Sulfation Patterns of Saccharides and Heavy Metal Ion Binding

Sulfated saccharides are an essential part of extracellular matrices, and they are involved in a large number of interactions. Sulfated saccharide matrices in organisms accumulate heavy metal ions in addition to other essential metal ions. Accumulation of heavy metal ions alters the function of the organisms and cells, resulting in severe and irreversible damage. The effect of the sulfation pattern of saccharides on heavy metal binding preferences is enigmatic because the accessibility to structurally defined sulfated saccharides is limited and because standard analytical techniques cannot be used to quantify these interactions. We developed a new strategy that combines enzymatic and chemical synthesis with surface chemistry and label-free electrochemical sensing to study the interactions between well-defined sulfated saccharides and heavy metal ions. By using these tools we showed that the sulfation pattern of hyaluronic acid governs their heavy metal ions binding preferences.     

High adsorptive γ-AlOOH(boehmite)@SiO2/Fe3O4 porous magnetic microspheres for detection of toxic metal ions in drinking water

γ-AlOOH(boehmite)@SiO(2)/Fe(3)O(4) porous magnetic microspheres with high adsorption capacity toward heavy metal ions were found to be useful for the simultaneous and selective electrochemical detection of five metal ions, such as ultratrace zinc(II), cadmium(II), lead(II), copper(II), and mercury(II), in drinking water.     

Soil-modified carbon paste electrode: a useful tool in environmental assessment of heavy metal ion binding interactions

Carbon paste electrodes (CPEs) modified with different soils in their native form were prepared to create a soil-like solid phase suitable for application in studies of heavy metal ion uptake and binding interactions. The preparation of CPEs modified with five different soils was examined and their heavy metal ion uptake behavior investigated using a model Cu(II) aqueous solution. Metal ions were accumulated under open circuit conditions and were determined after a medium exchange using differential pulse anodic stripping voltammetry, applying pre-electrolysis at –0.7 V. The soil-modified CPE accumulation behavior, including the linearity of the current response versus Cu(II) concentration, the influence of the pH on the solution, and the uptake kinetics, was thoroughly investigated. The correlation between the soil-modified CPE uptake capability and the standard soil parameters, such as ion exchange capacity, soil pH, organic matter and clay content, were evaluated for all five examined soils. The influence of selected endogenous cations (K(I), Ca(II), Fe(III)) on the transfer of Cu(II) ions from a solution to the simulated soil solid phase was examined and is discussed. Preliminary examinations of the soil-modified CPE uptake behavior with some exogenous heavy metal ions of strong environmental interest (Pb(II), Hg(II), Cd(II) and Ag(I)) are also presented. This work demonstrates some attractive possibilities for the application of a soil-modified CPE in studying soil-heavy metal ion binding interactions, with a further potential use as a new environmental sensor appropriate for fast on-site testing of polluted soils.     

Soil-modified carbon paste electrode: a useful tool in environmental assessment of heavy metal ion binding interactions

Carbon paste electrodes (CPEs) modified with different soils in their native form were prepared to create a soil-like solid phase suitable for application in studies of heavy metal ion uptake and binding interactions. The preparation of CPEs modified with five different soils was examined and their heavy metal ion uptake behavior investigated using a model Cu(II) aqueous solution. Metal ions were accumulated under open circuit conditions and were determined after a medium exchange using differential pulse anodic stripping voltammetry, applying preelectrolysis at -0.7 V. The soil-modified CPE accumulation behavior, including the linearity of the current response versus Cu(II) concentration, the influence of the pH on the solution, and the uptake kinetics, was thoroughly investigated. The correlation between the soil-modified CPE uptake capability and the standard soil parameters, such as ion exchange capacity, soil pH, organic matter and clay content, were evaluated for all five examined soils. The influence of selected endogenous cations (K(I), Ca(II), Fe(III)) on the transfer of Cu(II) ions from a solution to the simulated soil solid phase was examined and is discussed. Preliminary examinations of the soil-modified CPE uptake behavior with some exogenous heavy metal ions of strong environmental interest (Pb(II), Hg(II), Cd(II) and Ag(I)) are also presented. This work demonstrates some attractive possibilities for the application of a soil-modified CPE in studying soil-heavy metal ion binding interactions, with a further potential use as a new environmental sensor appropriate for fist on-site testing of polluted soils.     

