Metal Binding Characteristics Research Articles

Spectroscopic insights into the binding characteristics of heavy metals to dissolved organic matter in landfill leachate

Landfill leachate is produced in the process of sanitary landfilling, which contains significant amounts of dissolved organic matter (DOM) and heavy metal contaminants. Insights into the interactions between heavy metals and DOM in landfill leachate are beneficial for the understanding of heavy metal fates and optimization of landfill leachate treatment. In this work, the coherent structural changes of landfill leachate DOM during binding with various heavy metals were explored through the integration of molecular spectroscopic methods with chemometrics and statistic correlation analyses. The results indicate that protein substances, phenolic and discrete carboxyl groups in landfill leachate DOM were involved in the complexation with heavy metals, resulting in the formation of conjugated macromolecules/aggregates with high aromaticity and molecular weight/size. The fluorescent protein-like, fulvic acid-like, and humic-like fractions in DOM were engaged in the interaction, which were closely related to phenolic-like and carboxylic-like structure. Compared to membrane concentrates DOM, raw leachate DOM exhibited a higher binding affinity to heavy metals (especially for Cu2+, whilst the weakest was Cd2+). The integrated approach provides useful information in elucidating the binding processes of metals with landfill leachate DOM, including site heterogeneity, binding strength and functional group sequences.

Lithium in Greek Coal Fly Ashes: Contents and Characterization by Sequential Extraction

Lithium belongs to the critical elements and is used in a variety of high-tech applications. In the context of the circular economy, demand has arisen for technologies that are able to recover high-tech metals from wastes and byproducts. To achieve efficient recovery, apart from assessing metal enrichment, extensive knowledge of metal binding and leaching characteristics is required. The aim of the present study is to investigate the Li contents and mode of occurrence in Greek coal fly ashes. Eight coal fly ashes from different power plants in Greece were collected, and their major constituents were analyzed by X-ray fluorescence spectroscopy (XRF); their mineralogy was studied by X-ray powder diffraction (XRD), and their Li content was determined by ICP–MS. To identify Li binding and leaching characteristics, two sequential extraction methods (Tessier and BCR) were employed. The results showed that the Li content in the samples studied was between 95 and 256 μg/g and could be mainly attributed to the amorphous material encountered in the samples. The sequential extraction experiments revealed that 70–90% of Li is included in the residual fraction, indicating that it is strongly bound to the fly ash matrix.

Role of molecular weight-dependent spectral properties in regulating Cu(II) binding by dissolved organic matter from different sources

The complexation of metals with dissolved organic matter (DOM) under different compositions and molecular weights (MWs) will result in different environmental fate and toxicity, but the specific role and impact of DOM MWs remain less well understood. This study explored the metal binding characteristics by DOM with different MWs from different sources, including sea, river, and wetland waters. The results of fluorescence characterization showed that the >1 kDa high-molecular-weight (HMW)-DOM were mainly from terrestrial sources while the low-molecular-weight (LMW)-DOM fractions were mostly from microbial sources. Based on UV–Vis spectroscopic characterization, the LMW-DOM contained more unsaturated bonds than its HMW counterpart, and the substituents are generally dominated by polar functional groups. Summer DOM had more unsaturated bonds and a higher metal binding capacity than winter DOM. Furthermore, DOM with different MWs had significantly different Cu binding properties. In addition, Cu binding with microbially derived LMW-DOM mainly caused the change in the peak at 280 nm, while binding with terrigenous HMW-DOM resulted in the change of the 210 nm peak. Compared with the HMW-DOM, most of the LMW-DOM had stronger Cu-binding ability. Correlation analysis indicates that metal binding ability of DOM mainly depends on its concentration, number of unsaturated bonds and benzene rings, and types of substituents during interactions. This work provides an improved understanding of the metal-DOM binding mechanism, the role of composition- and MW-dependent DOM from different sources, and thus the transformation and environmental/ecological role of metals in aquatic systems.

