPLC-tandem MS analyses. Sample throughput is substantially improved over chromatography-based methods through parallel sample analysis in 1 h of electrophoretic separation. The method is versatile in that peptides ranging from glutathione to large proteins can be analysed by simple modification(s) of the extraction and electrophoretic conditions, and the nature of the method supports serendipitous detection of unexpected or novel thiol metabolites.     

金属结合蛋白基因及其在清除重金属污染中的应用

一些微生物和植物由于对毒性金属具有独特的抗性机制,使得利用它们来清除日益严重的环境污染已发展成为一种十分有效的技术——生物修复。研究表明,不同的金属结合蛋白（如MT 和PC）,在生物忍耐和降解过量重金属毒性机制中起重要作用。愈来愈多的MT 和PC基因被克隆,并已成功地应用于生物遗传转化,这些转基因生物在清除重金属污染方面已显示出潜在的应用价值。 Abstract:Heavy metal pollution has become a global environmental hazard.The use of microorganisms and plants for the decontamination of heavy metals is recognized as a low lost and high efficiency method for cleaning up metal contamination.It shows that various metal-binding proteins such as metallothioneins (MTs) or phytochelatines (PCs) play an important role in defense systems and detoxification to heavy metals in organisms.Many genes of MTs and PCs have been cloned and utilized successfully in genetically modified bacteria and plants for increasing remediation capacity.These transgenic organisms have been displayed a great potential in bioremediation and phytoremediation of heavy metals.     

Metallothionein research in terrestrial invertebrates: Synopsis and perspectives

While most of metallothionein research during the past years has been carried out on mammals or vertebrates, only relatively few studies have been directed towards invertebrates. Even fewer investigations have focussed on terrestrial invertebrates. The best studied metallothioneins and/or metallothionein genes among terrestrial invertebrates are those from an insect species (Drosophila melanogaster), a nematode (Caenorhabditis elegans) and some terrestrial gastropods (Helix pomatia, Arianta arbustorum). From these few examples it already appears that terrestrial invertebrate metallothioneins provide intriguing models to better understand the multiplicity of functions of these proteins and their evolution within the animal kingdom. Like in mammals, metallothioneins in terrestrial invertebrates seem to perform different functions simultaneously. This is exemplified by terrestrial gastropods, which are able to accumulate different metals in different tissues, in which metal-specific metallothionein isoforms or conformation forms are expressed, allowing these organisms to detoxify more efficiently nonessential trace elements such as cadmium, and at the same time to maintain the homeostasis of essential trace elements such as copper. A major proportion of metallothionein research in terrestrial invertebrates addresses the ecophysiological and ecotoxicological significance of these proteins with regard to the increasing risk due to chemical pollution. One promising aspect in this concern is the potential utilization of metallothioneins as biomarkers for risk assessment in terrestrial environments.     

Silver staining for carboxymethylated metallothioneins in polyacrylamide gels

A sensitive method for detecting metallothioneins (MTs) by use of silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs was developed. Carboxymethylation of metallothioneins is indispensable because it prevents their aggregation, thereby allowing each of them to be detected as a single band by SDS-PAGE. However, when the gel was subjected to the silver-staining method of C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert [(1981) Science 211, 1437-1438], the image of carboxymethylated purified MTs was totally negative. Pretreatment of the gel with 1% sodium thiosulfate just prior to the silver-staining procedure successfully reversed the negative image of carboxymethylated MTs. Further, they could be detected with a limit of nanogram levels per lane. This method can be applied to MTs in cell extracts from cultured cell lines treated with cadmium or to those from liver of cadmium-intoxicated mice.     

Comparison of the immunological properties of mammalian (rodent), bird,fish, amphibian (toad), and invertebrate (crab) metallothioneins

Summary Immunoassays such as rocket immunoelectrophoresis, western dot blot hybridization, and competitive ELISA analysis were used to estimate and compare homology among fishes (several species), crab, toad, chicken and rodent metallothioneins. The relative abundance of tissue-specific metallothionein in catfish was highest in liver followed by kidney and pancreas like mammalian systems. Immunologically, considerable homology exists among fish metallothioneins, although the extent of homology differs to some extent. No homology was found between fish or chicken metallothioneins. The amphibian (toad) and invertebrate (crab) metallothioneins showed only partial homology with fish metallothioneins in antigenic determinants.Abbreviations MT Metallothionein - PBS Phosphate Buffered Saline - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - ELISA Enzyme Linked Immunosorbent Assay - ME 2-Mercaptoethanol - BSA Bovine Serum Albumim     

Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip

An electrochemical detection method for chemical sensing has been developed using a DNA aptamer immobilized gold electrode chip. DNA aptamers specifically binding to 17beta-estradiol were selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random ssDNA library, composed of approximately 7.2 x 10(14) DNA molecules. Gold electrode chips were employed to evaluate the electrochemical signals generated from interactions between the aptamers and the target molecules. The DNA aptamer immobilization on the gold electrode was based on the avidin-biotin interaction. The cyclic voltametry (CV) and square wave voltametry (SWV) values were measured to evaluate the chemical binding to aptamer. When 17beta-estradiol interacted with the DNA aptamer, the current decreased due to the interference of bound 17beta-estradiol with the electron flow produced by a redox reaction between ferrocyanide and ferricyanide. In the negative control experiments, the current decreased only mildly due to the presence of other chemicals.     

