Identification of important residues in metal-chelate recognition by monoclonal antibodies

The molecular characterization of antibodies directed against metal-chelate complexes will provide important insights into the design and development of radiotherapeutic and radioimaging reagents. In this study, two monoclonal antibodies directed against different metal-chelate complexes were expressed as recombinant Fab fragments. Covalent modification and site-directed mutagenesis were employed to ascertain those residues important in antigen recognition. Antibody 5B2 was raised to a Pb(II)-loaded isothiocyanatobenzyl-diethylenetriamine pentaacetic acid (DTPA)-protein conjugate. The native antibody bound to complexes of Pb(II)-p-aminobenzyl-DTPA with an affinity of 4.6 x 10(-9) M. A monovalent Fab fragment prepared from the native protein and a bivalent recombinant fragment exhibited comparable affinities for the same Pb(II)-chelate complex, approximately 6-fold lower than that of the intact antibody. Covalent modification and molecular modeling predicted that Lys(58) in the heavy chain contacted the Pb(II)-chelate ligand. Mutational analysis supported a role for Lys(58) in ion pair or hydrogen bond formation with the carboxylate groups on the chelate. Antibody E5 was directed toward an isothiocyanatobenzyl-ethylenediamine tetraacetic acid (EDTA)-protein conjugate loaded with ionic Cd(II). The native immunoglobulin recognized Cd(II)-p-aminobenzyl-EDTA with an affinity of 8.2 x 10(-12) M. A proteolytically derived fragment and a bivalent recombinant fragment bound to the same Cd(II)-chelate complex with affinities that were comparable to that of the native antibody. Homology modeling and mutagenesis identified three residues (Trp(52) and His(96) in the heavy chain and Arg(96) in the light chain) that were important for Cd(II)-chelate recognition. His(96) likely mediates a direct ligation to the Cd(II) ion and Trp(52) appears to be involved in hydrophobic stacking with the benzyl moiety of the chelator. Arg(96) appeared to mediate an electrostatic or hydrogen bond to the chelate portion of the complex. These studies demonstrate that antibody recognition of metal-chelate haptens occurs through a limited number of molecular contacts and that these molecular interactions involve both direct ligation between the antibody and the metal ion and interactions between the antibody and the chelator.     

Binding properties of a monoclonal antibody directed toward lead-chelate complexes

A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution.     

Co-ordination of transition metal ions by galactaric acid: a potentiometric and spectroscopic study

A solution study on the ability of galactaric acid [GalaH(2), HOOC(CH)(4)COOH] in the complexation of biological metal ions such as Co(II) and Ni(II) and toxic metal ions such as Cd(II), Pb(II) and Hg(II), is reported. The stability constants of the complex species are determined by means of potentiometric measurements. Galactaric acid behaves as chelate ligand through carboxylic oxygen and alpha-hydroxy group towards Co(II) and Ni(II), while in the Pb(II) and Cd(II) containing system it co-ordinates the metal ion with carboxylic oxygen and two alcoholic hydroxy groups. The prevailing species at acidic or neutral pH is [MGala] which is also isolated in the solid state and characterized by means of IR spectroscopy. On increasing pH, the [MGalaH(-1)](-) species is also formed where the co-ordinated OH group undergoes deprotonation in all metal ion complexes except those with Hg(II), where the co-ordination of hydroxide ion is suggested as the precipitation of the metal hydroxide occurs at pH 7.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Surface treated Pteris vittata L. pinnae powder used as an efficient biosorbent of Pb(II), Cd(II), and Cr(VI) from aqueous solution

Biosorption is a surface-dependent phenomenon. Surface modifications by chemical treatment methods could either improve or reduce the biosorption capacity of potential biosorbents. In the present work, pristine Pteris vittata L. pinnae (PPV) powder was treated separately with sodium hydroxide (NaOH), calcium chloride (CaCl₂), and nitric acid (HNO₃). The pristine and treated biosorbents were used to assess the biosorption of Pb(II), Cd(II), and Cr(VI) as a function of pH. Kinetics and adsorption isotherms were studied. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and scanning electron microscope combined with energy dispersive x-ray (SEM-EDX) spectroscopic techniques were used to characterize the biosorbents before and after chemical treatments. The possible functional groups contributing to the metal sorption were identified. Results revealed favorable biosorption of Pb(II), Cd(II), and Cr(VI) described by pseudo-second order kinetics. NaOH-treated P. vittata (NPV) showed higher biosorption capacity for Pb(II) and Cd(II) compared to that of PPV. ATR-FTIR studies indicated that -OH, -COOH, and -NH₂ groups were mainly involved in Cr(VI) and -OH in Pb(II) and Cd(II) biosorption. The enhanced efficiency of NPV and CaCl₂ treated P. vittata (CPV) in the uptake of Pb(II) and Cd(II) compared to PPV can be associated with their altered physicochemical characters.     