Insight into the effects of biological treatment on the binding properties of copper onto dissolved organic matter derived from coking wastewater

Biological treatment can remove more than 89.8% of total organic carbon (TOC) and 94.4% of fluorescent dissolved organic matter (DOM) in the coking wastewater, thereby affecting the migration, transformation and bioavailability and binding characteristics of heavy metals (HMs). The results of parallel factor analysis (PARAFAC) show that protein-like materials accounted for 97.53% in the coking wastewater DOM, a large number of humic-like substances are produced and accounted for more than 55.40% after biological treatment. A new spectral data processing method, the 1/n-th power transformation after two-dimensional correlated spectroscopy (2D-COS) in combination with synchronous fluorescence spectra (SFS), can identify small features obscured by strong peaks, and reveal more binding sites as well as preserve the sequential order information. The result indicates that the preferential bonding of Cu(II) is at 306 nm (protein-like) for coking wastewater DOM, and at 514 nm (humic-like) for effluent DOM. The C−O group of esters and alcohols can preferentially complexate with Cu(II) in the coking wastewater and effluent DOM. The log KM values of PARAFAC components with Cu(II) are in the range of 3.59–5.06 for coking wastewater DOM, and in the range of 4.80–5.64 for the effluent DOM. Log KM values for protein-like materials with Cu(II) are higher than these for fulvic- and humic-like substances. Humic-like substances can form more stable complexes with Cu(II) in the effluent DOM. Biological treatment increases the chemical stability of DOM-Cu(II) complexes, thereby further reducing the environmental risk of Cu(II).

Novel insights into molecular composition of organic phosphorus in lake sediments

Organic phosphorus (Po) plays a key role in eutrophication and ecological equilibrium in lake systems. However, characterizing the composition of Po in lake sediments has been a bottleneck hindering further understanding of the biogeochemical cycle of Po. Here, multiple methods of 31P NMR spectroscopy and molecular weight (MW) ultrafiltration were combined to detect Po composition characteristics from a novel angle in ten lake sediments of China. The results showed that sediment Po mainly consisted of monoester (mono-P, 14±8.8% of the NaOH-EDTA total P on average), diester (di-P, 1.4±1.4%) and phosphonate (phos-P, 0.1±0.1%), while the abundance of Po was largely underestimated by 31P NMR methods. Some specific species of mono-P were successfully determined, and the contents of these species followed a decreasing order: inositol hexakisphosphate (IHP6) > RNA mononucleotides (RNA-mnP) > β-glycerophosphate (β-gly) > D-glucose 6-phosphate (Glu-6) > α-glycerophosphate (α-gly), which was largely dependent upon their bioreactivity. A significant relationship between MW and Po components was observed despite the great differences among sediment samples. For refractory Po components, IHP6 was mainly rich in the MW < 3 kDa while phos-P was almost only detected in the MW > 3 kDa, which largely attributed to their metal binding affinities and characteristics. The abundance of bioreactive Po species (α-gly, β-gly, Glu-6, di-P) in high MW (HMW, > 3 kDa) were all higher than that of low MW (LMW, < 3 kDa) due to microbial degradation and self-assembly. If the HMW organic molecules were biologically and chemically more reactive than its LMW counterparts, the high percentage of α-gly, β-gly, glu-6 and di-P in the HMW portion would highlights their high reactivity from the perspective of MW. These insights revealed the dynamics of the MW distribution of Po components and provide valuable information to better understand the Po composition and bioreactivity in sediments.

Insight into binding characteristics of copper(II) with water-soluble organic matter emitted from biomass burning at various pH values using EEM–PARAFAC and two-dimensional correlation spectroscopy analysis