Multiple forms of metallothionein from the digestive gland of naturally occurring and cadmium-exposed mussels, Mytilus galloprovincialis

Polymorphism of metallothioneins in the digestive gland of naturally occurring (control) and experimentally Cd-exposed mussels Mytilus galloprovincialis (200 μg Cd l^–1; 14 days) was studied by applying the conventional methods of Sephadex column liquid chromatography (G-75 and DEAE A-25), and by an electrochemical method (DPASV) for determination of Cd, Zn and Cu concentrations in chromatographic fractions. In both control and Cd-exposed mussels, two distinct molecular mass components of the metallothioneins, monomeric (MT-10) and dimeric (MT-20), were resolved by Sephadex G-75 gel filtration chromatography. In control mussels, the MT-10 component was predominantly expressed as containing markedly higher constitutive levels of Zn (100×) and Cu (10×) than of Cd. Each of these two molecular mass components was further resolved into seven metal-rich peaks by anion-exchange chromatography. In Cd-exposed mussels the larger proportion of Cd was bound to the MT-20 than to the MT-10 component, suggesting that the dimeric component may be considered as a primarily inducible metallothionein. The elution positions of metal-binding maxima of Cd-exposed and control mussels on the respective DEAE chromatographic profiles were comparable. A great similarity in elution positions of Cd maxima between the composite and single-specimen samples was also observed. Our study confirms a high multiplicity of MT forms in mussels from the Mytilus genus not only under the laboratory high-level metal exposure conditions, but also at a natural seawater metal exposure level. The ecotoxicological significance of dimeric and monomeric MT forms, as well as their possible application in the biomonitoring of seawater for trace metals, has been considered. Electronic Publication     

Some methodological aspects of metallothionein evaluation

The specificity of the methods used for metallothioneins (MTs) determination in both field and laboratory studies is crucial to a relevant interpretation of the results. The technique applied in the present study is commonly used, but several potential problems may limit its validity: (i). the stability of the metal-SH links during heating and the possibility of metal exchanges between MTs and others compounds; (ii). the presence of heat-stable thiol compounds with high molecular weight or very low molecular weight, which may subsist in the supernatant and interfere during global analysis of MTs; (iii). the subsistence of metals not bound to MT in the supernatant after heating; and (iv). the impact of reducing agents such as mercaptoethanol on the metal-binding ligands. Our investigations were conducted mainly on oysters (Crassostrea gigas) and polychaetes (Hediste/Nereis diversicolor) sampled, respectively, in two French metal-rich sites, the Gironde estuary and Boulogne harbour. This study, which allowed us to clarify most of the potential problems noted above, indicates the importance of dealing with methodological difficulties.     

Microfluidic electrochemical assay for rapid detection and quantification of Escherichia coli

Microfluidic electrochemical biosensor for performing Loop-mediated isothermal amplification (LAMP) was developed for the detection and quantification of Escherichia coli. The electrochemical detection for detecting the DNA amplification was achieved using Hoechst 33258 redox molecule and linear sweep voltametry (LSV). The DNA aggregation and minor groove binding with redox molecule cause a significant drop in the anodic oxidation of LSV. Unlike other electrochemical techniques, this method does not require the probe immobilization and the detection of the bacteria can be accomplished in a single chamber without DNA extraction and purification steps. The isothermal amplification time has a major role in the quantification of the bacteria. We have shown that we could detect and quantify 24 CFU/ml of bacteria and 8.6 fg/μl DNA in 60 min and 48 CFU/ml of bacteria in 35 min in LB media and urine samples. We believe that this microfluidic chip has great potential to be used as a point of care diagnostic (POC) device in the clinical/hospital application.     

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to alpha-helices whose content resulted in 5% of the total secondary structure of the protein. The short alpha-helix found in N. coriiceps metallothionein showed an onset of denaturation at 30 degrees C and a T(m) at 48 degrees C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the alpha-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins.     