Use of silica-immobilized humin for heavy metal removal from aqueous solution under flow conditions

Humin extracted from Sphagnum peat moss was immobilized in a silica matrix and column experiments were performed in order to evaluate the removal and recovery of metal ions from aqueous solution under flow conditions. These experiments also allowed testing the recycling capacity of the column. Single-element solutions of Cu(II) and Pb(II), and a multi-metal solution containing Cd(II), Cu(II), Pb(II), Ni(II), and Cr(III) were passed through the columns at a flow rate of 2 ml/min. A 0.5 M sodium citrate solution was used as the stripping agent in the metal-ion recovery process. Humin immobilized in the silica matrix exhibited a similar, and in some cases, even a higher capacity than other biosorbents for the removal of metal ions from aqueous solutions under flow conditions. The sodium citrate was effective in removing Cu(II), Pb(II), Cd(II), and Ni(II) from the metal saturated column. The selectivity of the immobilized biomass was as follows: Cr(III)>Pb(II)>Cu(II)>Cd(II)>Ni(II). This investigation provides a new, environmentally friendly and cost-effective possibility to clean up heavy-metal contaminated wastewaters by using the new silica-immobilized humin material.     

Metal(II) binding ability of a novel N-protected amino acid. A solution-state investigation on binary and ternary complexes with 2,2'-bipyridine

The binary and ternary systems 2,2'-bipyridine (bpy)-M(II)-NO2psglyH2 (M(II) = Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Pb(II); NO2psglyH2 = N-(2-nitrophenylsulfonyl)glycine) were investigated in aqueous solution by means of potentiometry and electron spectroscopy in order to identify the type, number and stability of the complex species as a function of pH and metal-to-ligand molar ratio. The aim is to evaluate the effect of a substituent on the phenyl ring of the N-sulfonyl amino acids on their coordination properties. The prevailing species in the binary systems is the [ML] (M = Co(II), Ni(II), Cu(II), Cd(II), Pb(II)) where the amino acid molecule is in the dianionic form and coordinates the metal ion through both carboxylic oxygen and deprotonated sulfonamidic nitrogen, while in the Mn(II)- and Zn(II)-containing binary system the only complex species found are those with the amino acid in the monoanionic form. In the ternary 2,2'-bipyridine-containing systems the chelating coordination mode of the dianionic amino acid is maintained with M(II) = Co(II), Ni(II), Cu(II), Cd(II), Pb(II) and the addition of the aromatic base also enables the Zn(II) ion to substitute for the sulfonamide nitrogen-bound hydrogen of NO2psglyH2.     

Reduction of Hexavalent Chromium by Immobilized Viable Cells of Arthrobacter sp. SUK 1201

Arthrobacter sp. SUK 1201, a potent isolate reported from chromite mine overburden of Orissa, India, has been evaluated for Cr(VI) reduction with immobilized whole cells. For whole-cell immobilization, Ba-alginate was found to be most effective, and the Cr(VI) reduction potential was maximum in minimal salts (MS) medium with cells immobilized in 2% alginate. Fourier transform infrared spectra of depolymerized cells has failed to detect any sign of complexation of Cr(VI) or its reduced products with the cell mass. Reduction efficiency of the beads increased with increase in cell load, but decreased with increase in Cr(VI) concentration in the medium. Glycerol was the most potent electron donor for chromate reduction, followed by glucose and peptone. Optimum pH for Cr(VI) reduction was 7.0, and the process was inhibited by metal ions such as Ni(II), Co(II), Cd(II), Zn(II), and Mn(II) but not by Cu(II) and Fe(III). Similarly, CCCP (carbonyl cyanide-m-chlorophenylhydrazone), DCC (N,N,-dicyclohexylcarbodiimide), sodium azide, and sodium fluoride were inhibitory in nature, whereas chromate reduction was unaffected in the presence of DNP (2,4-dinitrophenol). Moreover, immobilized cells of SUK 1201 remained biologically active for four consecutive cycles, accompanied with an initial increase in cell number in the beads, although a decline in chromate reduction was recorded from the second cycle onward. Immobilized cells of Arthrobacter sp. SUK 1201, therefore, could be a potential tool for long-term uses in chromium detoxification.     

