Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

Manganese(II) dynamics and distribution in glial cells cultured from chick cerebral cortex

The kinetics of manganese(II) ion uptake and efflux have been investigated using tracer⁵⁴Mn(II) with glial cells cultured from chick cerebral cortex in chemically defined medium. The initial velocity of Mn(II) uptake versus [Mn(II)] exhibit saturation, with an apparent S_0.518(±3) M. Both the rate and extent of Mn(II) uptake are inhibited by Ca(II), either added externally or preloaded into the glial cells. Preloading of glia with Mn(II) also inhibits the rate of external⁵⁴Mn(II) uptake. Zn(II) inhibits but Cu(II) activates Mn(II) uptake. Efflux of Mn(II) from preloaded cells occurs as a biphasic process, with rapid release of 30–40% of total cell Mn(II), then much slower release of the remainder. Permeabilization of cells with dextran sulfate also rapidly released ca. 30% of total cell Mn(II). High external Mn(II) enhanced both the rate and extent of Mn(II) efflux. CCCP, an uncoupler of oxidative phosphorylation, inhibited both Mn(II) uptake and efflux significantly, but addition of cyanide, ouabain, insulin, hydrocortisone, K⁺, or Nd(III) had no effect on either process. Taken together, these data suggest a model in which Mn(II) is brought across the plasma membrane by facilitated diffusion, binds to cytosolic protein sites, and is partitioned into the mitochondria by an active transport mechanism. The fact that the Mn(II) flux rates observed with cultured glia are much faster than those reported for overall uptake and efflux of brain Mn(II)in vivo suggests that the blood-brain barrier may play a significant role in determining these latter rates in whole animals.Supported in part by NIH grant GM-33358 and a Biomedical Research Support Grant from the NIH administered by Penn State.     

Kinetic,ESR, and trapping evidence for in vivo binding of Mn(II) to glutamine synthetase in brain cells

Mn(II) has been proposed as a potential modulator of various important CNS enzymes, particularly glutamine synthetase, which is compartmentalized in the cytoplasm of glia. Previous studies demonstrated that total glial Mn(II) was 50–57 M, of which 30–40% occurs in the cytoplasm. In the present study, electron spin resonance (ESR) was used to determine that the concentration of free cytoplasmic Mn(II) in cultured chick glial cells is 0.8 (±0.2) M, very near K_d for the GS-Mn(II) complex. No free Mn(II) could be detected in glial mitochondria. Association of Mn(II) with brain glutamine synthetase (GS) was assessed under in vivo conditions in the presence of millimolar Mg(II) by trapping bound⁵⁴Mn(II) ions in the active site with irreversible inhibitors, namely methionine-sulfoximine (MSOX) or specific analogues thereof plus ATP. Ovine brain tissue was lysed directly into buffer containing Mn(II), 3 mM Mg(II), 1 mM MSOX, 1 mM ATP, 200 mM KCl, and 20 mM NaCl. Alternatively, primary cultures of chick glial cells were permeabilized into these inactivation mixtures. -Methyl-d,l-prothionine-S,R-sulfoximine was used to specifically inhibit the mechanistically-related enzyme -glutamyl-cysteine synthetase prior to specific inactivation of GS by -ethyl-d,l-methionine-S,R-sulfoximine. Even inthe presence of 2–3 mM Mg(II), with only 5–10 M Mn(II) present, approximately 20–30% of GS subunits were trapped with bound Mn(II). These results indicate that brain GS exhibits a high degree of specificity for binding Mn(II) over Mg(II) and that Mn(II) binds to GS to a significant extent under in vivo conditions.     

