Cd Binding Research Articles

Synthesis, supramolecular complexation and DFTstudies of a bis(pyrene)-appended ‘capped’ triazole-linked calix[4]arene as Zn2+ and Cd2+ fluorescent chemosensors

ABSTRACT A new bimodal triazole-bridged pyrene-appended calix[4]arene 2g has been synthesised and characterised and complexation studies showed that the binding of Cd2+ and Zn2+ occurs as demonstrated by enhanced monomer and declining excimer emission fluorescence spectral changes. With several other metal ions, quenching of both monomer and excimer emissions occurred. The observed monomer formation in the fluorescence spectra seen with Zn2+ and Cd2+ is possibly linked not just to the diminished parallel orientations of the pyrene rings but with the magnitude of the resulting HOMO-LUMO gaps and other parameters measured by DFT calculations.

Molecular cloning, characterization and expression profiles of CD2AP in Nile tilapia (Oreochromis niloticus) responding to Streptococcus agalactiae infection and interaction with CD2 cytoplasmic segment

The interaction between CD2-associated protein (CD2AP) and CD2 plays a vital role in lymphocyte adhesion and T cells activation in mammals. In this study, a CD2AP gene (GenBank accession number: MK579862; designated as On-CD2AP) was identified from tilapia (Oreochromis niloticus). Sequence analysis showed that On-CD2AP protein shares high similarity with mammals, including three Src homology 3 (SH3) domains, a section of poly proline motif and a coiled coil region. Transcription levels of On-CD2AP were detected in nine tissues of healthy Nile tilapia, and the highest expression levels were detected in the spleen and gill. On-CD2AP were significantly up-regulated in thymus, head kidney and brain after infected by Streptococcus agalactiae, as well as in head kidney leukocytes (HKLs) with LPS and LTA stimulation. Moreover, a section conserved pro-rich motif that are responsible for binding of CD2 to CD2AP were found in the CD2 cytoplasmic sequence of Nile tilapia (On-CD2C). A weak interaction between On-CD2AP and On-CD2C was proved by yeast two-hybrid assay. In addition, the recombinant proteins of CD2AP-His (rOn-CD2AP-His) and GST-CD2C (GST-rOn-CD2C) were obtained through prokaryotic expression system. His pull-down assay showed that rOn-CD2AP-His and GST-rOn-CD2C could bind to each other. These findings indicate that CD2AP is crucial in immune response during S.agalactiae infection, and the mechanism of interaction between CD2AP and CD2 is conservative in Nile tilapia.

A novel morphological ion imprinted polymers for selective solid phase extraction of Cd(II): Preparation, adsorption properties and binding mechanism to Cd(II)

A Cd(II) ion-imprinted polymers (SOS@Cd(II)-IIPs) with specific recognition property and high adsorption capacity was synthesized on a novel spheres-on-sphere (SOS) particle surface. Then, the obtained SOS@Cd(II)-IIPs were characterized and applied in solid phase extraction (SPE) column for the preconcentration of cadmium in real samples. The affinity constant (KITC), reaction enthalpy (ΔH), and entropy (ΔS) of Cd(II) binding to monomers (3-mercaptopropyl trimethoxysilane, MPTMS) were also determined by isothermal titration calorimetry (ITC) to analyse their adsorption mechanism. The coordination binding of Cd(II) to MPTMS was demonstrated as an exothermic process with a high KITC value of 1.60×105 M−1 and an enthalpy value of −9.54 kJ∙mol−1. The binding reaction was mainly driven by both enthalpy and entropy, and can be completed spontaneously at any temperatures. The SOS@Cd(II)-IIPs synthesized on the SOS particle surface exhibited a maximum adsorption capacity of 41.212 mg∙g−1 for Cd(II). Meanwhile, the SOS@Cd(II)-IIPs also showed a specificity recognition ability for Cd(II) using analogues Cu(II), Pb(II), Cr(II), and Ni(II) as interference. In real samples, SOS@Cd(II)-IIPs based SPE can extract approximately 95–105% of Cd(II) with a RSD of 3.41%–8.12%, indicating that it could have wide application prospects in Cd(II) adsorption and detection.