Synthesis, electrochemistry and metal binding properties of monosubstituted ferrocenoyl peptides with thioether-containing sidechains

Ferrocenoyl peptides incorporating thioether functionality respond more strongly to mercury(II) than to other heavy metal ions in solution. Compounds reported previously in this context are all 1,1′-disubstituted, and all include two or more sulfur-containing amino acids. To test whether two thioether groups are required for effective mercury binding by these systems, we have prepared a series of singly-substituted ferrocenoyl peptides from ferrocenecarboxylic acid and l-methionine, S-methyl-l-cysteine or S-trityl-l-cysteine, and tested them as electrochemical probes for mercury(II). Nine ferrocenoyl peptides have been synthesised using a Boc-protecting group strategy and HBTU-mediated peptide coupling. The electrochemical properties of these compounds have been determined using cyclic voltammetry, and all show fully reversible one electron oxidation steps. Forward sweep half wave peaks (E_F), reverse sweep half wave peaks (E_R), peak separations (ΔE_P) and half wave potentials (E_1/2) are reported. Changes in the potential of the iron(II)/iron(III) redox couple of the ferrocene core have been used to quantify heavy metal-peptide interactions, revealing that these monotopic systems also respond more strongly to mercury(II) than to zinc(II), cadmium(II), silver(I) and lead(II). NMR experiments to characterise the peptide-mercury interaction implicate the thioether sulfur as the site of mercury binding and indicate 1:1 stoichiometry. The crystal structure of ferrocenoyl-S-methyl-l-cysteine methyl ester is also reported. The greater responsiveness of these systems to mercury(II) makes them interesting leads for the development of biologically inspired sensors for this toxic heavy metal.     

Reactivity of Thioglycolic Acid Physically and Chemically Bound to Silica Gel as New Selective Solid Phase Extractors for Removal of Heavy Metal Ions From Natural Water Samples

A method is presented for the immobilization of thioglycolic acid moiety on the surface of active silica gel via a simple and direct synthetic route and based on one step reaction procedure. Two-product solid phase extractors were successfully synthesized according to physical adsorption and chemical immobilization binding techniques, phases (I) and (II), respectively. The mode of interaction between the silanol group and the thioglycolic moiety was also discussed for both phases based on the infrared analysis studies. The thermal stability properties as well as the effect of buffer solutions on the percentage hydrolysis of the two silica gel phases were examined and revealed the high stability and superiority of silica phase (II) in these respects. The evaluation of the selectivity and metal uptake properties incorporated in these two silica gel phases were also studied and discussed for a series of divalent heavy metal ions under different controlling factors. The mmol/g values were found to be higher in case of phase (I). The selective removal and extraction of some heavy metal ions, viz . Cu(II), Zn(II), and Hg(II) from natural seawater samples was successfully accomplished with the percentage recovery values for the three tested metal ions in the range of 96.5-98.4 - 0.2-0.6%. The presence of higher concentrations of Na(I), K(I), Mg(II) and Ca(II) showed insignificant role or no matrix effect on such selective extraction process due to their 0% values of removal by these silica gel phases (I) and (II).     

Single Stage Tandem Mass Spectrometry Assignment of the C-5 Uronic Acid Stereochemistry in Heparan Sulfate Tetrasaccharides using Electron Detachment Dissociation

The analysis of heparan sulfate (HS) glycosaminoglycans presents many challenges, due to the high degree of structural heterogeneity arising from their non-template biosynthesis. Complete structural elucidation of glycosaminoglycans necessitates the unambiguous assignments of sulfo modifications and the C-5 uronic acid stereochemistry. Efforts to develop tandem mass spectrometric-based methods for the structural analysis of glycosaminoglycans have focused on the assignment of sulfo positions. The present work focuses on the assignment of the C-5 stereochemistry of the uronic acid that lies closest to the reducing end. Prior work with electron-based tandem mass spectrometry methods, specifically electron detachment dissociation (EDD), have shown great promise in providing stereo-specific product ions, such as the B₃ ^´ –CO₂, which has been found to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) in some HS tetrasaccharides. The previously observed diagnostic ions are generally not observed with 2-O-sulfo uronic acids or for more highly sulfated heparan sulfate tetrasaccharides. A recent study using electron detachment dissociation and principal component analysis revealed a series of ions that correlate with GlcA versus IdoA for a set of 2-O-sulfo HS tetrasaccharide standards. The present work comprehensively investigates the efficacy of these ions for assigning the C-5 stereochemistry of the reducing end uronic acid in 33 HS tetrasaccharides. A diagnostic ratio can be computed from the sum of the ions that correlate to GlcA to those that correlate to IdoA.