DNA cleavage promoted by trigonal-bipyramidal zinc(II) and copper(II) complexes formed by asymmetric tripodal tetradendate 2-[bis(2-aminoethyl)amino]ethanol

Asymmetric trigonal-bipyramidal Zn(II) complex 1 formed by 2-[bis(2-aminoethyl)amino]ethanol (L) was found to be able to promote the cleavage of supercoiled plasmid DNA pBR322 to the nicked and linear DNA via a hydrolytic manner but only in neutral Tris-HCl buffer, no cleavage was observed in HEPES or NaH₂PO₄/Na₂HPO₄ buffer. However, the copper complex 2 of L, possessing the similar coordination geometry, can only promote DNA cleavage via an oxidative mechanism in the presence of ascorbic acid. ESI-MS study implies that complex 1 exist mainly as [Zn(L)]²⁺/[Zn(L-H)]⁺ in neutral Tris-HCl buffer. Moreover, there is no discriminable species for complex 1 in HEPES or NaH₂PO₄/Na₂HPO₄ buffer. A phosphate activation mechanism via phosphate coordinating to Zn(II) center of [Zn(L)]²⁺/[Zn(L-H)]⁺ to form the stable trigonal-bipyramidal structure is proposed for the hydrolytic cleavage promote by complex 1. For complex 2, the abundance of [Cu(L)Cl]⁺ is higher than that of [Cu(L)]²⁺/[Cu(L-H)]⁺ in Tris-HCl buffer. The lower phosphate binding/activating ability of Cu(II) in complex 2 may be the origin for its incapability to promote the hydrolytic DNA cleavage. However, the readily accessible redox potential of Cu(II) makes complex 2 promote the oxidative DNA cleavage. Although the DNA cleavage promoted by complex 1 has no specificity, trigonal-bipyramidal Zn(II) complexes formed by asymmetric tripodal polyamine with ethoxyl pendent should be a novel potential model for practical artificial nuclease.     

Concentrations of physiologically important metal ions in glial cells cultured from chick cerebral cortex

Energy dispersive x-ray fluorescence and atomic absorption spectroscopy were used to determine the concentrations of Mg, Ca, Mn, Fe, Zn, and Cu in primary cultures of astroglial cells from chick embryo cortex in chemically defined serum-free growth medium. The intracellular volume of cultured glia was determined to be 8.34 l/mg protein. Intracellular Mn, Fe, Zn, and Cu in these cells were ca. 10–200 M, or 20–200 times the concentrations in the growth medium. Mg²⁺ was 7 mM in glial cells, only four-fold higher than in growth medium. Glutamine synthetase (GS), compartmentalized in glia, catalyzes a key step in the metabolism of neurotransmitterl-glutamate as part of the glutamate/glutamine cycle between neurons and glia. Hormones (insulin, hydrocortisone, and cAMP) added to growth medium differentially altered the activity of GS and the intracellular level of Mn(II), but not Mg(II). These findings suggest the possibility that glutamine synthetase activity could be regulated in brain by the intracellular levels of Mn(II) or the ratio of Mn(II)/Mg(II), which may in turn be controlled indirectly by means of transport processes that respond to hormones or secondary metabolic signals.     

Imidazolate-bridged dicopper(II) and copper(II)–zinc(II) complexes of macrocyclic ligand with methylimidazol pendants: Model study of copper(II)–zinc(II) superoxide dismutase

Two new homo- and hetero-dinuclear complexes, [Cu₂L(im)](ClO₄)₃4H₂O (1) and [CuZnL(im)](ClO₄)₃4H₂O (2) (where Im=1H-1midazole and L = 3, 6, 9, 16, 19, 22-hexaaza-6, 19-bis(1H-imidazol-4-ylmethyl)tricycle[22, 2, 2, 2^11,14]triaconta-1, 11, 13, 24, 27, 29-hexaene) were synthesized and characterized as model compounds for the active site of copper(II)–zinc(II) superoxide dismutase (Cu₂Zn₂–SOD). X-ray crystal structure analysis revealed that the metal centers in both complexes exhibit distorted trigonal-bipyramid coordination geometry and the CuCu and CuZn distances are both 6.02 Å. Magnetic and ESR spectral measurements of 1 showed antiferromagnetic exchange interactions between the imidazolate-bridged Cu(II) ions. The ESR spectrum of 2 displays typical signals of mononuclear Cu(II) complex, demonstrating the formation of heterodinuclear complex 2 rather than a mixture of homodinuclear Cu(II)/Zn(II) complexes. pH-dependent ESR and UV–visible spectral measurements manifest that the imidazolate exists as a bridging ligand from pH 6 to 11 for both complexes. The IC₅₀ values of 1.96 and 1.57 μM [per Cu(II) ion] for 1 and 2 suggest that they are good models for the Cu₂Zn₂–SOD.     