Study of the genotoxic potential of 48 inorganic derivatives with the SOS chromotest

The genotoxic potential of 48 inorganic derivatives was studied using the bacterial colorimetric assay: the SOS Chromotest. Some of these compounds are known as carcinogens (As, CR(VI), Cd, Ni) or suspected carcinogens for human beings (Hg, Pb), others are known as non-carcinogens. Among these 48 derivatives, only the two Cr(VI) compounds and the Sn(II) compounds gave positive results.     

Adsorption of heavy metal ions from aqueous solutions by activated carbon prepared from apricot stone

Apricot stones were carbonised and activated after treatment with sulphuric acid (1:1) at 200 degrees C for 24 h. The ability of the activated carbon to remove Ni(II), Co(II), Cd(II), Cu(II), Pb(II), Cr(III) and Cr(VI) ions from aqueous solutions by adsorption was investigated. Batch adsorption experiments were conducted to observe the effect of pH (1-6) on the activated carbon. The adsorptions of these metals were found to be dependent on solution pH. Highest adsorption occurred at 1-2 for Cr(VI) and 3-6 for the rest of the metal ions, respectively. Adsorption capacities for the metal ions were obtained in the descending order of Cr(VI) > Cd(II) > Co(II) > Cr(III) > Ni(II) > Cu(II) > Pb(II) for the activated carbon prepared from apricot stone (ASAC).     

Biosorption of heavy metals from aqueous solutions with tobacco dust

A typical lignocellulosic agricultural residue, namely tobacco dust, was investigated for its heavy metal binding efficiency. The tobacco dust exhibited a strong capacity for heavy metals, such as Pb(II), Cu(II), Cd(II), Zn(II) and Ni(II), with respective equilibrium loadings of 39.6, 36.0, 29.6, 25.1 and 24.5 mg of metal per g of sorbent. Moreover, the heavy metals loaded onto the biosorbent could be released easily with a dilute HCl solution. Zeta potential and surface acidity measurements showed that the tobacco dust was negatively charged over a wide pH range (pH > 2), with a strong surface acidity and a high OH⁻ adsorption capacity. Changes in the surface morphology of the tobacco dust as visualized by atomic force microscopy suggested that the sorption of heavy metal ions on the tobacco could be associated with changes in the surface properties of the dust particles. These surface changes appeared to have resulted from a loss of some of the structures on the surface of the particles, owing to leaching in the acid metal ion solution. However, Fourier transform infrared spectroscopy (FTIR) showed no substantial change in the chemical structure of the tobacco dust subjected to biosorption. The heavy metal uptake by the tobacco dust may be interpreted as metal–H ion exchange or metal ion surface complexation adsorption or both.     

Effects of cadmium and lead on ferric chelate reductase activities in sugar beet roots

The effects of the heavy metals Cd and Pb on the activity of the enzyme ferric chelate reductase (FC-R, E.C. 1.6.99.13) have been studied in excised sugar beet root tips. The activity of this enzyme is markedly increased by iron deficiency. Metals were used as chloride salts or chelated with EDTA, and chemical speciation was carried out to predict the metal chemical species in equilibrium both in the ferric reductase assay and in the nutrient solutions. Three different heavy metal treatments were used. First, effects of Cd and Pb on the functioning of the FC-R were assessed in Fe-deficient plants, by including metals in the enzyme assay medium only. Results indicate that 50 μM CdCl₂ or Cd-EDTA did not affect FC-R activities even when assay time was as long as 2 h, whereas Pb slightly decreased enzyme activity only at concentrations of 2 mM. Second, short-time Cd and Pb pre-treatments (30–60 min) were imposed on intact Fe-deficient plants before carrying out the assay of FC-R activity. These short-term treatments induced significant decreases in the FC-R activities previously induced by Fe deficiency. With Cd, effects were more pronounced at higher concentrations, and they were stronger when Cd was in the free ion form than when present in the form of Cd-EDTA chelate. Third, prolonged Cd and Pb treatments were imposed on plants grown on 45 μM Fe-EDTA to assess the long-term effects of heavy metals on the induction of the FC-R enzyme. These long-term heavy metal treatments caused a significant increase in the root FC-R activities, indicating that Cd and Pb induce a deficiency in Fe in sugar beet that in turn elicits FC-R activity. The increases, however, are not as large as those found in total absence of Fe.     