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Alginate Properties and Heavy Metal Biosorption by Marine Algae

The physical properties of the alginate component in four different brown seaweeds (Sargassumfluitans, Ascophyllum nodosum, Fucus vesiculo-sus, andLaminaria japonica) were characterized using potentiometric titration,¹³C-nuclear magnetic resonance (NMR), chemical analysis, and viscosity measurements. The heavy metal binding capacities of the corresponding seaweeds were directly proportional to their respective total carboxyl group content, and related to the electronegativity of the elements investigated (Ca, Zn, Cd, Cu, and Pb). The uronic acid composition or sequence of the alginate component did not affect the metal uptake properties of the biosorbents studied here. However, the alginate leaching owing to its solubilization by Na ions was observed to decrease with increasing intrinsic viscosity of the extracted alginate, related to its molecular weight, and with increasing apparent acidic dissociation constant, related to the alginate density inside the biomass.     

Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating (HexA-GalNAc sulfate) disaccharides, the functions of which depend on patterns of sulfation and uronic acid epimerization. The correlation of biological activities with structure requires a strategy to determine the sequences of CS oligosaccharides without the need for total isolation. Tandem mass spectrometry has enabled the development of proteomics, based on CID fragmentation of ions produced from complex mixtures of proteolytic peptides, and has the potential for rapid sequencing of CS and other glycosaminoglycan classes. The most challenging aspects of CS sequencing are to distinguish GalNAc residues sulfated at the 4- versus the 6-position and uronic acid epimers. This work describes the utility of (1) reducing terminal derivatives and (2) control of precursor ion charge state for tandem mass spectrometric strategies for determining GalNAc sulfation positional isomers of CS. The capability of tandem MS to differentiate uronic acid epimers is also shown, providing evidence that complete or nearly complete information on CS covalent structure may be obtained using tandem MS.     

Highly selectively monitoring heavy and transition metal ions by a fluorescent sensor based on dipeptide

Fluorescent sensor (DMH) based on dipeptide was efficiently synthesized in solid phase synthesis. The dipeptide sensor shows sensitive response to Ag(I), Hg(II), and Cu(II) among 14 metal ions in 100% aqueous solution. The fluorescent sensor differentiates three heavy metal ions by response type; turn on response to Ag(I), ratiometric response to Hg(II), and turn off detection of Cu(II). The detection limits of the sensor for Ag(I) and Cu(II) were much lower than the EPA's drinking water maximum contaminant levels (MCL). Specially, DMH penetrated live cells and detected intracellular Ag⁺ by turn on response. We described the fluorescent change, binding affinity, detection limit for the metal ions. The study of a heavy metal-responsive sensor based on dipeptide demonstrates its potential utility in the environment field.     

Novel dye-attached macroporous films for cadmium, zinc and lead sorption: Alkali Blue 6B-attached macroporous poly(2-hydroxyethyl methacrylate)

Alkali Blue 6B-attached poly(2-hydroxyethyl methacrylate) (poly(HEMA)) microporous films were investigated as chelate forming sorbents for heavy metal removal. Poly(HEMA) microporous films were prepared by UV-initiated photo-polymerization of HEMA in the presence of an initiator (azobisisobutyronitrile (AIBN)). Alkali Blue 6B was attached covalently. These films with a swelling ratio of 58%, and carrying 14.8 mmol Alkali Blue 6B m(-2) which were then used in the removal of Cd(II), Zn(II) and Pb(II) from aqueous media. Adsorption rates were very high, equilibrium was achieved in about 30 min. The maximum adsorption of heavy metal ions onto the Alkali Blue 6B-attached films were 41.4 mmol m(-2) for Cd(II), 52.4 mmol m(-2) for Zn(II), and 64.5 mmol m(-2) for Pb(II). When the heavy metal ions competed during the adsorption from a mixture the adsorption values for Cd(II), Zn(II) and Pb(II) were quite close. Heavy metal ions were desorbed by using 0.1 M HNO(3). A significant amount of the adsorbed heavy metal ions (up to 95%) could be desorbed in 30 min. Repeated adsorption/desorption cycles showed the feasibility of these novel dye-attached microporous films for heavy metal removal.     

