Preparation and metal-binding behaviour of chitosan functionalized by ester- and amino-terminated hyperbranched polyamidoamine polymers

A series of insoluble chitosan (CTS) derivatives were prepared by grafting ester- and amino-terminated dendrimer-like polyamidoamine (PAMAM) into CTS using a divergent method by repeating two processes: (1) Michael addition of methyl acrylate (MA) to surface amino groups, and (2) amidation of the resulting esters with ethylenediamine (EDA). Their structures were characterized by infrared spectra (IR) and wide-angle X-ray diffraction (WAXD). The adsorption capabilities of the products for Au(3+), Pd(2+), Pt(4+), Ag(+), Cu(2+), Zn(2+), Hg(2+), Ni(2+), and Cd(2+) were studied. The results showed that the products exhibited better adsorption capabilities for Au(3+) and Hg(2+) than for other metal ions, and the adsorption capabilities of amino-terminated products were higher than those of ester-terminated ones. Also it was observed that a high percentage of grafting of PAMAM into CTS does not ensure a high adsorption capacity.     

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).     

A new metal chelate affinity adsorbent for cytochrome C

We have prepared a novel metal-chelate adsorbent utilizing N-methacryloyl-L-histidine methyl ester (MAH) as a metal-chelating ligand. MAH was synthesized by using methacryloyl chloride and l-histidine methyl ester dihydrochloride. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Then, Cu(2+) ions were chelated directly on the chelating beads. Cu(2+)-chelated beads were used in the adsorption of cytochrome c (cyt c) from aqueous solutions. The maximum cyt c adsorption capacity of the Cu(2+)-chelated beads (658.2 micromol/g Cu(2+) loading) was found to be 31.7 mg/g at pH 10 in phosphate buffer. The nonspecific cyt c adsorption on the naked PHEMA beads was 0.2 mg/g. Cyt c adsorption increased with increasing Cu(2+) loading. Cyt c adsorption capacity was demonstrated for the buffer types with the effects in the order phosphate > HEPES > MOPS > MES > Tris-HCl. Cyt c molecules could be adsorbed and desorbed five times with these adsorbents without noticeable loss in their cyt c adsorption capacity.     

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads

Silica gel bead coated with macroporous chitosan layer (CTS-SiO₂) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu²⁺, Zn²⁺, Ni²⁺ ions, respectively on CTS-SiO₂, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu²⁺-CTS-SiO₂ with the value of 4980 ± 125 IU g⁻¹ of the adsorbent, while the maximal adsorption capacity for trypsin on Zn²⁺ and Ni²⁺ loaded adsorbent was 3762 ± 68 IU g⁻¹ and 2636 ± 53 IU g⁻¹, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO₂ beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO₂ could effectively digest BSA revealed by HPLC peptide mapping.     

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).     

Adsorption of Hg2+, Cu2+ and Zn2+ ions from aqueous solution using formaldehyde cross-linked modified chitosan-thioglyceraldehyde Schiff's base

A chitosan-thioglyceraldehyde Schiff's base cross-linked magnetic resin (CSTG) was prepared and characterized using various instrumental methods. Then, the prepared resin was used for comparative studies on the removal of toxic metal ions like: Hg(2+), Cu(2+) and Zn(2+) from aqueous solutions. The effects of the initial pH value of the solution, contact time, the initial metal ion concentration and temperature on the adsorption capacity of the composite were investigated. The kinetics data were analyzed by pseudo-first order and pseudo-second order equations. The adsorption kinetics was well described by the pseudo-second order equation, and the adsorption isotherms were better fitted by the Langmuir equation. The maximum theoretical adsorption capacities of the CSTG resin for Hg(2+), Cu(2+) and Zn(2+) were found to be 98±2, 76±1 and 52±1 mg g(-1), respectively. The negative values of Gibbs free energy of adsorption (ΔG(ads°) indicated the spontaneity of the adsorption of all metal ions on the novel resin.     