The metal-binding characteristics of water-soluble organic matter (WSOM) emitted from biomass burning (BB, i.e., rice straw (RS) and corn straw (CS)) with Cu(II) under various pH conditions (i.e., 3, 4.5, and 6) were comprehensively investigated. Two-dimensional correlation spectroscopy (2D-COS) and excitation–emission matrix (EEM) –PARAFAC analysis were applied to investigate the binding affinity and mechanism of BB WSOM. The results showed that pH was a sensitive factor affecting binding affinities of WSOM, and BB WSOMs were more susceptible to bind with Cu(II) at pH 6.0 than pH 4.5, followed by pH 3.0. Therefore, the Cu(II)-binding behaviors of BB WSOMs at pH 6.0 were then investigated in this study. The 2D-absorption-COS revealed that the preferential binding with Cu(II) was in the order short and long wavelengths (237–239 nm and 307–309 nm) > moderate wavelength (267–269 nm). The 2D-synchronous fluorescence-COS results suggested that protein-like substances generally exhibited a higher susceptibility and preferential interaction with Cu(II) than fulvic-like substances. EEM–PARAFAC analysis demonstrated that protein-like (C1) substances had a greater complexation ability than fulvic-like (C2) and humic-like (C3) substances for both BB WSOM. This indicated that protein-like substances within WSOM played dominant roles in the interaction with Cu(II). As a comparison, RS WSOM generally showed stronger complexation capacity than CS WSOM although they exhibited similar chemical properties and compositions. This suggested the occurrence of heterogeneous active metal-binding sites even within similar chromophores for different WSOM. The results enhanced our understanding of binding behaviors of BB WSOM with Cu(II) in relevant atmospheric environments.

Binding characteristics of cadmium and zinc onto soil organic matter in different water managements and rhizosphere environments

Soil organic matter (SOM) could immobilize most of metals, but it could promote the migration of a small part of metals in special environments. Heavy rainfall and drought makes wetlands affected by the alternation of drought and flood, altering the mobility of metals. Few studies have been conducted on the changes of binding characteristics of metals onto SOM which derived from different water conditions and rhizospheric environments. The objective of this paper was to explore the sequential differences of spectral variations of fluorescent groups and UV-Vis groups of metals onto SOM which derived from different water managements and rhizospheric environments. The method adopted was mainly two-dimensional correlation analysis (2DCOS). The results showed that flooding samples contained more aromatic substances compared to draining samples, which could promote metal binding. The binding characteristics were shown in the following: (1) Cd2+ and Zn2+ could react with aromatic substances, react with functional groups in SOM, and promote the formation of new groups such as carboxyl; (2) both Zn2+ and Cd2+ could bind with functional groups on proteins but relatively reductive environment can weaken the binding ability of Cd2+; (3) the protein-like or fulvic-like groups gave the fastest responses and then came the amide and carboxyl groups in nearly all flooding samples; (4) in flooding samples, Cd2+ was most easily to bind with fulvic-like groups, while Zn2+ was most easily to bind with protein-like groups. This work is conducive to the long-term management of heavy metal pollutants in wetlands.

Investigation on the effects of sediment resuspension on the binding of colloidal organic matter to copper using fluorescence techniques

Colloidal organic matter (COM), an important component of dissolved organic matter (DOM), plays a significant role in the transport and cycling process of the heavy metals. In this study, COM was fractionated from DOM using 0.2 μm, 100 kDa, and 2 kDa ultrafiltration membranes and the fluorescence spectra of the COM fractions were obtained. Excitation and emission matrix–parallel factor analysis and two-dimensional fluorescence correlation spectroscopy were applied to investigate the effect of sediment resuspension on the heavy metal binding characteristics of COM fractions with different molecular weights. Compared with the DOM fractions, COM exhibited stronger binding affinities and more binding sites for Cu(II), which was attributed to the significant binding effects of the components of COM. Our results suggested that the protein-like components were mainly responsible for binding heavy metals in the high-molecular-weight fraction (>100 kDa), whereas the humic-like components were responsible in the low-molecular-weight fraction (<100 kDa). Furthermore, sediment resuspension significantly influenced the composition and heavy metal binding characteristics of COM. Following resuspension, the binding affinity of COM decreased significantly, which might be attributed to the binding competition from inorganic colloids. Thus, COM plays an important role in the binding and transportation behavior of heavy metals, which is an important consideration in shallow lake ecosystems.