Synthesis and electrochemical studies of disubstituted ferrocene/dipeptide conjugates with sulfur-containing side chains

A series of 1,1′-disubstituted ferrocenoyl peptides incorporating dipeptide sidearms has been synthesized and studied electrochemically. The target peptides include ferrocene as an electrochemical reporter, sulfur-containing amino acids (l-methionine, S-methyl-l-cysteine, S-trityl-l-cysteine, S-benzhydryl-l-cysteine) as metal binding agents, and amino acids with non-polar side chains (l-alanine, l-valine, l-phenylalanine) as spacers between reporter and metal binding groups. Ferrocene/dipeptide conjugates were prepared using solution phase peptide synthesis methods employing a BOC-protecting group strategy and HBTU- (O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) mediated peptide coupling. The electrochemical properties of these 1,1′-substituted ferrocenoyl peptides have been characterized using cyclic voltammetry. All exhibit fully reversible one electron oxidation steps; forward sweep half wave peaks (E_F), reverse sweep half wave peaks (E_R), peak separations (ΔE_P) and half wave potentials (E_1/2) are reported. Finally, towards the goal of utilizing ferrocenoyl peptides to detect heavy metals in solution, the response of these ferrocene/dipeptide conjugates to metal cations (zinc(II), mercury(II), cadmium(II), lead(II), silver(I)) has been examined. Monitoring changes in the potential of the Fe(II)/Fe(III) redox couple to follow peptide/metal interactions, we have probed the influence of the spacer unit between the redox reporter and the metal-binding amino acid, and shown that these systems respond to mercury(II) more strongly than to other heavy metal ions.     

Long‐life Heavy Metal Ions Sensor Based on Graphene Oxide‐anchored Conducting Polymer

A long‐life electrochemical sensor for the continuous analysis of heavy metal ions (Zn(II), Cd(II), Pb(II), Cu(II), and Hg(II)) was developed using the graphene oxide (GO) anchored‐functionalized polyterthiophene (poly[3′‐(2‐aminopyrimidyl)‐2,2′:5′,2′′‐terthiophene], polyPATT) composite. The PATT monomer was synthesized and polymerized with GO to form the composite using a potential cycling method, followed by Nafion coating. The modified sensor surface was characterized employing electrochemical and surface analysis methods. Experimental variables affecting the analytical performance were optimized. Interference effects of other metal ions having similar redox potentials were also investigated. The performance of chronocoulometry (CC) without predeposition was compared with the results of square wave anodic stripping voltammetry (SWASV) with predeposition. The dynamic range of CC for the target ions were between 1 ppb and 10 ppm, respectively with the detection limits between 0.05 (±0.05) and 0.20 (±0.15) ppb for the CC method without predeposition, and between 0.08 (±0.05) and 0.30 (±0.12) ppb for the SWASV with 300 sec of deposition time (n=3 ). The reliability of the method was evaluated by continuously analysing environmental water samples using a single sensor probe in a flow system for 93 days.     