Variations in the coordination environment of Co, Cu and Zn complexes prepared from a tridentate (imino)pyridine ligand and their structural comparisons

The variations in the coordination environment of Co(II), Cu(II) and Zn(II) complexes with the neutral, tridentate ligand bis[1-(cyclohexylimino)ethyl]pyridine (BCIP) are reported. Analogous syntheses were carried out utilizing either the M(BF₄)₂ · xH₂O or MCl₂ · xH₂O metal salts (where M = Co(II), Cu(II) or Zn(II)) with one equivalent of BCIP. When the hydrated metal starting material was used, cationic, octahedral complexes of the type [M(BCIP)₂]²⁺ were isolated as the tetrafluoroborate salt (4, 5). Conversely, when the hydrated chloride metal salt was used as the starting material, only neutral, pentacoordinate [M(BCIP)Cl₂] complexes (1-3) formed. All complexes were characterized by X-ray diffraction studies. The three complexes that are five coordinate have distortions due mainly to the pyridine di-imine bite angle. The [Cu(BCIP)Cl₂] (2) also exhibits deviations in the Cu(II)-Cl bond distances with values of 2.4242(9) and 2.2505(9) Å, which are not seen in the analogous Zn(II) and Co(II) structures. Similarly, the two six coordinate complexes (5, 6) are also altered by the ligand frame bite angle giving rise to distorted octahedral geometries in each complex. The [Cu(BCIP)₂](BF₄)₂ (6) also exhibits Cu(II)-N_imine bond lengths that are on average 0.14 Å longer than those found in the analogous 5 coordinate complex, [Cu(BCIP)Cl₂]. In addition to X-ray analysis, all complexes were also characterized by UV/Vis and IR spectroscopy with ¹H NMR spectroscopy being used for the analysis of the Zn(II) analogue (3).     

Study of the coordination behaviour of (3,5-diphenyl-1H-pyrazol-1-yl)ethanol against Pd(II), Zn(II) and Cu(II)

A new easily synthetic route with a 96% yield of ligand 2-(3,5-diphenyl-1H-pyrazol-1-yl)ethanol (L) is obtained. The reactivity of L against Pd(II), Zn(II) and Cu(II) leads to [PdCl₂(L)₂] (1), [ZnCl₂(L)] (2) and [CuCl(L′)]₂ (3) (L′ is the ligand L without alcoholic proton), respectively. According to the different geometries imposed by the metallic centre and the capability of L to present various coordination links, it has been obtained complexes with square planar (1 and 3) or tetrahedral (2) geometry and different nuclearity: monomeric (1 and 2) or dimeric (3). Complete characterisation by analytical and spectroscopic methods, resolution of L and 1-3 by single-crystal X-ray diffraction and magnetic studies for complex 3 are presented.     

Removal and recovery of Cu(II) and Zn(II) using immobilized Mentha arvensis distillation waste biomass

The potential use of the immobilized Mentha arvensis distillation waste (IMADW) biomass for removal and recovery of Cu(II) and Zn(II) from aqueous was evaluated in the present study. Biosorption capacity of Cu(II) and Zn(II) on IMADW increased with increase in pH reaching a maximum at 5 for Cu(II) and 6 for Zn(II). The equilibrium sorption data agreed well with Langmuir isotherm model and pseudo-second-order kinetic model in batch mode. Cu(II) and Zn(II) uptake by IMADW was best described by pseudo-first-order kinetic model in continuous mode. Maximum Cu(II) and Zn(II) uptake by IMADW was 104.48 and 107.75 mg/g, respectively. Fourier Transform Infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were also carried out to investigate functional groups and surface changes of biomass. The results showed that IMADW biomass is a potential biomaterial to remove Cu(II) and Zn(II) ions with a high biosorption capacity from aqueous solutions.     