Screening strains for microbial biosorption technology of cadmium

Heavy metals contaminate the environment and provide a threat to public health through drinking water and food chain. Microbial biosorption technology provides a more economical and competitive solution for bioremediation of toxicants such as heavy metals, and microbial genetic modification may modify microbes towards optimal sorption. It is very important to screen suitable strains for this purpose. In this study, three different types of microorganisms Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae were isolated and identified, from uncontaminated soils, and compared their sorption differences with respect to cadmium (Cd2+). We evaluated the effects of contact time and initial concentration on Cd2+ uptake, and found pseudo-second-order kinetic models were more suitable to describe biosorption processes. Adsorption isotherms were used to reflect their biosorption capacity. The maximum biosorption capacities of three strains calculated by the Langmuir model were 37.764, 56.497, and 22.437 mg Cd/g biomass, respectively. In bacteria, Cd2+ biosorption mainly occurred on cell wall, while the difference in biosorption between yeast inside and outside the cell was not significant. We found that due to the structural differences, the removal rate of E. coli surface decreased at a high concentration, while S. cerevisiae still had a lower biosorption capacity. FTIR spectroscopy reflected the difference in functional groups involved in biosorption by three strains. SEM-EDS analysis showed the binding of Cd2+ to microorganisms mainly relied on ion exchange mechanism. Based on the above results, we suggested that B. subtilis is more suitable to get genetically modified for heavy metal biosorption.

Cadmium and proton adsorption onto a halophilic archaeal species: The role of cell envelope sulfhydryl sites

The adsorption of metal onto archaea could affect metal behavior and bioavailability in hypersaline environments where archaea represent the dominant life form. However, the mechanism and thermodynamics of metal adsorption onto halophilic archaea are not well understood. In this study, Cd adsorption experiments and potentiometric titrations were conducted to model metal adsorption by the halophilic archaeon Halobacterium sp.. Cd adsorption onto the archaeal cells exhibits slow adsorption kinetics, taking 8 h to reach steady-state. The extent of Cd adsorption depends strongly on pCH+ and Cd loading. Desorption of Cd is even slower than adsorption, but exposure of the Cd-adsorbed biomass to high concentrations of cysteine promotes desorption and demonstrates that the distribution of Cd is controlled by reversible adsorption reactions, likely involving sulfhydryl binding of Cd on the cell surface. We conducted potentiometric titration experiments with and without sulfhydryl site blocking to yield a total sulfhydryl site concentration of 91 ± 28 μmol/g within the archaeal cell envelope. Using a combination of the X-ray absorption fine structure analysis results from Showalter et al. (2016) and the Cd adsorption measurements and titration data from this study, we construct a surface complexation model of the Cd adsorption behavior. Because the Cd adsorption behavior is likely similar to that of a range of chalcophilic micronutrients, the importance of Cd-sulfhydryl binding suggests that the production of high affinity sulfhydryl binding sites by the archaea may represent an adaptive strategy for halophiles to obtain metal nutrients in environments in which aqueous metal-chloride complexation dominates the speciation for many metals.

Binding and adsorption energy of Cd in soils and its environmental implication for Cd bioavailability

AbstractThe interaction of Cd and other heavy metals with soil colloidal particles controls the sequestration, mobility and bioavailability of Cd in soils. In this study, the binding (△Gbi) and adsorption (△Gad) energies of Cd on colloidal particles of 18 soils were determined by the Wien effect method. The binding energy of Cd on soil colloidal particles varied from 5.3 to 9.9 kJ mol−1, depending on the soil characteristics including pH, Mn‐oxide content and dissolved organic carbon in the soil. The Cd adsorption energy correlated positively with Mn‐oxide content and pH. In parallel, the extended X‐ray absorption fine structure (EXAFS) spectroscopy was used to determine the speciation of Cd in Cd‐saturated soil samples, which revealed that the outer‐sphere Cd was the predominant species, accounting for 32.2–73.7% of the total adsorbed Cd, and positively correlated to the binding and adsorption energies. Humic acid‐Cd (10.4–42.2%) and montmorillonite‐Cd (2.5–51.2%) were also major species that were identified by EXAFS spectroscopy. The toxicity (log EC50) of Cd in soils to three organisms (earthworm, Collembola and Chinese cabbage) was found to correlate positively with the binding energies, indicating the predictive capability of using binding energies of Cd in different soils as an indicator for evaluating Cd bioavailability and toxicity in soils.

