Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

Equilibrium and kinetics of the dinuclear complex formation between N,N′-ethylenebis(salicylideneiminato)copper(II) and metal(II,I) ions in acetonitrile

The equilibrium constants and rate constants of the reactions between N,N′-ethylenebis(salicylideneiminato)copper(II) ([Cu(salen)]) and metal(II,I) ions in acetonitrile have been spectrophotometrically determined. [Cu(salen)] acts as a didentate ligand to form a dinuclear complex. The rate constants for the very labile Mn(II), Fe(II), and Zn(II) ions were directly evaluated using a variable flow-rate instrument that was newly constructed for this study. The rate constants of the dinuclear complex formation for a series of metal(II) ions vary in parallel with those of the acetonitrile solvent exchange on the corresponding metal(II) ions. This finding indicates that the dinuclear complex formation reaction of the metal(II) ions proceeds via almost the same reaction mechanism as for the acetonitrile solvent exchange reaction.     

Synthesis and possible applications of biotin-linked copper clusters

The trifunctional aziridine XAMA-7 (CAS 57116-45-7) has been used to form crosslinks between a deep red-violet copper cluster of the type Cu(I)8Cu(II)6pen12Cl5- (pen=penicillamine) and molecules with biological activity such as d-biotin and proteins. A complex containing biotin, bovine serum albumin and the copper cluster displayed activity toward affinity columns of avidin on Agarose, and the red-violet pigment was immobilized on the gel. This interaction was completely blocked in gels which had been pretreated with d-biotin carboxylic acid. The free and biologically active versions of the cluster have some potential for biomedical applications. For example, the short-lived positron emitter 64Cu (suitable for positron tomography) may be carried in the cluster's structure. The cluster is paramagnetic, but it is a relatively weak effector of water proton spin-lattice relaxation. Other members of this structural group of inorganic compounds may have better magnetic properties, and the crosslinking reaction with aziridines appears to be generally applicable to the group.     

Characterization and metal affinity of Tirofiban, a pharmaceutical compound used in acute coronary syndromes

The crystal and molecular structure of Tirofiban [N-(n-butanesulfonyl)-O-(4-(4-piperidinyl)-butyl)-(S)-tyrosine] is here reported. In the solid state the carboxylic group is in the anionic form while the piperidine molecule appear in the protonated form. By H NMR spectroscopy and potentiometric study three pKa are found: pKa(COOH) = 3.1 (1), pKa(NHPIP) = 11.6(1) and pKa(NHSO2) = 13.8(1). The complexing ability of Tirofiban towards various metal ions (Cu(II), Ni(II), Co(II), Cd(II), Pb(II), Zn(II) and Ca(II)) is also determined by means of potentiometric studies. The prevailing species are [M(TirH)2]2+ where the ligand coordinates the metal ion through carboxylic group, while the piperidine nitrogen is still protonated. The great stability of these complexes may be due to the presence of hydrogen bond interactions, as well as the formation of stacking interactions involving the phenyl ring of the tyrosine residue.     

Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.     

Formation of homogeneous unilamellar liposomes from an interdigitated matrix

Phospholipid-ethanol-aqueous mixtures containing bilayer-forming lipids and 20-50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171-183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid-ethanol-water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.     

Formation of homogeneous unilamellar liposomes from an interdigitated matrix

Phospholipid-ethanol-aqueous mixtures containing bilayer-forming lipids and 20-50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171-183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid-ethanol-water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.     

Spectroscopic behavior of copper complexes of nonsteroidal anti-inflammatory drugs

The proposed curative properties of Cu-based nonsteroidal anti-inflammatory drugs (NSAIDs) have led to the development of numerous Cu(II) complexes of NSAIDs with enhanced anti-inflammatory activity. Crystalline complexes, Cu(II)-NSAID (ibuprofen, naproxen, tolmetin, and diclofenac), with a carboxylic function have been studied by means of infrared and Raman spectroscopy. All NSAIDs bind the metal through the carboxylate group. On the basis of the comparison between the wavenumber of the COO(-) group vibrations and Delta nu (nu(asimm)COO(-) - nu(simm)COO(-)) between Na salts and Cu(II) complexes, conclusions on the probable structure of the complexes have been drawn. The spectroscopic data support the formation of dimeric [Cu(2)L(4)(H(2)O)(2)] complexes in which the COO(-) group behaves as a bridging bidentate ligand. The low wavenumber region of the Raman spectrum provided information on Cu-O and Cu-Cu bonds in the complexes. Thermogravimetric results gave further support to the vibrational data.     