Binding characteristics of heavy metals to humic acid before and after fractionation by ferrihydrite

The fractionation of humic substances (HS) at the mineral and water interface can change the constituents and reactivity of HS, but there is still a lack of the understanding of the effects of HS fractionation on the binding characteristics of heavy metals to HS. In this study, the binding characteristics of five heavy metals (Cd, Cu, Ni, Pb, and Zn) to humic acid (HA) before and after adsorption by ferrihydrite were investigated by employing two-dimensional correlation spectroscopy (2D COS) integrated with synchronous fluorescence and Fourier transform infrared (FTIR) spectroscopies. 2D COS analyses of the fluorescence results indicated that the susceptibility of the fluorescence of humic-like fraction to heavy metals significantly decreased after the adsorption of HA by ferrihydrite, which may be due to the fact that humic-like components were preferentially adsorbed by ferrihydrite. However, the fractionation processes did not alter the metal binding sequence and affinity to different HA components. 2D COS analyses of the FTIR results suggested that fractionation processes decreased the susceptibility of COO− groups to heavy metals, and changed the metal binding sequence to polysaccharides C-O and aryl groups, with the exception of Pb. Furthermore, model calculations showed that the binding ability of heavy metals to both humic-like and fulvic-like fractions decreased after the adsorption of HA by ferrihydrite. The results of this study contribute to predicting heavy metal behavior in the environment.

Nature differences of humic acids fractions induced by extracted sequence as explanatory factors for binding characteristics of heavy metals

The composition and structure of Humic acid (HA) is so heterogeneous that it brings significant barriers to investigate the interaction between HA and heavy metal ions. The isolation of HA with relatively homogeneity is a key to reveal the binding mechanisms between HA and heavy metals. In this work, ten HA fractions (HAs) were obtained by sequential alkali extraction procedure and nature differences of the extracted HAs were considered as explanatory factors for binding characteristics of Cu2+, Pb2+ and Cd2+. The results indicate that more large molecular weight (MW) HA subunits, less carboxyl and phenolic group contents, weaker aromaticity and polarity were measured with increasing extractions, inducing weaker binding capacity of HAs. Ligand binding and bi-Langmuir models indicated that the sorption capacity and binding affinity of earlier extracted HAs were higher than the latter ones. The peak area changes at 3427, 1599, and 619 cm−1 pre- and post-adsorption in FTIR spectra suggested carboxyl, phenolic and nitrogen-containing groups were involved in the adsorption process. At the same time, the peak area difference between HAs and HAs-metal (ΔS) of phenolic groups were 8.22–20.50, 6.81–21.11 and 10.66–19.80% for Cu2+, Pb2+ and Cd2+, respectively, ΔS of carboxyl groups 6.64–17.03, 8.96–16.82 and 9.45–17.85% for Cu2+, Pb2+ and Cd2+, respectively, ΔS of nitrogen-containing groups 0.33–0.48, 0.20–1.38 and 0.31–0.59% for Cu2+, Pb2+ and Cd2+, respectively. ΔS of phenolic and carboxyl groups were larger than those of nitrogen-containing groups, implying that these two groups were the predominant binding sites suppliers for metal ions, which were also supported by the results of correlation analysis. This work is helpful to insight the environmental impacts of natural organic matter and the fate of heavy metals in natural environment.

Using two-dimensional correlation size exclusion chromatography (2D-CoSEC) to explore the size-dependent heterogeneity of humic substances for copper binding

Knowledge of the heterogeneous distribution of humic substances (HS) reactivities along a continuum of molecular weight (MW) is crucial for the systems where the HS MW is subject to change. In this study, two dimensional correlation spectroscopy combined with size exclusion chromatography (2D-CoSEC) was first utilized to obtain a continuous and heterogeneous presence of copper binding characteristics within bulk HS with respect to MW. HS solutions with varying copper concentrations were directly injected into a size exclusion chromatography (SEC) system with Tris-HCl buffer as a mobile phase. Several validation tests confirmed neither structural disruption of HS nor competition effect of the mobile phase used. Similar to batch systems, fluorescence quenching was observed in the chromatograms over a wide range of HS MW. 2D-CoSEC maps of a soil-derived HS (Elliot soil humic acid) showed the greater fluorescence quenching degrees with respect to the apparent MW on the order of 12500 Da > 10600 Da > 7000 Da > 15800 Da. The binding constants calculated based on modified Stern-Volmer equation were consistent with the 2D-CoSEC results. More heterogeneity of copper binding affinities within bulk HS was found for the soil-derived HS versus an aquatic HS. The traditional fluorescence quenching titration method using ultrafiltered HS size fractions failed to delineate detailed distribution of the copper binding characteristics, exhibiting a much shorter range of the binding constants than those obtained from the 2D-CoSEC. Our proposed technique demonstrated a great potential to describe metal binding characteristics of HS at high MW resolution, providing a clear picture of the size-dependent metal-HS interactions.