Overexpression of a Defensin-Like Gene CAL2 Enhances Cadmium Accumulation in Plants

Accumulation and detoxification of cadmium in rice shoots are of great importance for adaptation to grow in cadmium contaminated soils and for limiting the transport of Cd to grains. However, the molecular mechanisms behind the processes involved in this regulation remain largely unknown. Defensin proteins play important roles in heavy metal tolerance and accumulation in plants. In rice, the cell wall-localized defensin protein (CAL1) is involved in Cd efflux and partitioning to the shoots. In the present study, we functionally characterized the CAL2 defensin protein and determined its contribution to Cd accumulation. CAL2 shared 66% similarity with CAL1, and its mRNA accumulation is mainly observed in roots and is unaffected by Cd stress, but its transcription level was lower than that of CAL1 based on the relative expression of CAL2/Actin1 observed in this study and that reported previously. A promoter-GUS assay revealed that CAL2 is expressed in root tips. Stable expression of the CAL2-mRFP fusion protein indicated that CAL2 is also localized in the cell walls. An in vitro Cd binding experiment revealed that CAL2 has Cd chelation activity. Overexpression of CAL2 increased Cd accumulation in Arabidopsis and rice shoots, but it had no effect on the accumulation of other essential elements. Heterologous expression of CAL2 enhanced Cd sensitivity in Arabidopsis, whereas overexpression of CAL2 had no effect on Cd tolerance in rice. These findings indicate that CAL2 positively regulates Cd accumulation in ectopic overexpression lines of Arabidopsis and rice. We have identified a new gene regulating Cd accumulation in rice grain, which would provide a new genetic resource for molecular breeding.

Pb and Cd chemisorption by acid mineral soils with variable Mn and organic matter contents

Adsorption of Pb2+ and Cd2+ on acidic A and B horizon mineral soils of New York (MP) and Vermont (BH) were determined in 0.2 M Na acetate buffered at pH 5.5 to determine strength and capacity of the soils for chemisorption of these trace metal ions. Adsorption isotherms over a very wide concentration range were better modeled using the linearized Freundlich compared to the linearized Langmuir equation. Adsorption of Pb was much stronger than that of Cd for all soils, and A horizon soils adsorbed Pb and Cd more strongly than their B horizon counterparts. The logarithms of the Freundlich binding coefficients (log KF) were greater by about 1.0 unit for Pb than for Cd for all but the B-horizon of the MP soil, which was the least strongly adsorptive of all soils tested. Pb binding constants were significantly correlated to hydroxylamine- and NH4 oxalate-extractable Fe and Mn in addition to soil CEC, but the Cd binding constants were correlated significantly to extractable Fe only. Partition coefficients (KP) were calculated for Pb and Cd from the adsorption experiments, and for the lowest additions of Pb to the A-horizon soils, ranged from more than 4000 L kg−1 in the BH soils to about 300 in the more acidic MP soils. Cd partition coefficients were much lower than those of Pb in all soils, and the MP soils retained Cd particularly weakly, with a KP in the range of 3–20 for the A-horizon and even lower for the B-horizon. Pb and Cd were adsorbed more strongly on A-horizon than B-horizon soils, indicating the importance of organic matter in both Pb and Cd retention by chemisorption at low pH. For both Pb and Cd, partition coefficients diminished markedly with increasing metal addition levels.Adsorption of Pb and Cd ions caused release of alkaline earth metals and Mn into solution during the adsorption process on the high-Mn BH soils, whereas the more strongly acidic MP soils released Al. Decreasing solubility of P and S corresponding to increasing Pb adsorption on the soils suggested that, in addition to chemisorption, precipitation of Pb phosphate and Pb sulfate may have been involved in Pb removal from solution.

Speciation of heavy metals in bottom sediments of a drinking water reservoir for Gdańsk, Poland – changes over the 14 years

There was conducted a comparative study of the concentrations of heavy metals (Zn, Cu, Pb, and Cd) in the surface layer of sediments collected in two investigation periods: 1999–2000 and 2013. The results demonstrated that the degree of metals pollution decreased over time. The contamination factors (Cf) calculated for Zn, Cu, Pb, and Cd were respectively 3.63, 3.86, 3.23, and 3.25 in the first period of research and 1.35, 3.42, 1.98, and 1.43 in the second one. The metal speciation analysis indicated that Zn was mainly associated with the Fe–Mn oxides fraction and the residual fraction, and Cu – with the residual fraction and the organic fraction in both periods of investigation, while the percentage of geochemical fractions in binding of Pb and Cd changed in time. The share of fractions Fe–Mn oxides/carbonate increased in the case of Pb, and carbonate/exchangeable fractions in the case of Cd. The risk assessment code (RAC) decreased in the following order: Cd > Zn > Pb > Cu and was higher in 2013 - achieved the very high-risk category in the case of Cd. Thus, despite the decrease of sediment pollution, RAC for Cd increased due to changed geochemical speciation.

