Heavy Metal Binding Properties Research Articles

Extrapolymeric substances (EPS) in Mucilaginibacter rubeus P2 displayed efficient metal(loid) bio-adsorption and production was induced by copper and zinc

Strains of the genus Mucilaginibacter, belonging to the phylum Bacteroidetes, have been noted for exhibiting high genome plasticity and for the vigorous production of extracellular polymeric substances (EPS). Here we analyzed the composition and properties of EPS generated by M. rubeus P2, isolated from a gold-copper mine and exhibiting extremely high resistance to multiple heavy metals. Production of EPS increased significantly upon exposure to elevated concentrations of Cu(II) and Zn(II), but not Au(III). In addition, the EPS produced by M. rubeus P2 displayed extremely high bio-adsorption of As(III), Cu(II) and Au(III), but not of Zn(II). Moreover, EPS production in Mucilaginibacter rubeus P2 exposed to 1 mM of Cu(II) was 8.5 times higher than EPS production in the same strain without metal (loid)-exposure. These findings constitute the basis for a future use of these EPS-overproducing bacteria in bioremediation of heavy metal contaminated environments. The functional groups, especially –SH, CO, and N–H/C–N in the fingerprint zone of glutathione (GSH) and polysaccharides-like components of EPS, were the main components of EPS involved in both Zn(II) and Cu(II) binding and removal. Around 31.22% and 5.74% of Cu(II)-treated EPS was shown to exist as (CO) structures and these structures were converted into C–OH and O–C–O upon exposure to Cu(II), respectively. In contrast, (C–OH/C–O–C/P–O–C) groups in EPS were observed to be positively correlated to increasing concentrations of Zn(II) in strain P2. Furthermore, the complete genome of M. rubeus P2 helped us to identify 350 genes involved in carbohydrate metabolism, some of which are predicted to be involved in EPS production and modification. This work describes the first detailed biochemical and biophysical analysis of EPS from any strain of Mucilaginibacter with unique heavy metal binding properties. The results will be useful for a better understanding of how microorganisms such as M. rubeus P2 adapt to heavy metal polluted environments and how this knowledge can potentially be harnessed in biotechnological applications such as industrial waste water purification, bioremediation of heavy metal contaminated soil and beneficial plant microbe interactions. The toolbox provided in this paper will provide a valuable basis for future studies.

Insight into the heavy metal binding properties of dissolved organic matter in mine water affected by water-rock interaction of coal seam goaf

The water-rock interaction has a significant effect on the binding characteristics of dissolved organic matter and heavy metals when mine water flows in goaf. This study used fluorescence excitation-emission matrix (EEM) quenching combined with parallel factor (PARAFAC) analysis to characterize the binding properties of DOM with Fe (Ⅲ), Fe (Ⅱ+Ⅲ) and Mn (Ⅱ) in mine water under rock-influenced conditions. Two protein-like components and two humic-like components were identified by PARAFAC, in which protein-like components dominated (75.9%). The fluorescence intensity of each component can all be weakened, especially the stability constant (logKM) value of Fe (Ⅱ+Ⅲ) with fulvic-like acid and humic-like acid was the largest and the binding was more stable. Clay minerals and iron-bearing minerals in rocks had significant effects on the binding characteristics of DOM and metal ions under water-rock interaction. Iron ions released by the oxidation of pyrite and siderite in sandstone can reduce the fluorescence intensity of the derived components. The competitive adsorption effect of clay minerals on metal ions made the fluorescence intensity of the derived components under the action of sandstone containing less clay minerals (19.5%) be lower than that of mudstone (31.3%). Meanwhile, the process of water-rock interaction was accompanied by microbial activities to convert protein-like components into fulvic-like and humic-like components, or higher levels of stable substances. This study shows that when assessing the potential ability of mine water DOM and metal ions binding and migration during the flow of water in goaf it is crucial to take into account the presence of water-rock interaction.

Characterization of the heavy metal binding properties of periplasmic metal uptake protein CLas-ZnuA2.