Preparation, structural, magnetic and thermal properties of two heterobimetallic cyano-bridged nickel(II)-copper(II)/platinum(II) coordination polymers

Two unique bimetalic Pt(II) coordination polymers of composition [Ni(hydeten)₂Pt(CN)₄] (Ni-Pt) and [Cu(hydeten)₂Pt(CN)₄] (Cu-Pt) [hydeten = N-(2-hydroxyethyl-ethylenediamine) or 2-(2-aminoethylamino)ethanol] have been synthesized and structurally characterized by various methods in this study. The crystal structure of Cu-Pt was determined by single-crystal X-ray diffraction analysis. The structure of Cu-Pt forms of infinite 2,2-TT type [-Cu(hydeten)₂-NC-Pt(CN)₂-CN-] chains containing paramagnetic copper atoms bridged by tetracyanoplatinate species. In this complex, Cu(II) centers display an axially elongated octahedron with two chelating hydeten molecules in the equatorial positions and N-bonded bridging cyano groups in the axial positions, whereas Pt(II) centers are four coordinate with four cyanide-carbon atoms in a square-planar arrangement. The decrease of the moments of these complexes in temperature range of 50 305 K can assigned to the antiferromagnetic interactions in the structures. The thermal decomposition of Cu-Pt comprise of five distinguished stages, while the thermal decomposition of Ni-Pt take place four different stages.     

Co(II), Zn(II) and Cu(II) compounds with 1,4; 1,3 or 1,2-bis-(benzimidazole-1-yl-methylene)-benzene as a flexible spacer ligand: Synthesis, characterization and X-ray structures of some polynuclear species

New dinuclear and polynuclear Co(II), Zn(II) and Cu(II) compounds with the ligands 1,4-bis-(benzimidazole-1-yl-methylene)-benzene (bbpx), 1,3-bis-(benzimidazole-1-yl-methylene)-benzene (bbmx) and 1,2-bis-(benzimidazole-1-yl-methylene)-benzene (bbox) are reported. With Co(II) and Zn(II) and the ligands bbpx, bbmx and bbox six new dinuclear and polynuclear compounds are described, i.e. [CoCl₂(bbpx)]_n(DMF)_2n (1), [CoCl₂(bbmx)]₂(DMF)₂ (2), [ZnCl₂(bbmx)]₂(DMF)₂ (3), [CoCl₂(bbmx)]_n(DMF)_n/2(CH₃OH)_n/4 (4), [CoCl₂(bbox)]_n(DMF)_3n/2 (5) and [ZnCl₂(bbox)]_n(DMF)_3n/2 (6). Also one polynuclear Cu(II) compound is reported, i.e. [Cu(bbpx)₂(C₂N₃)_1.5(OH)_0.5]₂(DMF)_4.15(CH₃OH)_1.75(H₂O)_3.5. The X-ray structures, physical and electronic properties are discussed.     

Synthesis and characterization of a crosslinked chitosan derivative with a complexing agent and its adsorption studies toward metal(II) ions

A new chitosan derivative has been synthesized by crosslinking a metal complexing agent, [6,6′-piperazine-1,4-diyldimethylenebis (4-methyl-2-formyl) phenol] (L), with chitosan (CTS). The resulting material (CCTSL) was characterized by elemental (CHN), spectral (FTIR and solid-state NMR), thermal (TGA and DTA), and structural (powder XRD and SEM) analyses. Adsorption experiments (pH dependency, kinetics, and equilibrium) of CCTSL toward various metal ions such as Mn(II), Fe(II), Co(II), Cu(II), Ni(II), Cd(II), and Pb(II) were carried out at 25 °C. The results showed that the adsorption was dependent on the pH of the solution, with a maximum capacity between pHs 6.5 and 8.5. The kinetics was evaluated by applying the pseudo-first-order and pseudo-second-order equation models and the equilibrium data were analyzed by Langmuir isotherm model. The maximum adsorption capacity was 1.21 mmol g⁻¹ for Cu(II) and the order of adsorption capacities for the metal(II) ions studied was found to be Cu(II) > Ni(II) > Cd(II)  Co(II)  Mn(II)  Fe(II)  Pb(II).     