Impact of drying/wetting conditions on the binding characteristics of Cu(ii) and Cd(ii) with sediment dissolved organic matter.

The biogeochemical processing of dissolved organic matter (DOM) in bottomland sediment under drying/wetting conditions regulates the environmental behavior of heavy metals. Although moisture is a critical factor, the structural characteristics of DOM and its reactivity with heavy metals under drying/wetting conditions are not well known. Herein, the response of DOM to drying/wetting conditions and its influence on the binding of Cu(ii) and Cd(ii) onto DOM were clarified via various multi-spectroscopic techniques. Ultraviolet-visible spectra (UV-Vis) showed that higher aromatic, hydrophobic, and molecular weight fractions were observed in sediment DOM under drying conditions than those under wetting conditions. The binding abilities for Cd(ii) with DOM under drying/wetting conditions are lower than those for Cu(ii). The stability constants between Cu(ii) and DOM were found to decrease under drying/wetting conditions; however, the binding capacities for Cu(ii) increased, especially under wetting conditions. Two-dimensional correlation spectroscopy based on Fourier-transform infrared (FTIR) and synchronous fluorescence spectra (SFS) showed that Cu(ii) and Cd(ii) have different binding sequences and binding sites and that Cu(ii) has more binding sites under drying and wetting conditions; however, Cd(ii) shows the opposite behavior. These results clearly demonstrate that the binding of sediment DOM with Cu(ii) is more prevalent and stable compared with Cd(ii) under drying and wetting conditions. Because of its relatively low binding capacity and binding stability, Cd(ii) can exhibit a high environmental hazard for migration and transformation with DOM due to water flow under wetting conditions. This study helps reveal the impact of drying/wetting conditions on the environmental behavior of heavy metals in bottomland wetlands.

Chronic exposure of soybean plants to nanomolar cadmium reveals specific additional high-affinity targets of cadmium toxicity.

Solving the global environmental and agricultural problem of chronic low-level cadmium (Cd) exposure requires better mechanistic understanding. Here, soybean (Glycine max) plants were exposed to Cd concentrations ranging from 0.5 nM (background concentration, control) to 3 µM. Plants were cultivated hydroponically under non-nodulating conditions for 10 weeks. Toxicity symptoms, net photosynthetic oxygen production and photosynthesis biophysics (chlorophyll fluorescence: Kautsky and OJIP) were measured in young mature leaves. Cd binding to proteins [metalloproteomics by HPLC-inductively coupled plasma (ICP)-MS] and Cd ligands in light-harvesting complex II (LHCII) [X-ray absorption near edge structure (XANES)], and accumulation of elements, chloropyll, and metabolites were determined in leaves after harvest. A distinct threshold concentration of toxicity onset (140 nM) was apparent in strongly decreased growth, the switch-like pattern for nutrient uptake and metal accumulation, and photosynthetic fluorescence parameters such as Φ RE10 (OJIP) and saturation of the net photosynthetic oxygen release rate. XANES analyses of isolated LHCII revealed that Cd was bound to nitrogen or oxygen (and not sulfur) atoms. Nutrient deficiencies caused by inhibited uptake could be due to transporter blockage by Cd ions. The changes in specific fluorescence kinetic parameters indicate electrons not being transferred from PSII to PSI. Inhibition of photosynthesis combined with inhibition of root function could explain why amino acid and carbohydrate metabolism decreased in favour of molecules involved in Cd stress tolerance (e.g. antioxidative system and detoxifying ligands).