Candidatus Liberibacter asiaticus (CLas), a phloem-limited unculturable Gram-negative bacterium, causes citrus greening disease. The proteome analysis of CLas showed the presence of a heavy metal permease and Co/Zn/Cd cation exporter system. However, there is no designated metal uptake protein specific for the heavy metal permease in CLas. One of the metal uptake proteins, designated as CLas-ZnuA2, in our previous studies, showed a lower metal-binding affinity for Mn2+ and Zn2+ and was postulated to bind and transport metals rather non-specifically. The present study focused on the characterization of the heavy metal binding properties of CLas-ZnuA2 using SPR, CD, DSC and crystallographic studies. The crystal structure analysis of Cd2+ bound CLas-ZnuA2 showed octahedral geometry for Cd2+ binding as compared to a non-preferred square-pyramidal geometry for Mn2+ and Zn2+ binding in earlier reported crystal structures. In SPR analysis, the binding affinities of 4.7 × 10-6 M, 7.2 × 10-6 M, 5.3 × 10-5 M and 4.3 × 10-5 M for Hg2+, Cd2+, Ba2+ and Co2+ respectively were higher as compared to earlier reported values for Mn2+ and Zn2+. Likewise, CD and DSC analysis showed relatively higher thermal stability for CLas-ZnuA2 on heavy metal binding. Taken together with the expression of the permease and exporter system for heavy metals, our results indicate that CLas-ZnuA2 may be involved in sequestering and transport of various transition divalent metals in environmentally stressed conditions.

Isolation, characterization and optimization of EPSs produced by a cold-adaptedMarinobacterisolate from Antarctic seawater

AbstractMarinobactersp. W1-16 from Antarctic surface seawater was analysed for the production of extracellular polymeric substances (EPSs). Enhancement of the EPS biosynthesis was carried out by evaluating the influences of the carbon source (type and concentration), temperature, pH and salinity. EPS yields varied strongly depending on sugar substrate and temperature, while pH and salinity did not strongly affect levels of EPS production.Marinobactersp. W1-16 produced the highest quantity of EPSs when growing at 15°C and pH 8, in the presence of 2% glucose and 3% NaCl. The EPS chemical characterization revealed a molecular weight of about 260 kDa. Colorimetric assays determined a higher quantity of carbohydrate than of proteins and uronic acids, as well as the presence of sulphate, in the extracted EPSs. The monosaccharidic composition resulted in Glc:Man:Gal:GalN:GalA:GlcA in relative molar proportions of 1:0.9:0.2:0.1:0.1:0.01. Some biotechnological potentialities (i.e. emulsifying and cryoprotective actions, and heavy metal binding properties) of the EPSs were proved, suggesting possible industrial and bioremediation applications.

Characterization analysis and heavy metal-binding properties of CsMTL3 in Escherichiacoli.

Members of the metallothionein (MT) superfamily are involved in coordinating transition metal ions. In plants, MT family members are characterized by their arrangement of Cys residues. In this study, one member of the MT superfamily, CsMTL3, was characterized from a complementary DNA (cDNA) library from young cucumber fruit; CsMTL3 is predicted to encode a 64 amino acid protein with a predicted molecular mass of 6.751 kDa. Phylogenetic analysis identified it as a type 3 family member as the arrangement of N‐terminal Cys residues was different from that of MT‐like 2. Heterologous expression of CsMTL3 in Escherichia coli improved their heavy metal tolerance, particularly to Cd2+ and Cu2+, and led to increased uptake of Cd2+ and Cu2+; increased uptake was also observed for cells expressing Arabidopsis thaliana metallothionein 3 (AtMT3) and phytochelatin‐like (PCL), with greatest uptake in PCL‐expressing cells. These findings demonstrate that CsMTL3 can improve metal tolerance, especially for Cd2+ ions, when heterologously expressed in E. coli, and suggest that the composition and arrangement of N‐terminal Cys residues are associated with binding capacity and preference for different metal ions.