Dimerization of human growth hormone in the presence of metal ions

Zn(2+) and Co(2+) ions are known to promote human growth hormone reversible dimerization. In these studies, dimerization was also shown to be initiated by nine other metal ions: Cd(2+), Hg(2+), Cu(2+), Ag+, Au(3+), Au+, Pd(2+), Ni(2+), and Pt(4+). In some cases (Hg(2+), Ag(+), Au(3+), and Ni(2+)) formation of higher oligomers also took place. In addition further detailed investigation of dimerization in the presence of Zn(2+) ions was carried out.     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals

Transition metal ions, although maintained at low concentrations, play diverse important roles in many biological processes. Two assays useful for the rapid quantification of a range of first-row transition metal ions have been developed. The colorimetric assay extends the 4-(2-pyridylazo)resorcinol assay of Hunt et al. (J. Biol. Chem. 255, 14793 (1984)) to measure nanomole quantities of Co(2+), Ni(2+), and Cu(2+) as well as Zn(2+). The fluorimetric assay takes advantage of the coordination of a number of metal ions (Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+)) by Fura-2 and can also be used to measure nanomole quantities of these ions. The assays developed here have the advantage of not requiring the extensive sample preparation necessary for other methodologies, such as atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy (ICPES), while being comparable in accuracy to the detection limits of ICPES for the first-row transition metal ions. To demonstrate the effectiveness of these assays, we determined the affinity of carbonic anhydrase II (CA II), a prototypical zinc enzyme, for Ni(2+) and Cd(2+). These data indicate that CA II binds transition metals with high affinity and is much more selective for Zn(2+) over Ni(2+) or Cd(2+) than most small-molecule chelators or other metalloenzymes.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

First- and second-row transition metal oxa-aza macrocyclic complexes: a DFT study of an octahedral conformation

A theoretical study of structures of the 1,7,1 l,17-tetraoxa-2,6,12,16-tetraaza-cycloeicosane ligand ([20]AneN(4)O(4)) coordinated to Fe(2+), Co(2+), Ni(2+), Ru(2+), Rh(2+), and Pd(2+) transition metals ions was carried out with the DFT/B3LYP method. Complexes were fully optimized in C(s) symmetry with the metal ions coordinated either to nitrogen (1a) or oxygen atoms (1b). For all the cases performed in this work, 1a was always more stable than 1b. Considering each row it is possible to see that the binding energy increases with the atomic number. The M(2+) cation binding energies increase in the following order: Fe(2+)相似文献   

Cobalt- and nickel-binding property of cullin-2.

Treatment with divalent metal ions such as cobalt (Co(2+)) or nickel (Ni(2+)) result in the stabilization of hypoxia-inducible factor-1alpha (HIF1alpha). Recently, HIF1alpha was shown to be ubiquitinated by an E3-ligase complex and be subsequently targeted for proteasomal degradation. In this study, we demonstrated that Co(2+) and Ni(2+) specifically bind to cullin-2. Mutant analysis revealed that cullin-2 possesses at least three sites for the binding. Furthermore, fluorescence spectroscopy revealed that only Co(2+) and Ni(2+) have the binding activity to cullin-2, but other metal ions, including Cu(2+), Ca(2+), Mg(2+), Mn(2+), and Zn(2+), did not. Finally, we found that Co(2+) and Ni(2+) do not bind to any components of the E3-ligase other than cullin-2, suggesting that cullin-2 is a key target of Co(2+) and Ni(2+). Interestingly, Co(2+) did not affect the complex formation of the ligase, suggesting that the metal binding to cullin-2 affects the function, but not the assembly of the E3-ligase.     

Adsorptive removal of copper and nickel ions from water using chitosan coated PVC beads

A new biosorbent was developed by coating chitosan, a naturally and abundantly available biopolymer, on to polyvinyl chloride (PVC) beads. The biosorbent was characterized by FTIR spectra, porosity and surface area analyses. Equilibrium and column flow adsorption characteristics of copper(II) and nickel(II) ions on the biosorbent were studied. The effect of pH, agitation time, concentration of adsorbate and amount of adsorbent on the extent of adsorption was investigated. The experimental data were fitted to Langmuir and Freundlich adsorption isotherms. The data were analyzed on the basis of Lagergren pseudo first order, pseudo-second order and Weber-Morris intraparticle diffusion models. The maximum monolayer adsorption capacity of chitosan coated PVC sorbent as obtained from Langmuir adsorption isotherm was found to be 87.9 mg g(-1) for Cu(II) and 120.5 mg g(-1) for Ni(II) ions, respectively. In addition, breakthrough curves were obtained from column flow experiments. The experimental results demonstrated that chitosan coated PVC beads could be used for the removal of Cu(II) and Ni(II) ions from aqueous medium through adsorption.     