Biomass valorization of walnut shell into biochar as a resource for electrochemical simultaneous detection of heavy metal ions in water and soil samples: Preparation,characterization, and applications

Lately, due to its accessibility and eco-friendliness, walnut shell biochar (WS-BC) is gaining attention as an electrode material component in the electrochemical detection of water pollutants. The overall performance of WS-BC is reliant on the nature of raw biomass and the production methods as well. In our concept, biochar, prepared from raw walnut shell (WS) by pyrolysis, was added to a carbon paste electrode (CPE), and poly-tyrosine (p-Tyr) was electrodeposited on the surface of the BC-doped electrode. The conditions of the elaboration of the electrode, such as pH, potential, and the number of deposition cycles, pH were optimized. The obtained p-Tyr-BC-CPE platform was tested for the determination of cadmium, lead, copper, and mercury ions in water and soil samples, using square wave voltammetry (SWV). The raw WS biomass and its BC were examined by thermal analysis (TG-DSC), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM/EDX) techniques. The synergistic effects of the coexistence of the WS-BC and the thin film of p-Tyr, for the detection of traces of heavy metal ions were investigated by electrochemical tests. The electrochemical characterization of the unmodified and modified electrodes was performed using the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods, while the Cd²⁺, Pb²⁺, Cu^2+, and Hg²⁺ detection experiments were studied using the CV and SWV techniques. The optimized experimental conditions for the p-Tyr-BC-CPE platform were evaluated. The obtained electrochemical results showed that the p-Tyr-BC-CPE platform produced excellent sensitivity toward the heavy metal ions: LOD of 0.086, 0.175, 0.246, and 0.383 nM for Cd(II), Pb(II), Cu(II) and Hg(II), respectively. The modified electrode platform displayed high selectivity, stability, and good reproducibility.     

Selective Electrochemical Analysis of Various Metal Ions at an EDTA Bonded Conducting Polymer Modified Electrode

An EDTA‐bonded conducting polymer modified electrode was prepared and characterized by FT‐IR. The modified electrode was used for the selective electrochemical analysis of various trace metal ions such as, Cu(II), Hg(II), Pb(II), Co(II), Ni(II), Fe(II), Cd(II), and Zn(II) at the different pHs by linear sweep and square wave voltammetry. Dynamic ranges were obtained using square wave voltammetry from 0.1 μM to 10.0 μM for Co(II), Ni(II), Cd(II), Fe(II), and Zn(II) and 0.5 nM to 20 nM for Cu(II), Hg(II), and Pb(II) after 10 min of preconcentration. The detection limits were determined to be 0.1 nM, 0.3 nM, 0.4 nM, 50.0 nM, 60.0 nM, 65.0 nM, 80.0 nM, and 90.0 nM for Cu(II), Hg(II), Pb(II), Co(II), Ni(II), Cd(II), Fe(II), and Zn(II), respectively. The technique offers an excellent way for the selective trace determination of various heavy metal ions in a solution.     

Application of Crosslinked Acrylamidoxime/2-Acrylamido-2-methylpropane Sulfonic Acid Copolymer in Wastewater Treatment

Crosslinked acrylonitrile/acrylamidoxime/2-acrylamido-2-methylpropane sulfonic acid (AN/AAx/AMPS) based hydrogels was prepared by radical solution polymerization technique. The structures of hydrogels were characterized by FTIR analysis and the results were consistent with the expected structures. These hydrogels were used for the separation of Cd(II), Cu(II), and Fe(III) ions from their aqueous solutions. The influence of the uptake conditions such as pH, time, and initial feed concentration on the metal ion binding capacity of hydrogel was also tested. The selectivity of the hydrogel toward the different metal ions tested was Cd(II)>Fe(III)>Cu(II). The recovery of metal ions was also investigated in acid media.     

Understanding metalloprotein folding using a de novo design strategy

Metal ions play significant roles in most biological systems. Over the past two decades, there has been significant interest in the redesign of existing metal binding sites in proteins/peptides and the introduction of metals into folded proteins/peptides. Recent research has focused on the effects of metal binding on the overall secondary and tertiary conformations of unstructured peptides/proteins. In this context, de novo design of metallopeptides has become a valuable approach for studying the consequence of metal binding. It has been seen that metal ions not only direct folding of partially folded peptides but have at times also been the elixir for properly folding random-coil-like structures in stable secondary conformations. Work in our group has focused on binding of heavy metal ions such as Hg(II) to de novo designed alpha-helical three stranded coiled coil peptides with sequences based on the heptad repeat motif. Removal from or addition of a heptad to the parent 30-residue TRI peptide with the amino acid sequence Ac-G(LKALEEK)(4)G-NH(2) generated peptides whose self-aggregation affinities were seen to be dependent on their lengths. It was noted that adjustment in the position of the thiol from an "a" position in the case of the shorter BabyL9C to a "d" position for BabyL12C resulted in a peptide with low association affinities for itself, weaker binding with Hg(II), and a considerably faster kinetic profile for metal insertion. Similar differences in thermodynamic and kinetic parameters were also noted for the longer TRI peptides. At the same time, metal insertion into the prefolded and longer TRI and Grand peptides has clearly demonstrated that the metal binding is both thermodynamically as well kinetically different from that to unassociated peptides.     