Anti-Amyloid Aggregation Activity of Black Sesame Pigment: Toward a Novel Alzheimer's Disease Preventive Agent.

Black sesame pigment (BSP) represents a low cost, easily accessible material of plant origin exhibiting marked antioxidant and heavy metal-binding properties with potential as a food supplement. We report herein the inhibitory properties of the potentially bioaccessible fraction of BSP following simulated gastrointestinal digestion against key enzymes involved in Alzheimer’s disease (AD). HPLC analysis indicated that BSP is transformed under the pH conditions mimicking the intestinal environment and the most abundant of the released compounds was identified as vanillic acid. More than 80% inhibition of acetylcholinesterase-induced aggregation of the β-amyloid Aβ1-40 was observed in the presence of the potentially bioaccessible fraction of BSP, which also efficiently inhibited self-induced Aβ1-42 aggregation and β-secretase (BACE-1) activity, even at high dilution. These properties open new perspectives toward the use of BSP as an ingredient of functional food or as a food supplement for the prevention of AD.

Evaluation of the heavy metal binding properties of wood and soil humic acids by the NICA–Donnan Model

Evaluation of the heavy metal binding properties of wood and soil humic acids by the NICA–Donnan Model

Heavy metal binding properties of Pinus sylvestris mycorrhizas from industrial wastes

Mycorrhizas of <em>Pinus sylvestris</em>, collected from zinc wastes in Poland and France were investigated using transmission electron microscope (TEM) and scanning electron microscope (SEM) equipped with energy dispersion spectroscopy (EDS) and electron energy loss spectroscopy (EELS). At both sites, mycorrhizas of <em>Hebeloma</em> were the most frequent, however, they were often characterised by a sparse or only locally developed fungal mantle. Mycorrhizas formed by suilloid fungi were much less frequent, and usually produced a clearly defined fungal mantle characterised by abundant formation of pigments and crystals covering the hyphae of the outer mantle. These two groups of mycorrhizas differed in their heavy metal binding properties. A biofiltering effect of Pb and Zn by the fungal mantle was observed only in the case of suilloid mycorrhizas, which represented up to 10% of the total number of mycorrhizas. No statistical differences between the mantle, the cortical cell walls and the vascular tissue were demonstrated in mycorrhizas formed by other fungi dominating on industrial wastes. In the case of <em>Hebeloma</em> and <em>Inocybe</em>, however, elements such as Cu and Cd were present in higher amounts in the extra-matrical mycelium, whereas no or only low amounts of these elements were detected within fungal mantles, mainly in mycorrhizas from the French waste. Analysis of the root systems has shown relatively high percentage of nonmycorrhizal short roots, suggesting the inhibition of mycorrhiza formation or a decreased number of mycorrhizal propagules. The role of dead roots and mycorrhizas in biosorption and immobilization of heavy metals was discussed.

Anomalous N-Glycan Structures with an Internal Fucose Branched to GlcA and GlcN Residues Isolated from a Mollusk Shell-Forming Fluid

This report describes the structural details of a unique N-linked valence epitope on the major protein within the extrapallial (EP) fluid of the mollusk, Mytilus edulis. Fluids from this area are considered to be responsible for shell expansion by a self-assembly process that provides an organic framework for the growth of CaCO3 crystals. Previous reports from our laboratories have described the purification and amino acid sequence of this EP protein, which was found to be a glycoprotein (EPG) of approximately 28 KDa with 14.3% carbohydrate on a single N-linked consensus site. Described herein is the de novo sequence of the major glycan and its glycomers. The sequence was determined by ion trap sequential mass spectrometry (ITMS(n)) resolving structure by tracking precursor-product relationships through successive rounds of collision induced disassociation (CID), thereby spatially resolving linkage and branching details within the confines of the ion trap. Three major glycomers were detected, each possessing a 6-linked fucosylated N-linked core. Two glycans possessed four and five identical antennae, while the third possessed four antennas, but with an additional methylfucose 2-linked to the glucuronic acid moiety, forming a pentasaccharide. The tetrasaccharide structure was: 4-O-methyl-GlcA(1-4)[GlcNAc(1-3)]Fuc(1-4)GlcNAc, while the pentasaccharide was shown to be as follows: mono-O-methyl-Fuc(1-2)-4-O-methyl-GlcA(1-4)[GlcNAc(1-3)]Fuc(1-4)GlcNAc. Samples were differentially deuteriomethylated (CD3/CH3) to localize indigenous methylation, further analyzed by high resolution mass spectrometry (HRMS) to confirm monomer compositions, and finally gas chromatography mass spectrometry (GC-MS) to assign structural and stereoisomers. The interfacial shell surface location of this major extrapallial glycoprotein, its calcium and heavy metal binding properties and unique structure suggests a probable role in shell formation and possibly metal ion detoxification. A closely related terminal tetrasaccharide structure has been reported in spermatozoan glycolipids of freshwater bivalves.