A zinc(II)/lead(II)/cadmium(II)-inducible operon from the Cyanobacterium anabaena is regulated by AztR, an alpha3N ArsR/SmtB metalloregulator

A novel Zn(II)/Pb(II)/Cd(II)-responsive operon that consists of genes encoding a Zn(II)/Pb(II) CPx-ATPase efflux pump (aztA) and a Zn(II)/Cd(II)/Pb(II)-specific SmtB/ArsR family repressor (aztR) has been identified and characterized from the cyanobacterium Anabaena PCC 7120. In vivo real time quantitative RT-PCR assays reveal that both aztR and aztA expression are induced by divalent metal ions Zn(II), Cd(II), and Pb(II) but not by other divalent [Co(II), Ni(II)] or monovalent metal ions [Cu(I) and Ag(I)]. The introduction of a plasmid containing the azt operon into a Zn(II)/Cd(II)-hypersensitive Escherichia coli strain GG48 functionally restores Zn(II) and Pb(II) resistance with a limited effect on Cd(II) resistance. Gel mobility shift assays and aztR O/P-lacZ induction experiments confirm that AztR is the metal-regulated repressor of this operon. In vitro biochemical and mutagenesis studies indicate that AztR contains a sole metal-binding site, designated the alpha3N site, that binds Zn(II), Cd(II), and Pb(II) with a high affinity. Optical absorption spectra of Co(II)- and Cd(II)-substituted AztR and (113)Cd NMR spectroscopy of (113)Cd(II)-substituted AztR reveal that the sole alpha3N site in AztR is a CadC-like distorted tetrahedral S(3)(N,O) metal site. The first metal-coordination shell in the AztR alpha3N site differs from other alpha3N family members that sense Cd(II)/Pb(II) and those alpha5 repressors that sense Zn(II)/Co(II). Our results reveal that the alpha3N site in AztR mediates derepression of the azt operon in the presence of Zn(II), as well as Cd(II) and Pb(II); this might have provided Anabaena with an evolutionary advantage to adapt to heavy-metal-rich environments, while maintaining homeostasis of an essential metal ion, Zn(II).     

Monoclonal antibodies that recognize minimal differences in the three-dimensional structures of metal-chelate complexes

Immunization of BALB/c mice with a cadmium-chelate-protein conjugate resulted in the isolation of two hybridoma cell lines (A4 and E5) that synthesized antibodies with different variable regions, but similar metal-chelate affinity. The ability of these two monoclonal antibodies to interact with 12 different metal-chelate complexes was studied using the KinExA 3000 immunoassay instrument. The two antibodies showed the highest affinity for cadmium and mercury complexes of ethylenediamine N,N,N',N'-tetraacetic acid (EDTA). The E5 antibody bound to EDTA complexes of cadmium and mercury with equilibrium dissociation constants (K(d)) of 1.62 x 10(-)(9) M and 3.64 x 10(-)(9) M, respectively. The corresponding values for the A4 antibody were 14.7 x 10(-)(9) M and 3.56 x 10(-)(9) M. Addition of a cyclohexyl ring to the EDTA backbone increased the affinity of E5 for the metal-chelate haptens, while decreasing the binding of A4 to the same haptens. Based on available crystal structures, molecular models were constructed for five different divalent metal-chelate complexes. The models were compared to determine structural features of the haptens that may influence antibody recognition. Difference distance matrixes were used to identify areas of the metal-chelate haptens that differed in three-dimensional space. Antibody affinity correlated well with the extent of total structural difference for these metal-EDTA complexes.     

Comparison of the toxicity of heavy metals to cabbage growth

Summary Cabbage plants were grown for 55 days with a nutrient solution containing 1 and 10 ppm of V, Cr(III), Cr(VI), Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg(I), orHg(II). A comparison of the plant growth and chemical analysis revealed that Cr(VI), Cu, Cd, and Hg(II) in the solution are most toxic to the plant growth (hence detrimental to the cabbage-head formation) and Mn, Fe, and Zn are less toxic than other heavy metals, and that Mn, Zn, Co, Ni, and Cd and translocated into all the plant organs while V, Cr(III), Cr(VI), Fe, Cu, Hg(I), and Hg(II) are accumulated in the roots.