The importance of stereochemically active lone pairs for influencing Pb(II) and As(III) protein binding

The toxicity of heavy metals, which is associated with the high affinity of the metals for thiolate rich proteins, constitutes a problem worldwide. However, despite this tremendous toxicity concern, the binding mode of As(III) and Pb(II) to proteins is poorly understood. To clarify the requirements for toxic metal binding to metalloregulatory sensor proteins such as As(III) in ArsR/ArsD and Pb(II) in PbrR or replacing Zn(II) in δ-aminolevulinc acid dehydratase (ALAD), we have employed computational and experimental methods examining the binding of these heavy metals to designed peptide models. The computational results show that the mode of coordination of As(III) and Pb(II) is greatly influenced by the steric bulk within the second coordination environment of the metal. The proposed basis of this selectivity is the large size of the ion and, most important, the influence of the stereochemically active lone pair in hemidirected complexes of the metal ion as being crucial. The experimental data show that switching a bulky leucine layer above the metal binding site by a smaller alanine residue enhances the Pb(II) binding affinity by a factor of five, thus supporting experimentally the hypothesis of lone pair steric hindrance. These complementary approaches demonstrate the potential importance of a stereochemically active lone pair as a metal recognition mode in proteins and, specifically, how the second coordination sphere environment affects the affinity and selectivity of protein targets by certain toxic ions.     

Dynamic chelation ion chromatography of transition and heavy metal ions using a mobile phase containing 4-chlorodipicolinic acid

The chromatographic behaviour of selected transition and heavy metal ions, the lanthanides, uranium and aluminium, on a neutral polystyrene-divinylbenzene (PS-DVB) stationary phase (7 microm Hamilton PRP-1) dynamically modified with 4-chlorodipicolinic acid, was investigated to evaluate retention characteristics. Complicated retention factor against pH plots were found for these metals demonstrating changes in retention order. It was concluded that complexation between the metal ions and the ligand adsorbed on the resin was strongly influenced by the decrease in dynamic loading with increase in pH, coinciding with changes in the metal-to-ligand ratio in the mobile phase. Possible reversed-phase interactions between metal-chlorodipicolinic acid complexes and the hydrophobic PS-DVB stationary phase also could not be ruled out. An eluent of 0.25 mM chlorodipicolinic acid, I M potassium nitrate at pH 2.2 was suitable for the separation of seven transition and heavy metal ions in under 20 min on a 250 x 4.6 mm column (with 50-mm guard column), determined in a certified water sample with good accuracy (R2 > or = 0.994) and reproducibility (RSD 1-4.2%). Pb(II), Cd(II) and Cu(II) were additionally analysed in <10 min in a more complicated certified rice flour matrix, using the same eluent but adjusted to pH 1.5, again with good accuracy (R2 > or = 0.998) and reproducibility (RSD 0.48-1.38%).     

Ion exchange properties of monolithic and particle type iminodiacetic acid modified silica

A 10 cm silica monolith has been modified with iminodiacetic acid (IDA) groups and characterised for its selectivity toward alkali, alkaline earth, and selected transition metal cations. Physical characterisation of the modified monolith found non-homogeneous modification along the length of the monolith, although sufficient capacity was achieved to facilitate significant retention of alkaline earth and transition/heavy metal ions over a range of eluent pH and ionic strength conditions. For alkaline earth and transition/heavy metal ions, selectivity of the 10 cm IDA monolith closely matched that seen with a 25 cm IDA modified silica gel particle packed column, although the separation of alkali metal ions was noticeably poorer on the monolithic column. Peak efficiencies for most metal ions were of a similar order for both column types, except for Zn(II), which showed significant peak broadening on the IDA monolithic column.