Isolation and purification treatments change the metal-binding properties of humic acids: effect of HF/HCl treatment

Environmental contextStudying the mechanism of binding between metals and natural organic matter is fundamental to understanding the transport and availability of these contaminants in the environment. The influence of sample treatment on the purification of organic matter showed significant differences in the interaction with metals. The results will contribute to improved modelling of metal binding to organic matter in soils, thereby providing a basis for a more realistic risk assessment. AbstractWe studied the changes in metal binding characteristics of extracted humic acids induced by HF/HCl treatment followed by dialysis, i.e. the last step of the International Humic Substances Society (IHSS) extraction protocol. We performed metal binding experiments with both the alkaline-extracted material (AE) and the fully purified (FP) humic acid using the electrochemical stripping technique (AGNES) and modelled the results using the NICA-Donnan model. The results showed an increase of free Zn, Cd and Pb concentrations of ~1 order of magnitude for the AE compared with the FP. These differences may be mostly explained by the different carbon content (51.3 % FP and 36.5 % AE) associated with an AE/FP carboxyl ratio of 0.5. Simulations using the NICA-Donnan model showed that halving the amount of carboxylic groups (Qmax,1) for the FP reduced this difference to 0.25 log units for Cd and Zn and to 0.15 log unit for Pb. There is a clear need for further research on the differences between purified v. less-disturbed natural organic material, which will contribute to improved modelling of metal binding to organic matter in soils, hence providing a basis for a more realistic risk assessment.

Cucumber Metallothionein-Like 2 (CsMTL2) Exhibits Metal-Binding Properties.

We identified a novel member of the metallothionein (MT) family, Cucumis sativus metallothionein-like 2 (CsMTL2), by screening a young cucumber fruit complementary DNA (cDNA) library. The CsMTL2 encodes a putative 77-amino acid Class II MT protein that contains two cysteine (Cys)-rich domains separated by a Cys-free spacer region. We found that CsMTL2 expression was regulated by metal stress and was specifically induced by Cd2+ treatment. We investigated the metal-binding characteristics of CsMTL2 and its possible role in the homeostasis and/or detoxification of metals by heterologous overexpression in Escherichia coli cells. Furthermore, we produced a deletion mutant form of the protein, CsMTL2m, that contained the two Cys-rich clusters but lacked the spacer region, in E. coli. We compared the metal-binding properties of CsMTL2 with those of CsMTL2m, the β domain of human metallothionein-like protein 1 (HsMTXb), and phytochelatin-like (PCL) heterologously expressed in E. coli using metal-binding assays. We found that E. coli cells expressing CsMTL2 accumulated the highest levels of Zn2+ and Cd2+ of the four transformed cell types, with levels being significantly higher than those of control cells containing empty vector. E. coli cells expressing CsMTL2 had a higher tolerance for cadmium than for zinc ions. These findings show that CsMTL2 improves metal tolerance when heterologously expressed in E. coli. Future studies should examine whether CsMTL2 improves metal tolerance in planta.

Metagenomics analysis reveals a new metallothionein family: Sequence and metal-binding features of new environmental cysteine-rich proteins

Metallothioneins are cysteine-rich proteins, which function as (i) metal carriers in basal cell metabolism and (ii) protective metal chelators in conditions of metal excess. Metallothioneins have been characterized from different eukaryotic model and cultivable species. Presently, they are categorized in 15 families but evolutionary relationships between these metallothionein families remain unresolved. Several cysteine-rich protein encoding genes that conferred Cd-tolerance in Cd-sensitive yeast mutants have previously been isolated from soil eukaryotic metatranscriptomes. They were called CRPs for “cysteine-rich proteins”. These proteins, of unknown taxonomic origins, share conserved cysteine motifs and could be considered as metallothioneins. In the present work, we analyzed these CRPs with respect to their amino acid sequence features and their metal-binding abilities towards Cd, Zn and Cu metal ions. Sequence analysis revealed that they share common features with different known metallothionein families, but also exhibit unique specific features. Noticeably, CRPs display two separate cysteine-rich domains which, when expressed separately in yeast, confer Cd-tolerance. The N-terminal domain contains some conserved atypical Cys motifs, such as one CCC and two CXCC ones. Five CRPs were expressed and purified as recombinant proteins and their metal-binding characteristics were studied. All these CRPs chelated Cd(II), Zn(II) and Cu(I), although displaying a better capacity for Zn(II) coordination. All CRPs are able to confer Cd-tolerance, and four of them confer Zn-tolerance in the Zn-sensitive zrc1Δ yeast mutant. We designated these CRPs as environmental metallothioneins belonging to a new formerly undescribed metallothionein family.