Preparation and characterization of chitosan-grafted-poly(2-amino-4,5-pentamethylene-thiophene-3-carboxylic acid N'-acryloyl-hydrazide) chelating resin for removal of Cu(II), Co(II) and Ni(II) metal ions from aqueous solutions

The graft copolymerization of ethylacrylate (EA) onto chitosan initiated by potassium persulphate and Mohr's salt combined redox initiator system in limited aqueous medium was carried out in heterogeneous media. Moreover, modification of the grafted chitosan was carried out by reaction of the ester group (-COOEt) with 2-amino-4,5-pentamethylene-thiophene-3-carboxylic acid hydrazide which eventually produce chitosan-grafted-poly(2-amino-4,5-pentamethylene-thiophene-3-carboxylic acid N'-acryloyl-hydrazide) (chitosan-g-ATAH) chelating resin. The application of the modified resin for metal ion uptake was studied using Cu(2+), Co(2+) and Ni(2+) ions. The modified chelating resins were characterized using FTIR spectroscopy, SEM and X-ray diffraction.     

Select metal adsorption by activated carbon made from peanut shells

Agricultural by-products, such as peanut shells, contribute large quantities of lignocellulosic waste to the environment each growing season; but few, if any, value-added uses exist for their disposal. The objective of this study was to convert peanut shells to activated carbons for use in adsorption of select metal ions, namely, cadmium (Cd2+), copper (Cu2+), lead (Pb2+), nickel (Ni2+) and zinc (Zn2+). Milled peanut shells were pyrolyzed in an inert atmosphere of nitrogen gas, and then activated with steam at different activation times. Following pyrolysis and activation, the carbons underwent air oxidation. The prepared carbons were evaluated either for adsorption efficiency or adsorption capacity; and these parameters were compared to the same parameters obtained from three commercial carbons, namely, DARCO 12x20, NORIT C GRAN and MINOTAUR. One of the peanut shell-based carbons had metal ion adsorption efficiencies greater than two of the three commercial carbons but somewhat less than but close to Minotaur. This study demonstrates that peanut shells can serve as a source for activated carbons with metal ion-removing potential and may serve as a replacement for coal-based commercial carbons in applications that warrant their use.     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma

Metal-chelating ligand and/or comonomer 2-methacrylolyamidohistidine (MAH) was synthesized by using methacryloyl chloride and L-histidine methyl ester. MAH was characterized by NMR and FTIR. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of methylmethacrylate (MMA) and MAH carried out in an aqueous dispersion medium. Poly(MMA-MAH) beads had a specific surface area of 37.5 m(2)/g. Poly(MMA-MAH) beads were characterized by water uptake studies, FTIR, SEM and elemental analysis. Elemental analysis of MAH for nitrogen was estimated as 34.7 microM/g of polymer. Then, Cu(2+) ions were chelated on the beads. Cu(2+)-chelated beads with a swelling ratio of 38% were used in the adsorption of human-immunoglobulin G (HIgG) from both aqueous solutions and human plasma. The maximum adsorption capacities of the Cu(2+)-chelated beads were found to be 12.2 mg/g at pH 6.5 in phosphate buffer and 15.7 mg/g at pH 7.0 in MOPS. Higher adsorption value was obtained from human plasma (up to 54.3 mg/g) with a purity of 90.7%. The metal-chelate affinity beads allowed one-step separation of HIgG from human plasma. The adsorption-desorption cycle was repeated 10 times using the same beads without noticeable loss in their HIgG adsorption capacity.     

Characterization of structure and metal ions specificity of Co2+-binding RNA aptamers

Studies on RNA motifs capable of binding metal ions have largely focused on Mg(2+)-specific motifs, therefore information concerning interactions of other metal ions with RNA is still very limited. Application of the in vitro selection approach allowed us to isolate two RNA aptamers that bind Co(2+) ions. Structural analysis of their secondary structures revealed the presence of two motifs, loop E and "kissing" loop complex, commonly occurring in RNA molecules. The Co(2+)-induced cleavage method was used for identification of Co(2+)-binding sites after the determination of the optimal cleavage conditions. In the aptamers, Co(2+) ions seem to bind to N7 atoms of purines, inducing cleavage of the adjacent phosphodiester bonds, similarly as is the case with yeast tRNA(Phe). Although the in vitro selection experiment was carried out in the presence of Co(2+) ions only, the aptamers displayed broader metal ions specificity. This was shown by inhibition of Co(2+)-induced cleavages in the presence of the following transition metal ions: Zn(2+), Cd(2+), Ni(2+), and Co(NH(3))(6)(3+) complex. On the other hand, alkaline metal ions such as Mg(2+), Ca(2+), Sr(2+), and Ba(2+) affected Co(2+)-induced cleavages only slightly. Multiple metal ions specificity of Co(2+)-binding sites has also been reported for other in vitro selected or natural RNAs. Among many factors that influence metal specificity of the Co(2+)-binding pocket, chemical properties of metal ions, such as their hardness as well as the structure of the coordination site, seem to be particularly important.     

Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献