Added Metal Ions Research Articles

The potential use of manganese oxidation in treating metal effluents

A technique of using microbially formed manganese oxides to takeup and immobilise metal effluents is described. An algal facilitated bacterial oxidation of manganese (Mn 2+, which overcomes slow rates of bacteria-only manganese oxidation, was used to produced high yields of oxides using 1.6 g/L Mn 2+ at neutral pH. Metals such as Cu, Zn, Co and Ni can be immobilised in stable co-precipitated manganese oxides. This effect was achieved either by the direct addition of metals during oxidation or by pretreating algal cells with metals prior to oxidation. Acid leaching (pH 2 H 2SO 4) showed that the immobilised metals were tightly bound by the manganese oxides. Transmission electron microscopy and X-ray analysis (TEM/EDAX) confirmed that bioformed manganese oxides had physically encapsulated the added metal ions, thereby preventing their release.

Electrochemiluminescence from Tunicate, Tunichrome–Metal Complexes and Other Biological Samples

Low level intrinsic electrochemiluminescence (ECL) was induced from body fluids and homogenized tissues of oysters and several species of tunicates. No significant ECL was detected in human blood cell lysates, or bovine haematin, but minor ECL was observed in avian blood cell lysates. Both terrestrial grass and seagrass exhibited ECL, which is probably attributable to chlorophyll, since dead (brown) grass did not demonstrate ECL. It was postulated that organic–metal complexes in marine invertebrates were, at least in part, responsible for the intrinsic ECL, since such animals are known to be rich in organically bound metals. However, alternative biochemical mechanisms for the observed ECL, which do not involve metal chelates, are possible. Various metal ions were added to the invertebrate preparations to determine whether exogenous metals could enhance or inhibit the ECL reactions. Strongly oxidizing metal ions such as Ag+, Au+, Cu2+, Hg2+ and Sb2+ at ≥100 ppm severely inhibited the intrinsic ECL response. No statistically significant ECL enhancement due to addition of metal ions was noted. ECL ‘profiles’ were generated which demonstrated differences in the ECL responses of individual tunicate preparations to the presence of various exogenous metal ions. Differences in ECL profiles may represent differences in types or levels of endogenous metal chelates or other biochemical constituents. In addition, synthetic tunichromes (tunicate pigments) were analysed for ECL in the presence and absence of various added metal ions. One synthetic tunichrome isomer demonstrated a specific ECL interaction with Hg2+, while the other demonstrated broader ECL activity with several metal ions.

Electrochemiluminescence from tunicate, tunichrome-metal complexes and other biological samples.

Low level intrinsic electrochemiluminescence (ECL) was induced from body fluids and homogenized tissues of oysters and several species of tunicates. No significant ECL was detected in human blood cell lysates, or bovine haematin, but minor ECL was observed in avian blood cell lysates. Both terrestrial grass and seagrass exhibited ECL, which is probably attributable to chlorophyll, since dead (brown) grass did not demonstrate ECL. It was postulated that organic-metal complexes in marine invertebrates were, at least in part, responsible for the intrinsic ECL, since such animals are known to be rich in organically bound metals. However, alternative biochemical mechanisms for the observed ECL, which do not involve metal chelates, are possible. Various metal ions were added to the invertebrate preparations to determine whether exogenous metals could enhance or inhibit the ECL reactions. Strongly oxidizing metal ions such as Ag+, Au+, Cu2+, Hg2+ and Sb2+ at > or = 100 ppm severely inhibited the intrinsic ECL response. No statistically significant ECL enhancement due to addition of metal ions was noted. ECL "profiles' were generated which demonstrated differences in the ECL responses of individual tunicate preparations to the presence of various exogenous metal ions. Differences in ECL profiles may represent differences in types or levels of endogenous metal chelates or other biochemical constituents. In addition, synthetic tunichromes (tunicate pigments) were analysed for ECL in the presence and absence of various added metal ions. One synthetic tunichrome isomer demonstrated a specific ECL interaction with Hg2+, while the other demonstrated broader ECL activity with several metal ions.