Efficient and very selective dual mode chemosensor for Cd(II) ions in live cells

A sensitive dual mode chemosensor NS-1 comprising Nitrobenzoxadiazole and salicylhydrazide conjugate has been synthesized via single step reaction. The probe NS-1 is characterized by analytical techniques such as multi nuclear NMR techniques and Mass spectrometry. The probe is showing a strong change in color from yellow to red on treatment of Cd(II) ions, interestingly its shows bright “Switch-ON” fluorescence state upon binding of Cd2+ ions in buffer solution whereas other cations did not showed any color change as well as fluorescent change. Interestingly the probe NS-1 did not results in the any color and fluorescence change with the adding together of Zn(II) ions, hence the probe is able to differentiate between Cd(II) ions from Zn(II). Further the color change and turn-on fluorescence can be rationalized by the interruption of internal charge transfer upon binding of Cd(II) ions with NS-1. The Internal charge transfer disturbance led to fluorescence change of the receptor NS-1 upon addition of Cd2+ has been further supported by TD-DFT calculations. The limit of detection was found to be 6.31 nano molar. The association constant was found to be 7.97*104 M−1 using Benesi-Hildebrand plot method. MTT assay suggesting that the probe NS-1 is least toxic to cells and it will be widely applicable to image Cd(II) ions in living cells via fluorescence imaging.

Terrestrial-derived soil protein in coastal water: metal sequestration mechanism and ecological function

Terrestrial fungi, especially arbuscular mycorrhizal (AM) fungi, enhance heavy metal sequestration and promote ecosystem restoration. However, their ecological functions were historically overlooked in discussions regarding water quality. As an AM fungi-derived stable soil protein fraction, glomalin-related soil protein (GRSP) may provide insights into the ecological functions of AM fungi associated with water quality in coastal ecosystems. Here, we first assessed the metal-loading dynamics and ecological functions of GRSP transported into aquatic ecosystems, characterized the composition characteristics, and revealed the mechanisms underlying Cu and Cd sequestration. Combining in situ sampling and in vitro cultures, we found that the composition characteristics of GRSP were significantly affected by the element and mineral composition of sediments. In situ, GRSP-bound Cu and Cd contributed 18.91–22.03% of the total Cu and 2.27–6.37% of the total Cd. Functional group ligands and ion exchange were the principal mechanisms of Cu binding by GRSP, while Cd binding was dominated by functional group ligands. During the in vitro experiment, GRSP sequestered large amounts of Cu and Cd and formed stable complexes, while further dialysis only released 25.74 ± 3.85% and 33.53 ± 3.62% of GRSP-bound Cu and Cd, respectively.

Assessment of biochar and/or nano zero-valent iron for the stabilisation of Zn, Pb and Cd: A temporal study of solid phase geochemistry under changing soil conditions.

The remediation of a soil contaminated with Zn, Pb and Cd was tested by using biochar (BC), nano zero-valent iron (nZVI) and a combination of these two (BC+nZVI). Each amendment was individually applied to the soil at 2wt%. We tested the influence of (i) the used amendments, (ii) time, and (iii) soil moisture conditions on the metal availability and soil physico-chemical parameters using various extraction methods, as well as soil pore water samplings. We found that metal availability was mainly affected by pH under the influence of time and water content. Among the tested treatments, BC was the most successful, resulting in the lowest amounts of the target metals in the pore water and the smallest temporal changes in metal concentrations and pH in the soil. The use of nZVI efficiently decreased water-extractable Pb in the short- and long-term. The BC+nZVI treatment also yielded promising results regarding the immobilisation of the studied metals. Time provoked a general decrease in pH, which occasionally increased the available metal concentrations. Raising the soil water content increased the pH and subsequently lowered the available metal concentrations in the pore water. The mechanisms of metal stabilisation were further investigated by SEM/EDS. The results indicated that the used soil amendments enhanced the binding of Zn, Pb, and Cd on Fe/Mn/Al oxides/hydroxides, which in turn resulted in the stabilisation of the target metals.

Characteristics of cadmium immobilization in the cell wall of root in a cadmium-safe rice line (Oryza sativa L.).

Cultivating cadmium (Cd)-safe rice lines, which show low Cd accumulation in brown rice, is generally beneficial to ensure food safety. The Cd retention in root of Cd-safe rice line D62B plays an important role in its low Cd translocation from root to shoot. To understand the mechanism of Cd retention in root, a hydroponic experiment was conducted to investigate the subcellular distribution of Cd and the contribution of polysaccharides to Cd binding to the root cell wall of a Cd-safe rice line D62B with a common rice line Luhui17 as a control material. D62B retained more Cd in the root by sequestrated a higher proportion of Cd in the cell wall, further it transferred less Cd to shoot. Close to half of the Cd in the root cell wall of D62B was accumulated in the hemicellulose 1 (HC1), and the proportions of HC1 in it were 1.2-1.7 times higher than these of Luhui17. The proportion of Cd in the pectin showed a dose-dependent increase in two rice lines. D62B contained significantly higher uronic acid concentrations of the pectin and greater pectin methyl esterase (PME) activities than Luhui17 in the root cell wall. These results indicated that a superior Cd binding capacity of the cell wall polysaccharides in D62B played an important role in its Cd retention in root.