Oxidation Protection in Metal-Binding Peptide Motif and Its Application to Antibody for Site-Selective Conjugation.

Here, we demonstrate that a metal ion binding motif could serve as an efficient and robust tool for site-specific conjugation strategy. Cysteine-containing metal binding motifs were constructed as single repeat or tandem repeat peptides and their metal binding characteristics were investigated. The tandem repeats of the Cysteine-Glycine-Histidine (CGH) metal ion binding motif exhibited concerted binding to Co(II) ions, suggesting that conformational transition of peptide was triggered by the sequential metal ion binding. Evaluation of the free thiol content after reduction by reducing reagent showed that metal-ion binding elicited strong retardation of cysteine oxidation in the order of Zn(II)>Ni(II)>Co(II). The CGH metal ion binding motif was then introduced to the C-terminus of antibody heavy chain and the metal ion-dependent characteristics of oxidation kinetics were investigated. As in the case of peptides, CGH-motif-introduced antibody exhibited strong dependence on metal ion binding to protect against oxidation. Zn(II)-saturated antibody with tandem repeat of CGH motif retains the cysteine reactivity as long as 22 hour even with saturating O2 condition. Metal-ion dependent fluorophore labeling clearly indicated that metal binding motifs could be employed as an efficient tool for site-specific conjugation. Whereas Trastuzumab without a metal ion binding site exhibited site-nonspecific dye conjugation, Zn(II) ion binding to antibody with a tandem repeat of CGH motif showed that fluorophores were site-specifically conjugated to the heavy chain of antibody. We believe that this strong metal ion dependence on oxidation protection and the resulting site-selective conjugation could be exploited further to develop a highly site-specific conjugation strategy for proteins that contain multiple intrinsic cysteine residues, including monoclonal antibodies.

Influence of ocean acidification on the complexation of iron and copper by organic ligands in estuarine waters

The uptake of anthropogenic atmospheric CO2 by the oceans causes a shift in the carbonate chemistry system which includes a lowering of pH; this process has been termed ocean acidification. Our understanding of the specific effects of ocean acidification on chemical speciation of trace metals, in particular on organic–metal interactions, is limited. In this study we have experimentally determined the effects of changing pH from 8.3 to 6.8 (on the NBS scale) on the speciation of iron and copper in estuarine waters. Our experimental results indicated that complexation of iron and copper would decrease and inorganic iron and copper concentrations increase, as pH decreased, although it was not possible to confidently quantify changes in speciation at lower pH due to constraints of the analytical technique. In addition to our experimental approach, we used a non-ideal competitive adsorption (NICA)–Donnan model to determine the chemical speciation of iron and copper as a function of pH. The NICA–Donnan model was optimised in order to produce similar metal binding characteristics to those observed in our sample across the pH range examined in our study. The model allowed for simultaneous modelling of solubility and organic complexation. Model results indicated that a decrease in iron and copper binding by organic matter at lower pH, coupled with increased solubility (for iron), resulted in a 3 fold increase in inorganic iron concentration and a 6 fold increase in inorganic copper concentration at pH of 7.41 compared to a pH of 8.18 (expressed on the total scale). This compared to a 10 fold increase in inorganic iron concentration, and a 5 fold increase in inorganic copper concentration, obtained at pH8.18, when the dissolved organic carbon (DOC) concentration was halved. Variability in DOC might thus be expected to have a greater impact on metal speciation in seawater, than projected variability in pH resulting from increases in atmospheric CO2. Our study therefore suggests that increases in the concentrations of the more bioavailable inorganic iron and copper species in estuarine waters resulting from increased pCO2 are likely to occur, but that such changes will be moderate in magnitude.

Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis

Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834-840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746-2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological activities to fully understand the mechanisms of Cu tolerance and toxicity in plants.

Molecular cloning of manganese superoxide dismutase gene in the cladoceran Daphnia magna: Effects of microcystin, nitrite, and cadmium on gene expression profiles

Superoxide dismutases (SODs) are metalloenzymes that represent one important line of defense against oxidative stress produced by reactive oxygen species in aerobic organisms. Generally, waterborne pollutants caused by irregular anthropogenic activities often result in oxidative damage in aquatic organisms. The aim of this study was to molecularly characterize the manganese superoxide dismutase gene (Dm-MnSOD) in the waterflea, Daphnia magna, and evaluate the mRNA expression patterns quantified by real-time PCR after exposure to three common waterborne pollutants (microcystin-LR, nitrite, and cadmium). The results showed that the full-length Dm-MnSOD sequence consists of 954bp nucleotides, encoding 215 amino acids, showing well-conserved domains that are required for metal binding and several common characteristics, such as two MnSOD domains. The deduced amino acid sequence of Dm-MnSOD shared over 70% similarity with homologues from Bythograea thermydron, Dromia personata, Cancer pagurus, and Scylla paramamosain. Dm-MnSOD gene expression was up-regulated in response to exposure to the three chemicals tested. The overall results indicated that Dm-MnSOD gene is an inducible gene and potential biomarker indicating these pollutants in the environment.

Mixed Ligand Copper (II) Complex with Isoniazid and Pyrazinamide

The fact that the pharmacological action of certain drugs was due to the formation of chelates with essential or trace metal ions of biological significance prompted many research workers all over the world to investigate the structural features of the metal chelates of drug substances both in solid state and in solution. So it was considered interesting and useful to study the metal complexes of drugs using various physicochemical methods. It is proposed to study the metal binding characteristics of some selected pharmaceuticals mentioned below. Isoniazid, a common antituberculosis drug, was shown to form complexes with metal ions like, Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ and Zn 2+ . (1) . Isoniazid was shown to form both 1:1 and 1:2 complexes with copper, binding through oxygen and amino nitrogen. (2-3) Pyrazinamide, another antituberculosis drug was reported to form metal complexes of the type MX2, 4L where M= Co(II), Ni(II), Cu(II) and X = Cl 1 , Br  , I  . Besides thermal and spectral studies of these complexes, ESR spectral study was also made for the copper complex. (4) Nickel was also shown to form neutral

Copper/Zinc Superoxide Dismutase from the CladoceranDaphnia magna: Molecular Cloning and Expression in Response to Different Acute Environmental Stressors

The copper/zinc superoxide dismutase (Cu/Zn-SOD) is a representative antioxidant enzyme that is responsible for the conversion of superoxide to oxygen and hydrogen peroxide in aerobic organisms. Cu/Zn-SOD mRNAs have been cloned from many species and employed as useful biomarkers of oxidative stresses. In the present study, we cloned Cu/Zn-SOD cDNA from the cladoceran Daphnia magna, analyzed its catalytic properties, and investigated mRNA expression patterns after exposure to known oxidative stressors. The full-length Cu/Zn-SOD of the D. magna (Dm-Cu/Zn-SOD) sequence consisted of 703 bp nucleotides, encoding 178 amino acids, showing well-conserved domains that were required for metal binding and several common characteristics. The deduced amino acid sequence of Dm-Cu/Zn-SOD showed that it shared high identity with Daphnia pulex (88%), Alvinella pompejana (56%), and Cristaria plicata (56%). The phylogenetic analysis indicated that Dm-Cu/Zn-SOD was highly homologous to D. pulex. The variation of Dm-Cu/Zn-SOD mRNA expression was quantified by real-time PCR, and the results indicated that the expression was up-regulated after 48-h exposure to copper, un-ionized ammonia, and low dissolved oxygen. This study shows that the Dm-Cu/Zn-SOD mRNA could be successfully employed as a biomarker of oxidative stress, which is a common mode of toxicity for many other aquatic hazardous materials.