Ion-exchange and adsorption of Fe(III) by Sepia melanin

Sepia eumelanin is associated with many metal ions, yet little is known about its metal binding capacity and the chemical nature of the binding site(s). Herein, the natural concentrations of metal ions are presented and the ability to remove metals by exposure of the melanin granules to EDTA is quantified. The results reveal that the binding constants of melanin at pH 5.8 for Mg(II), Ca(II), Sr(II) and Cu(II) are, respectively, 5, 4, 14 and 34 times greater than the corresponding binding constants of these ions with EDTA. By exposing Sepia eumelanin to aqueous solutions of FeCl(3), the content of bound Fe(III) can be increased from a natural concentration of approximately 180 ppm to a saturation limit of approximately 80 000 ppm or 1.43 mmol/g of melanin. Similar saturation limits are found for Mg(II) and Ca(II). Exposure of Sepia melanin granules to aqueous solutions containing Ca(II) results in the stoichiometric replacement of the initially bound Mg(II), arguing that these two ions occupy the same binding site(s) in the pigment. The pH-dependent binding of Mg(II) and Ca(II) suggests coordination of these ions to carboxylic acid groups in the pigment. Mg(II) and Ca(II) can be added to a Fe(III)-saturated melanin sample without affecting the amount of Fe(III) pre-adsorbed, clearly establishing Fe(III) and Mg(II)/Ca(II) occupy different binding sites. Taking recent Raman spectroscopic data into account, the binding of Fe(III) is concluded to involve coordination to o-dihydroxyl groups. The effects of metal ion content on the surface morphology were analyzed. No significant changes were found over the full range of Fe(III) concentration studied, which is supported by the Brunauer-Emmett-Teller surface area analysis. These observations imply the existence of channels within the melanin granules that can serve to transport metal ions.     

Effect of zinc ion on the interaction of some amino acid compounds of copper(II) with hydrogen peroxide.

Reaction of elemental copper and zinc powder mixtures with glycine (NH2.CH2COOH; HA) or aspartic acid (NH2CHCOOHCH2COOH; H2B) (in 1:1:2 ratio, respectively) in the presence of excess hydrogen peroxide (H2O2) at 50 degrees C, results in the formation of a new mixed metal peroxy carbonate compound corresponding to formula [Cu(Zn)2(O2(2-) (CO3)2(H2O)4], while the same reaction with elemental copper powder alone yields merely peroxy amino acid compounds having the formula [Cu(O2(2-)) (HA)2(H2O)] and [Cu(O2(2-)) (H2B) (H2O)2] for glycine and aspartic acid, respectively. These compounds have been characterized by elemental analysis, ESR, and electronic and IR spectra. It is interesting to note that both amino acids are converted to carbonate in the presence of zinc alone. A method analogous to that described above, for the reaction of elemental copper, zinc powder mixtures with succinic acid [(CH2COOH)2] or acetic acid (CH3COOH) in excess H2O2, on the other hand, gave a product essentially comprising copper succinate or acetate, respectively. These observations suggest an interesting and perhaps important phenomenon by which only the simple amino acids such as glycine and aspartic acid are converted to carbonates while their corresponding carboxylic acids form only their respective salts.     

Amino-modified silicate gels prepared by sol-gel processing as metal-ion sorbents

Two series of amino-modified silicate gels prepared by sol-gel processing were used to absorb Cu(II), Ni(II), Co(II), Mn(II) and Cr(III) from aqueous solutions. These easily prepared sorbents with various content of primary amino groups in series (A) or primary and secondary amino groups in series (AA) have reasonable stability. The gel composition, time and concentration dependence of the uptake of the metal ions by these materials were studied systematically. These materials would be further used as supports to disperse catalytically active phases by conventional wet chemical procedures. Apart from this they demonstrate potential for the preconcentration aid for transition metal analysis.     