Metal-binding capacity of metallothioneins of the liver of rats poisoned with heavy metals

The functioning of metallothioneins in the liver of rats, poisoned with copper sulfate and cadmium sulfate has been investigated. By sequential chromatography on sephadex G-50 and DEAE-cellulose the authors obtained metallothioneins (MT-1, MT-1A, MT-2, MT-2a), which differ in molecular weight and composition of associated metals. Heavy metal poisoning leads to activation of synthesis and metal-binding function of metallothioneins, as well as to changes in the composition of their isoforms.     

Separation methods that are capable of revealing blood-brain barrier permeability

The objective of this review is to emphasize the application of separation science in evaluating the blood-brain barrier (BBB) permeability to drugs and bioactive agents. Several techniques have been utilized to quantitate the BBB permeability. These methods can be classified into two major categories: in vitro or in vivo. The in vivo methods used include brain homogenization, cerebrospinal fluid (CSF) sampling, voltametry, autoradiography, nuclear magnetic resonance (NMR) spectroscopy, positron emission tomography (PET), intracerebral microdialysis, and brain uptake index (BUI) determination. The in vitro methods include tissue culture and immobilized artificial membrane (IAM) technology. Separation methods have always played an important role as adjunct methods to the methods outlined above for the quantitation of BBB permeability and have been utilized the most with brain homogenization, in situ brain perfusion, CSF sampling, intracerebral microdialysis, in vitro tissue culture and IAM chromatography. However, the literature published to date indicates that the separation method has been used the most in conjunction with intracerebral microdialysis and CSF sampling methods. The major advantages of microdialysis sampling in BBB permeability studies is the possibility of online separation and quantitation as well as the need for only a small sample volume for such an analysis. Separation methods are preferred over non-separation methods in BBB permeability evaluation for two main reasons. First, when the selectivity of a determination method is insufficient, interfering substances must be separated from the analyte of interest prior to determination. Secondly, when large number of analytes is to be detected and quantitated by a single analytical procedure, the mixture must be separated to each individual component prior to determination. Chiral separation in particular can be essential to evaluate the stereo-selective permeation and distribution of agents into the brain. In conclusion, the usefulness of separation methods during BBB permeability evaluation is immense and more application of these methods is foreseen in the future.     

Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches.

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X(n)-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan alphabeta-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of alphabeta-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the alpha-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four alpha-domains and that lack a beta-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.     

Bivalve metallothionein as a biomarker of aquatic ecosystem pollution by trace metals: limits and perspectives.

Owing to their induction by metals metallothioneins (MTs) have been proposed as biomarkers of the metallic contamination of the environment. On the other hand, bivalves are regarded as very convenient bioindicators of the aquatic ecosystems and an extensive literature has been dedicated to their response to metals. Among studies supporting the involvement of MTs in metal detoxification some discrepancy appears due to inter- and intra-specific variations, or to heterogeneous exposure conditions. A lesser number of papers are dealing with the use of metallothionein levels as biomarkers, and sometimes they evidence that natural factors influencing metallothionein synthesis have to be taken into account before final conclusions can been drawn. Moreover, there is still a large number of non-intercalibrated protocols used to quantify amounts of metallothioneins in organisms. As comparisons are necessary to assess the relative abundance of metallothioneins in a studied species, more work has to be completed before such comparisons could be validated. In the present paper we wish to establish the limits of the use of mollusc metallothioneins as a biomarker of aquatic ecosystem contamination by trace metals, using published and recent data as support for our conclusions and perspectives.     

Erratum

The biochemical features of metallothioneins and their functional role in the cell are described. On this basis, the potential role of MTs as a biomarker of exposure in aquatic organisms, such as fishes and molluscs, is evaluated in the light of recent knowledge about MT gene regulation and inducibility. It appears that in fish MTs should be considered as a kind of stress protein which is particularly responsive to heavy metals. In molluscs, in particular in mussels, MTs seem more specifically involved in responses to heavy metals and they should therefore be considered a biomarker of exposure to heavy metal pollution. Common techniques for MT evaluation are listed and a simple spectrophotometric method recently developed is also reported. Finally, the correct approach to the use of MTs as a biomarker of exposure in biomonitoring programmes for an assessment of the physiological status of aquatic organisms is discussed.     

Metallothionein as a tool in biomonitoring programmes

The biochemical features of metallothioneins and their functional role in the cell are described. On this basis, the potential role of MTs as a biomarker of exposure in aquatic organisms, such as fishes and molluscs, is evaluated in the light of recent knowledge about MT gene regulation and inducibility. It appears that in fish MTs should be considered as a kind of stress protein which is particularly responsive to heavy metals. In molluscs, in particular in mussels, MTs seem more specifically involved in responses to heavy metals and they should therefore be considered a biomarker of exposure to heavy metal pollution. Common techniques for MT evaluation are listed and a simple spectrophotometric method recently developed is also reported. Finally, the correct approach to the use of MTs as a biomarker of exposure in biomonitoring programmes for an assessment of the physiological status of aquatic organisms is discussed.