Coordinative versatility of 2,3-bis(2-pyridyl)-5,8-dimethoxyquinoxaline (L) to different metal ions: syntheses, crystal structures and properties of [Cu(I)L]2 and [ML] (M=Cu(II), Ni(II), Zn(II) and Co(II))

The complexes of Cu(I), Cu(II), Ni(II), Zn(II) and Co(II) with a new polypyridyl ligand, 2,3-bis(2-pyridyl)-5,8-dimethoxyquinoxaline (L), have been synthesized and characterized. The crystal structures of these complexes have been elucidated by X-ray diffraction analyses and three types of coordination modes for L were found to exist in them. In the dinuclear complex [Cu(I)L(CH₃CN)]₂·(ClO₄)₂ (1), L acts as a tridentate ligand with two Cu(I) centers bridged by two L ligands to form a box-like dimeric structure, in which each Cu(I) ion is penta-coordinated with three nitrogen atoms and a methoxyl oxygen atom of two L ligands, and an acetonitrile. In [Cu(II)L(NO₃)₂]·CH₃CN 2, the Cu(II) center is coordinated to the two nitrogen atoms of the two pyridine rings of L which acts as a bidentate ligand. The structures of [Ni(II)L(NO₃)(H₂O)₂]·2CH₃CN·NO₃ (3), [Zn(II)L(NO₃)₂ (H₂O)]·2CH₃CN (4) and [Co(II)LCl₂(H₂O)] (5) are similar to each other in which L acts as a tridentate ligand by using its half side, and the metal centers are coordinated to a methoxyl oxygen atom and two bipyridine nitrogen atoms of L in the same side. The formation of infinite quasi-one-dimensional chains (1, 4 and 5) or a quasi-two-dimensional sheet (2) assisted by the intra- or intermolecular face-to-face aryl stacking interactions and hydrogen bonds may have stabilized the crystals of these complexes. Luminescence studies showed that 1 exhibits broad, structureless emissions at 420 nm in the solid state and at 450 nm in frozen alcohol frozen glasses at 77 K. Cyclic voltammetric studies of 1 show the presence of an irreversible metal-centered reduction wave at approximately −0.973 V versus Fc^+/0 and a quasi-reversible ligand-centered reduction couple at approximately −1.996 V versus Fc^+/0. The solution behaviors of these complexes have been further studied by UV-Vis and ESR techniques.     

Different crystal forms of Zn(II) compound with diethyl (pyridin-3-ylmethyl)phosphonate (3-pmpe) ligand: Zn(3-pmpe)Cl2

The synthesis and characterization of the new didentate ligand diethyl (pyridin-3-ylmethyl) phosphonate (3-pmpe) and three of its Zn(II) complexes are described. IR and X-ray analyses show that in the reaction of ZnCl₂ with 3-pmpe in methanol three crystalline polymorphs are formed: [Zn(3-pmpe)Cl₂]₂ (1) and [Zn(3-pmpe)Cl₂]_n (2 and 3). In these crystals 3-pmpe acts as a didentate N,O-bridging ligand and Zn(II) are in a slightly distorted tetrahedral ZnNOCl₂ environment. Zn²⁺ ions in 1 are doubly bridged by the 3-pmpe ligands, resulting in the formation of dinuclear species. In polymeric compounds 2 and 3 Zn²⁺ ions are singly bridged by the 3-pmpe, resulting in the formation of one-dimensional chains. Small differences in the conformation of the ligand in 1 and 2 have been found. The infrared spectra are in agreement with the structural data.     

Copper(II) complexes of 3-amino-1,2,4-triazine and 2-aminopyrazine: Strategies for designing crystalline materials using coordination polymers

Reaction of 3-amino-1,2,4-triazine (3-atz) or 2-aminopyrazine (2-apz) with Cu(hfac)₂·xH₂O led to the formation of the monometallic and trimetallic complexes Cu(hfac)₂(3-atz)₂ (1), Cu₃(hfac)₆(3-atz)₂ (2), Cu(hfac)₂(2-apz)₂ (3) and Cu₃(hfac)₆(2-apz)₂ (4). The azine molecules behave as both monodentate and bridging bidentate ligands. The Cu(II) ions exhibit a range of coordination geometries. In 1 and 3, the complex is distorted octahedral with the Jahn-Teller axis lying along one of the O-Cu-O axes. In 2 and 4, the central Cu(II) ion is also distorted octahedral with the Jahn-Teller axis lying along the N-Cu-N axis, while the terminal Cu(II) ions are five-coordinate. Structure analysis reveals that addition of the amino-substituent makes the ligands more coordinating, leading to shorter CuN bonds. In the case of 4, this results in a stronger magnetic superexchange pathway and the complex exhibits antiferromagnetic behavior at low temperatures.     