Characterization on a cementitious material composed of red mud and coal industry byproducts

This research was to investigate the possibility of incorporating red mud and coal industry byproducts as the raw material for producing cementitious material. Systematic mechanical strength tests were conducted to evaluate the performance of this cementitious material. Results showed that the designed red mud–coal industry byproducts based cementitious material had higher strength in the middle to late curing age (47.5MPa in 180days and 48.7MPa in 360days) than the OPC control group. The series of durability tests indicated that the cementitious material met with the ASTM requirement. Moreover, the toxicity characteristic leaching tests demonstrated that this cementitious material had good stabilization/solidification ability to bind the heavy metal in the red mud as raw material. Microanalysis revealed that the amorphous gel was the dominant structure of the material at the middle to late curing age, which possibly played a significant role on the heavy metal binding properties through the polymerization during the hydration process of this cementitious material. In essence, this designed red mud–coal industry byproducts based cementitious material not only meet with the physical and mechanical requirements of the ASTM standards, but also meet with the EPA regulation on the environmental heavy metal leaching limitation. This proves the designed material can possibly be used as a clean technology to recycle the red mud from alumina industries and byproduct from coal industries.

New insight into the molecular pathways of metallothionein‐mediated neuroprotection and regeneration

There is a large body of evidence demonstrating that metallothioneins (MTs) expressed in astrocytes following CNS injury, exhibit both neuroprotective and neuroregenerative properties and are critical for recovery outcomes. As these proteins lack signal peptides, and have well characterized free radical scavenging and heavy metal binding properties, the neuroprotective functions of MTs have been attributed to these intracellular roles. However, there is an increasing realization that the neuroprotective functions of MTs may also involve an extracellular component. In this issue of Journal of Neurochemistry, Ambjørn et al. reveal considerable insight into this novel function of MTs. In this review, we examine the seminal work of Ambjørn et al. in the context of our current understanding of the role of MT in astrocyte-neuron interactions in the injured brain, and also discuss the significant therapeutic potential of their work.

Utilizing a chronopotentiometric sensor technique for metallothionein determination in fish tissues and their host parasites

Metallothionein (MT) are a class of intracellular proteins possessing cysteine-rich and heavy metal-binding properties. They can be utilized as biomarkers for the detection and assessment of heavy metals in environmental pollution. This paper addresses the development of a sensor technique suitable for the determination of MT in the tissue samples of perches and their parasites. The work suggest a heavy metal biosensor based on interaction of heavy metals (lead(II), cadmium(II), copper(II) and silver(I)) with MT using adsorptive transfer stripping (AdTS) technique in combination with chronopotentiometric stripping analysis. The samples were investigated with various heavy metals (lead(II), cadmium(II), copper(II) and silver(I)), prior to sensor applications to understand the interaction between MT and the metals. The results obtained from this experiment clearly show that cadmium(II) had the highest affinity for MT followed by silver(I), copper(II) and lead(II). Based on these results, we attempted to quantify MT content with respect to content of a heavy metal in the samples of interest. The highest re-evaluated concentration of MT (495 ng/g of fresh weight) was determined in samples of Acanthocephalus anguillae.