Construction a hybrid biosorbent using Scenedesmus quadricauda and Ca-alginate for biosorption of Cu(II), Zn(II) and Ni(II): kinetics and equilibrium studies

The potential use of the immobilized fresh water algae (in Ca-alginate) of Scenedesmus quadricauda to remove Cu(II), Zn(II) and Ni(II) ions from aqueous solutions was evaluated using Ca-alginate beads as a control system. Ca-alginate beads containing immobilized algae were incubated for the uniform growth at 22 degrees C for 5d ays. Adsorption of Cu(II), Zn(II) and Ni(II) ions on the immobilized algae showed highest values at around pH 5.0. Adsorption of Cu(II), Zn(II) and Ni(II) ions on the immobilized algae increased as the initial concentration of metal ions increased in the medium. The maximum adsorption capacities of the immobilized algal biosorbents for Cu(II), Zn(II) and Ni(II) were 75.6, 55.2 and 30.4 mg/g (or 1.155, 0.933 and 0.465 mmol/g) biosorbent, respectively. When the heavy metal ions were in competition, the amounts of adsorbed metal ions were found to be 0.84 mol/g for Cu(II), 0.59 mol/g for Ni(II) and 0.08 mol/g for Zn(II), the immobilised algal biomass was significantly selective for Cu(II) ions. The adsorption-equilibrium was also represented with Langmuir, Freundlich and Dubinin-Radushkevich adsorption isotherms. The adsorption of Cu(II), Zn(II) and Ni(II) ions on the immobilized algae followed second-order kinetic.     

Independent and mutual interactions of copper(II) and zinc(II) ions with phytic acid

The interactions of phytic acid with Cu(II) and Zn(II) ions were examined as functions of metal ion concentrations and pH. Cu(II) ion-selective potentiometric and electron spin resonance (ESR) experiments provide strong evidence for the binding of Cu(II) ions to the phytic acid molecule at low pH (2.4–3.4) values. The relative stabilities of the copper and zinc phytates at low pH values were found to be very similar. For systems with metal ion:phytic acid molar ratios of 1:1–4:1 and 5:1–6:1 and pH values in the 3.4–5.9 and 3.4–5.0 ranges, respectively, Zn(II) ions were found to form complexes with phytic acid that were more stable than those of Cu(II) ions with phytic acid. The phytic acid molecule, however, was found to accommodate Cu(II) ions more readily than Zn(II) ions. For example, in systems containing equal amounts of Cu(II) and Zn(II) ions, 2 Zn(II) ions and 2, 3, 4, or 4.5 Cu(II) ions were found per phytic acid molecule depending upon metal ion:phytic acid molar ratios in the systems and pH. Total metal ion:phytic acid molar ratios and pH affected resultant metal ion solubilities and were factors influencing the effects of Zn(II) and Cu(II) ions on the binding of each other by phytic acid. Zn(II) and Cu(II) ions were observed to potentiate the binding of each other by phytic acid in some systems and compete with each other for phytate binding sites in others.     

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy.

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(III) (S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.     

Separation and properties of three forms of cathepsin H-like cysteine proteinase from rat spleen

Three forms of cathepsin H-like cysteine proteinase were purified from rat spleen by a method involving acid treatment and chromatography on pepstatin-Sepharose, Sephadex G-75, DEAE-Sephacel, CM-Toyopearl, and concanavalin A-Sepharose. The final preparations of these forms all migrated as single protein bands on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS). The molecular weights of the three forms were estimated to be 28,000 (form I), 26,000 (form II), and 22,000 (form III). The optimal pH was 6.5 for forms I and III and was 7.0 for form II with L-leucine 2-naphthylamide (Leu-NA) or with alpha-N-benzoyl-DL-arginine 2-naphthylamide (BANA). All of the forms consisted of two major species having isoelectric points of 7.1 and 6.5 on isoelectric focusing gels. They were all stable when incubated at pH values between 5.0 and 9.0 for 1 h at 22 degrees C. They were strongly inhibited by iodoacetic acid and E-64, but not by metal ions or pepstatin. Form III was not affected by leupeptin, chymostatin, antipain or elastatinal, which gave essentially complete inhibition of cathepsin B purified from rat spleen. Forms I and II were slightly inhibited by these compounds at the same concentrations. The properties of these forms were compared with those of the known enzymes cathepsin H and BANA-hydrolase.     