Transition metal-saccharide chemistry: d-glucose complexes of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II)

Metal complexes of d-glucose (d-Glc) from large cation containing dibromo-dichloro salts of dipositive metals [NEt₄]₂[MBr₂Cl₂] (M = Mn, Co, Ni, Cu and Zn) and the disodium salt of glucose were synthesized from a MeOH:MeCN mixture. The complexes were characterized by UV-vis absorption, circular dichroism, IR and proton magnetic resonance spectroscopies, and by elemental analysis, and were found to be Na[M(d-Glc)(OMe)Cl]. Cyclic voltammetric studies of these complexes, in the acidic to neutral pH range, indicated no dissociation, even in highly acidic conditions.This paper is dedicated to Professor Richard H. Holm (Harvard University) on the occasion of his 60th birthday.     

Synthesis,physico-chemical characterization and antimicrobial activity of cobalt(II), nickel(II), copper(II), zinc(II) and cadmium(II) complexes with some acyldihydrazones

Complexes of the type [M(bssdh)]Cl and [M(dspdh)]Cl, where M = Co(II), Ni(II), Cu(II), Zn(II) and Cd(II); Hbssdh = benzil salicylaldehyde succinic acid dihydrazone, Hdspdh = diacetyl salicylaldehyde phthalic acid dihydrazone have been synthesized and characterized with the help of elemental analyses, electrical conductance, magnetic susceptibility measurements, electronic, ESR and IR spectra and X–ray diffraction studies. Magnetic moment values and electronic spectral transitions indicate a spin free octahedral structure for Co(II), Ni(II) and Cu(II) complexes. IR spectral studies suggest that both the ligands behave as monobasic hexadentate ligands coordinating through three > C = O, two > C = N– and a phenolate group to the metal. ESR spectra of Cu(II) complexes are axial type and suggest as the ground state. X–ray powder diffraction parameters for [Co(bssdh)]Cl and [Co(dspdh)]Cl complexes correspond to an orthorhombic crystal lattice. The ligands as well as their metal complexes show a significant antifungal and antibacterial activity against various fungi and bacteria. The metal complexes are more active than the parent ligands.     

Manganese(II) coordination polymers with bis(5-tetrazolyl)methane: Synthesis, structure and magnetic properties

Two new Mn(II) coordination polymers with bis(5-tetrazolyl)methane (H₂btm), [Mn(btm)(phen)(H₂O)] · H₂O (1) and [Mn(btm)(2,2′-bpy)] · 1.5H₂O (2), have been synthesized and their structures determined by X-ray diffraction. In complex 1, the btm ligands assume the μ₂-1,1′:4 coordination mode and interlink Mn(II) ions into infinite one-dimensional chains. The chains are assembled into a three-dimensional architecture via hydrogen bonds and π-π interactions. For 2, Mn(II) ions are connected by btm ligands in the μ₃-1,1′:2:3′ mode to produce two-dimensional (6,3) coordination network. Magnetic investigations revealed that interactions through the btm bridges in both 1 and 2 are antiferromagnetic.     

Bioaccumulation of copper(II), lead(II) and chromium(VI) by growing Aspergillus niger

The effect of copper(II), lead(II) and chromium(VI) ions on the growth and bioaccumulation properties of Aspergillus niger was investigated as a function of initial pH and initial metal ion concentration. The optimum pH values for growth and metal ion accumulation were determined as 5.0, 4.5 and 3.5 for copper(II), lead(II) and chromium(VI) ions, respectively. Although all metal ion concentrations caused an inhibition effect on the growth of A. niger, it was capable of removing of copper(II) and lead(II) with a maximum specific uptake capacity of 15.6 and 34.4 mg g⁻¹ at 100 mg dm⁻³ initial copper(II) and lead(II) concentration, respectively. Growth of A. niger was highly effected by chromium(VI) ions and inhibited by 75 mg dm⁻³ initial chromium(VI) concentration since some inhibition occurred at lower concentrations.     

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.