Characterization of Cd2+ biosorption by Pseudomonas sp. strain 375, a novel biosorbent isolated from soil polluted with heavy metals in Southern China

Water pollution with heavy metals is a global problem. Using microbial adsorbents to remediate water bodies contaminated with heavy metals has been garnering considerable attention. In this study, a cadmium (Cd2+)-resistant bacterium, isolated from soil polluted with heavy metals, was characterized as Pseudomonas sp. 375 based on its biochemical characteristics and 16S rRNA gene. The minimum inhibitory concentration (MIC) of Cd2+ for strain 375 was 6 mM. We evaluated the effects of different parameters, such as initial pH, contact time, and initial Cd2+ concentration, on Cd2+ uptake. The data acquired using nonliving biomass were fitted to a Langmuir isotherm model; however, the Freundlich isotherm model showed better fit for data acquired using living biomass. The maximum biosorption capacities were 92.59 mg g−1 and 63.29 mg g−1 for living and nonliving cells, respectively. The kinetics of biosorption was described using a pseudo-second order kinetic model. The tightly bound Cd on the cell wall played a major role in Cd2+ adsorption for both biosorbents. SEM-EDX analysis also showed that Cd2+ was bound to the cell wall. FTIR spectral analysis showed that –CH2, –OH, –SO3, CO, N–H, C–N, phosphate, or sulfate functional groups were the main functional sites for the binding of Cd2+ ions. Effectively Cd2+ removal from Cd2+ contaminated water suggested Pseudomonas sp. 375 was an (a) inexpensive, effective, and promising biosorbent that can be used for bioremediation Cd2+-contaminated wastewater.

Phosphate-solubilizing bacteria will not significantly remobilize soil cadmium remediated by weathered coal.

Cadmium (Cd) re-mobilize by phosphate-solubilizing bacteria (PSB) from immobilization contaminated soil has drawn great attention due to its serious threat to human health through food chain. However, Cd binding with weathered coal (WC), an effective Cd immobilization material, will be re-mobilized by PSB or not is still unclear. In this study, the soil and sand pots with Cd were respectively mixed with the weight fractions of 0‰, 2‰, and 3‰ WC, inoculated with or without PSB, and planted with Amaranthus mangostanus L. The experimental results indicated that: (i) Cd in soil was transformed into organic fraction with WC, which has been led to the Cd accumulation concentrations in roots and shoots reduced by 38.8% and 20.5%, respectively; (ii) PSB could promote the concentration of exchangeable-Cd fraction and soil Cd uptake by amaranth in all treatments; and (iii) WC application in sand pot respectively reduced the Cd accumulation by 47.5% in roots and 24.1% in shoots, but PSB inoculation showed no significant effect on Cd accumulation in plants under WC application. SEM, zeta potential, and FT-IR results showed that PSB inoculation after Cd immobilized by WC had no influence on the microstructure, amount of negative charge, type, and content of functional groups in WC, indicating that organic fraction Cd in WC was not re-mobilized by PSB. Therefore, the application of WC in contaminated soil was conducive to transforming Cd in organic-bound forms and intensifying Cd immobilization effects.

Ultratrace Metal Speciation Analysis by Coupling of Sector-Field ICP-MS to High-Resolution Size Exclusion and Reversed-Phase Liquid Chromatography.