γ‐Polyglutamic Acid Produced by Bacillus Subtilis (Natto): Structural Characteristics, Chemical Properties and Biological Functionalities

Abstractγ‐(D,L)‐Polyglutamic acid [γ‐(D,L)‐PGA, or γ‐PGA] produced by Bacillus subtilis (natto) both on laboratory and pilot scale fermenter systems has been characterized. γ‐PGA in its free acid form (H+) is insoluble in water. The salt forms of K+, Na+, NH4+, Ca2+, and Mg2+ of γ‐polyglutamates are fully soluble in water. The structural characteristics of the salts of γ‐Polyglutamates (Na+, K+, NH4+, Ca2+, and Mg2+) were determined with 1H‐ and 13C‐NMR spectroscopy and FT‐IR spectroscopy. The thermal properties were determined with thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The typical physical and chemical properties including pH‐titration curve, concentration‐dependent viscosity temperature‐dependent viscosity, and heavy metal binding properties with Pb2+, Cd2+, and Cu2+ were also determined. The biological functionalities were partially characterized with in vivo feeding studies with broilers and egg‐layers, and activation of GTF (Glucose tolerance factor) activity with rat 3T3‐L1 cell culture studies. γ‐(D,L)‐Polyglutamic acid [γ‐(D,L)‐PGA] appears to have five different conformations depending on the environmental conditions. They are the α‐helix, β‐sheet, helix‐to‐random coil transition, random‐coil and enveloped aggregate. The conformational states, hydrogen bonding and polyanionic nature make γ‐PGA a versatile multi‐functional biopolymer possessing many useful biological functionalities.

Structural Characterization of the Major Extrapallial Fluid Protein of the MolluscMytilus edulis: Implications for Function

The major protein component of the extrapallial fluid of the mollusc Mytilus edulis has been previously isolated and partially characterized. It was postulated to play a role in shell mineralization because of its intriguing property of Ca(2+)-binding-induced self-assembling. However, it also binds other divalent ions, including Cd(2+), Cu(2+), Mn(2+), and Mg(2+). Herein is the initial report on the characterization of the primary structure of the extrapallial (EP) protein by RT-PCR and cDNA sequencing methods and by de novo peptide sequencing with mass spectrometry. The EP protein is comprised of 213 amino acids postcleavage of a signal peptide of 23 amino acids. The protein is rich in His, Glu, and Asp residues. The site of N-glycosylation, "NHTE", at amino acid positions 54-57 and the intramolecular disulfide bond between Cys 139 and Cys 171 of the protein have been characterized also. Sequence comparisons reveal that the EP protein possesses little homology to any presently known matrix proteins previously isolated from mollusc shells but rather it highly resembles a heavy metal binding protein and a histidine-rich glycoprotein, both from the hemolymph of M. edulis. The predicted domain profile and amino acid composition suggest that its N-terminus may be involved in calcium binding. The abundance of histidine residues of the protein may account for its heavy metal binding properties. Thus, the EP protein perhaps has multiple functions, serving as a Ca(2+)-transport protein, a shell matrix protein, and a heavy metal detoxification protein.

The Comparison of Heavy Metal Binding Properties and Tolerance between Transgenic Strains and Wild Type in Chlamydomonas reinhardtii

The Comparison of the heavy metal binding properties and tolerance was studied between wild type and transgenic strains which expressed a foreign MT like gene from Festuca rubraL Merlin in Chlamydomonas reinhardtii. The results showed that the cadmium binding capacity of transgenic strain was 1.5 fold of the wild type in low cadmium concentration(5-10μmol/L) , however, there was not marked difference of heavy metal binding capacity in high cadmium concentrtion (30-50μmol/L). It indicated that the difference of heavy metal binding capacity was from active uptake between transgenic strain and wild type. On the other hand, transgenic strain also showed more higher tolerance than wild type when they were cultured in 150μmol/L copper solution medium and 600μmol/L cadmium agar medium. Transgenic strain was used to treat wastewater, it could enhance more than 10% of copper removal efficiency than wild type.