Ion‐Exchange and Adsorption of Fe(III) by Sepia Melanin

Sepia eumelanin is associated with many metal ions, yet little is known about its metal binding capacity and the chemical nature of the binding site(s). Herein, the natural concentrations of metal ions are presented and the ability to remove metals by exposure of the melanin granules to EDTA is quantified. The results reveal that the binding constants of melanin at pH 5.8 for Mg(II), Ca(II), Sr(II) and Cu(II) are, respectively, 5, 4, 14 and 34 times greater than the corresponding binding constants of these ions with EDTA. By exposing Sepia eumelanin to aqueous solutions of FeCl₃, the content of bound Fe(III) can be increased from a natural concentration of ～180 ppm to a saturation limit of ～80 000 ppm or 1.43 mmol/g of melanin. Similar saturation limits are found for Mg(II) and Ca(II). Exposure of Sepia melanin granules to aqueous solutions containing Ca(II) results in the stoichiometric replacement of the initially bound Mg(II), arguing that these two ions occupy the same binding site(s) in the pigment. The pH‐dependent binding of Mg(II) and Ca(II) suggests coordination of these ions to carboxylic acid groups in the pigment. Mg(II) and Ca(II) can be added to a Fe(III)‐saturated melanin sample without affecting the amount of Fe(III) pre‐adsorbed, clearly establishing Fe(III) and Mg(II)/Ca(II) occupy different binding sites. Taking recent Raman spectroscopic data into account, the binding of Fe(III) is concluded to involve coordination to o‐dihydroxyl groups. The effects of metal ion content on the surface morphology were analyzed. No significant changes were found over the full range of Fe(III) concentration studied, which is supported by the Brunauer–Emmett–Teller surface area analysis. These observations imply the existence of channels within the melanin granules that can serve to transport metal ions.     

Utilization in alginate beads for Cu(II) and Ni(II) adsorption of an exopolysaccharide produced by <Emphasis Type="Italic">Chryseomonas luteola</Emphasis> TEM05

Copper and nickel adsorption onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and the immobilized C. luteola TEM05 from aqueous solutions were studied. After that, the multi metal ions containing these ions together were prepared and partial competitive adsorptions of these mixtures were also investigated. The metal adsorption of gel beads were carried out at pH 6.0, 25 °C. The maximum adsorption capacities in Langmuir isotherm for calcium alginate, calcium alginate + EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 1.505, 1.989, 1.976, 1.937 mmol/g dry weight for Cu(II) and 0.996, 1.224, 1.078, 1.219 mol/g dry weight for Ni(II), respectively.The competitive biosorption capacities of the carrier for all metal ions were lower than single conditions.     

High-performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides

The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.     

Cleavage of recombinant proteins at poly-His sequences by Co(II) and Cu(II)

Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0 degrees C to 70 degrees C. The work described here can also have implications for understanding protein stability in vitro and in vivo.     

Cooperative cluster formation in metallothionein

An ion-exchange chromatography procedure was used to resolve apometallothionein from the metallo- form in a study of metal-thiolate cluster formation. Chromatography of metallothionein reconstituted with Cd(II), Zn(II), or Cu(I) at neutral pH on carboxymethyl-cellulose led to removal of apoprotein from a solution without effect on recovery of the metalloprotein. Analysis of the effluent revealed apparent cooperative binding of these metal ions to the protein. Addition of 1-4 mol eq Cd(II) ions led to the recovery of metallothionein with around 4 mol eq Cd bound. The yield of this form increased with increasing starting metal ion equivalency. These results were obtained with two different ion-exchange resins. The cooperativity of binding was not total, but was initially confined to the carboxyl-terminal alpha domain. The results of metal and protein yields are inconsistent with random, noninteractive binding. Similar data were obtained with Zn(II) and Cu(I) ions although Cu(I) exhibited initial cooperative binding within the amino-terminal beta domain with over 5 mol eq Cu(I) bound.