Techniques for metal speciation analysis with subnanomolar (ppt) detection limits in complex matrices, with simultaneous quantification of matrix elements, have become a necessity for investigating targets of trace metal binding to macromolecules and pigments at environmentally relevant concentrations. In this work we optimized the analysis of such metal binding in a custom-built HPLC-ICP-MS system. Key elements of the optimization were the choice of components for the metal-free HPLC-DAD system and sector-field ICP-MS detection (ICF-sfMS) with desolvating injection and optimization of sample handling. Protein analysis was done using ammonium bicarbonate buffer and size exclusion chromatography (SEC-ICP-sfMS), with possible addition of anion exchange chromatography. Detection of metal exchange in pigments (chlorophylls and bacteriochlorophylls) was based on reversed-phase chromatography with a methanol-acetone gradient and coupling to the ICP-sfMS via a dedicated organic matrix interface (RPC-ICP-sfMS). The resulting HPLC-DAD-ICP-sfMS system has detection limits in the picomolar range in protein buffer, limited by the maximal achievable purity of buffers/solvents and not by system sensitivity. Tests for method optimization showed that sonication, meant to increase protein solubilization, leads to artifacts of metal loss from metalloproteins. Examples for Cd binding to soybean proteins and chlorophyll, Cr binding to Arabidopsis thaliana proteins, La binding to Desmodesmus quadricauda proteins, and Cu binding to Rhodospirillum rubrum proteins and pigments are shown. These application examples demonstrate that the system is sensitive enough to detect binding of metals to proteins and pigments at background concentration levels of typical nutrient solutions made from analytical grade chemicals, equivalent to ultratrace metal concentrations in nonpolluted environments.

Heterologous expression of Zn-binding peptide RaZBP1 from Russula bresadolae does not overcome Zn and Cd detoxification mechanisms in Hebeloma mesophaeum.

Homeostatic mechanisms preventing the toxicity of heavy metal ions in cells involve, among others, compartmentalization and binding with peptidaceous ligands, particularly the cysteinyl-rich metallothioneins (MTs). We have previously shown that in natural conditions Zn-overaccumulating ectomycorrhizal (EM) fungus Russula bresadolae stores nearly 40% of Zn bound with cysteinyl- and hystidyl-containing RaZBP peptides, which resemble MTs, while the detoxification of Zn and Cd in EM Hebeloma mesophaeum relies upon compartmentalization in small vesicles and vacuoles, respectively. Here, we examined the performance of RaZBP1 gene expressed in H. mesophaeum mycelium with respect to handling of Zn and Cd. Expression of RaZBP1 impaired growth of the mycelium on low-Zn medium by 60%, the growth was partly ameliorated upon the addition of Zn and remained considerable up to 2mmol/L Zn, while the growth of the wild-type and control mycelia transformed with empty T-DNA was severely reduced in the presence of 0.5mmol/L Zn; furthermore, RaZBP1 slightly added to Cd tolerance in the range of Cd concentrations of 0.625 to 8μmol/L. Staining of Zn- or Cd-exposed hyphal cells with Zn- or Cd-specific fluorescent tracers did not indicate that the expression of RaZBP1 would redirect the flow of the metals away from their innate sinks. Size exclusion chromatography of extracted metal species revealed that the complexes corresponding to Zn/Cd-RaZBP1 are present only in minute levels. Considering that RaZBP1 inhibited growth at low Zn, and despite the benefit that it provided to H. mesophaeum in the presence of high Zn and moderate Cd, these data indicate that the binding of excess Zn and Cd with RaZBP1 is not a trait that would be outright transmitted to H. mesophaeum.

Biosorption of cadmium by potential probiotic Pediococcus pentosaceus using in vitro digestion model.

An exponential increase of heavy metal toxicant in the human body is a growing health-related problem. In the present study, Pediococcus pentosaceus FB145 and FB181 strains were isolated from fermented seafood and served as highly Cd-resistant strains. The unchanged structural cells of P. pentosaceus strains after treatment with Cd cations were investigated using a scanning electron microscope energy dispersive X-ray analysis. Furthermore, both P. pentosaceus strains showed strong human gastrointestinal tract resistance properties. The Cd biosorption results fit the pseudo-second-order kinetics with capacities for P. pentosaceus FB145 and FB181 for Cd were 52.8 and 50.3 mg/g, respectively, whereas the maximum biosorption capacities were 52.65 and 50.35 mg/g, respectively. The equilibrium data were well fitted to the Freundlich isotherm model. The binding of Cd to bacterial cells may be caused by the presence of different functional groups such as carboxyl, amide, and phosphate on cell surface, which was confirmed by the Fourier transform infrared-attenuated total reflectance spectra. Moreover, these strains decreased the Cd bioaccessibility by 44.7-46.8% in the in vitro digestion model. These findings indicate that P. pentosaceus FB145 and FB181 are novel potent biosorbent for preventing cadmium toxicity and reducing its absorption into the human body.