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant ( Silene vulgaris ) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.

Molecular size of native proteins of Mytilus serum which contains a dominant fraction with heavy metal-binding properties

Native serum of Mytilus edulis was investigated by gradient PAGE. Protein composition varied between individual mussels. However, six major bands were obtained relatively constantly in electropherograms revealing molecular weights of 35±1.8 kDa, 136±4.0 kDa, 287±16.9 kDa, 423±26.3 kDa, 498±7.5 kDa and 667±24.8 kDa. The serum protein band 1 (SPB1) of ∼35 kDa occurred in all mussels at a relatively high concentration. From two very different experimental approaches information was expected about a physiological function of this prominent protein fraction: (1) No support was obtained for its postulated vitellogenin activity as it was not found in eggs. (2) On the other hand, SPB1 reacted strongly with heavy metals. It was precipitated by cadmium acetate and bound to organomercurial agarose. Furthermore, evidence was obtained that most of the Cd in serum from contaminated mussels was complexed with humoral components as about 75% of the serum-metal was retained by an ultrafiltration membrane (molecular weight cutoff 3 kDa). In contrast, a seawater solution of cadmium passed the filter pores as determined by atomic absorption chromatography. It is suggested that SPB1 could be involved in heavy metal transport between gills, considered to be the primary sites of metal entry, and other organs accumulating the uptaken water pollutants.

Investigation of the binding properties of heavy-metal-peptide complexes in plant cell cultures using HPLC-ICP-MS

Metal-induced, sulfhydryl-rich peptides (phytochelatins) found in plants, algae, yeasts and fungi have been described as sequesters and detoxifiers of heavy metal ions. High-performance liquid chromatography (HPLC) coupled on-line with inductively coupled plasma mass spectrometry (ICP-MS) has been used for the determination of heavy metal binding properties of phytochelatins in Silene vulgaris cell cultures. The induction of phytochelatins and the binding of heavy metals to these complexes were investigated by exposure of cell cultures with different concentrations of Cd, Cu, Pb and Zn. An in vitro heavy metal saturation assay and in vivo stress experiments with those elements able to bind to phytochelatins were carried out in order to characterize the binding affinity and binding stability of these compounds. It is shown that of the metals investigated, Cu binds most stably to phytochelatins under in vitro and in vivo conditions.

Amino acids and peptides. XXXIV. Synthesis of mouse metallothionein I. (1). Synthesis of dotriacontapeptide corresponding to C-terminal sequence 30-61 (alpha-fragment) of mouse metallothionein I and related peptides and examination of their heavy metal-binding properties.

A nonacosapeptide corresponding to the N-terminal sequence 1-29 (beta-fragment) of mouse metallothionein I and related peptides were synthesized by the conventional solution method and their heavy metals (Cu2+, Cu+ and Cd2+)-binding properties were examined. The Cu(2+)- or Cu(+)-binding activities of various peptides were not greatly dependent on the peptide structure, so far as examined, as in the cases of N. crassa MT and A. bisporus MTs. On the contrary, the Cd(2+)-binding activities of these peptides were fairly structure-dependent.

Amino acids and peptides. XXXV. Synthesis of mouse metallothionein I. (2). Synthesis of a nonacosapeptide corresponding to N-terminal sequence 1-29 (beta-fragment) of mouse metallothionein I and related peptides and examination of their heavy metal-binding properties.

A nonacosapeptide corresponding to the N-terminal sequence 1-29 (beta-fragment) of mouse metallothionein I and related peptides were synthesized by the conventional solution method and their heavy metals (Cu2+, Cu+ and Cd2+)-binding properties were examined. The Cu(2+)- or Cu(+)-binding activities of various peptides were not greatly dependent on the peptide structure, so far as examined, as in the cases of N. crassa MT and A. bisporus MTs. On the contrary, the Cd(2+)-binding activities of these peptides were fairly structure-dependent.