Study of the additions of cadmium and zinc to the cadmium, zinc metallothioneins using differential pulse polarography Influence of the pH

The binding properties and metal ion exchanges of Cd, Zn Metallothioneins (MT) from rabbit liver and horse kidney were studied using differential pulse polarography. The influence of the additions of cadmium or/and zinc to the non-saturated MT was investigated. The addition of cadmium does not remove the zinc initially bound to the protein, as one would expect from the values of stability constants of CdT and ZnT complexes, but produces the splitting of the peak attributed to the Cd-thionein complex into two: one corresponding to the initial one, CdT, and another peak, CdT', at a less cathodic potential. The addition of zinc provokes the transformation of the CdT peak, found for the native MT, into CdT'. Assuming that cadmium forms two complexes with the thionein the equilibrium between these two species CdT /ai CdT' was studied as a function of added metal ion concentration and of the solution pH. The pH plays a very important role in the stabilization of each of the two cadmium complexes. All ion exchange equilibria are quickly reached.

Enhancement of Oxidative Cleavage of DNA by the Binding Sites of Two Anti-double-stranded DNA Antibodies

Nucleic acid specificity was tested for two monoclonal anti-double-stranded DNA autoantibodies, 2C10 and H241, derived from two lupus-prone MRL/Mp-lpr/lpr mice. Antibody 2C10 bound double-stranded oligonucleotides with a preference for dA-dT over dG-dC base pairs and did not bind single-stranded oligonucleotides. Distamycin A, an antibiotic that binds to the minor groove, inhibited 2C10 binding of double-stranded DNA, suggesting that this antibody interacts with dA-dT base pairs in the minor groove. Antibody H241 binding was previously shown to have a dG-dC preference and to involve both major and minor grooves. In attempted footprinting assays, both 2C10 and H241 markedly en- hanced rather than protected against cleavage of DNA by hydroxyl radical-generating systems. With 2C10, this enhancement effect was observed only when hydroxyl radical generation was associated with oxidation of Fe(II). In contrast, H241 enhancement occurred in the presence of H2O2 and ascorbate or UV light irradiation and did not depend on added metal ion. The enhancement sites were related to the antibody binding specificities. The oligonucleotide 5'-AAAATATATATTT-3' was a much more effective inhibitor of the 2C10 enhancement than of the H241 effect, whereas the oligonucleotide 5'-GGGGCGCGCGCCC-3' was a much more effective inhibitor of the H241 enhancement. In addition, the enhanced cleavage occurred preferentially at dA-dT-rich regions with 2C10 and at dG-dC-rich regions with H241. These findings raise the possibility that anti-DNA autoantibodies could enhance DNA damage in inflammatory lesions in which hydroxyl radicals are generated.

Sequence-specific hydrolysis of yeast tRNA(Phe) mediated by metal-free bleomycin.

Bleomycin A2 (BLM) was found to mediate sequence specific hydrolysis of tRNA(Phe) in the absence of added metal ions. BLM A2 promoted phosphodiester bond hydrolysis 3' to the pyrimidine residue at all resolved Py-Pu sites not involving modified bases, as demonstrated by high-resolution electrophoretic analysis of 5'- and 3'-32P-end-labeled substrates. The reaction proceeded with surprising facility, approaching efficiency that of oxidative strand scission mediated by the FeII.BLM A2 complex. By the use of a number of BLM congeners, as well as a study of the time, temperature, and salt dependence of the hydrolysis, it was shown that in many respects the hydrolytic reaction parallels the oxidative degradation of RNA and DNA mediated by metallobleomycins. Thus, in contrast to the well-characterized oxidative degradation of DNA and RNA by bleomycin studied for two decades, the present report documents the ability of certain metal-free bleomycins to mediate RNA hydrolysis.

Identification of a Flavin:NADH Oxidoreductase Involved in the Biosynthesis of Actinorhodin: PURIFICATION AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

The biosynthesis of the polyketide antibiotic actinorhodin by Streptomyces coelicolor involves the oxidative dimerization and hydroxylation of a precursor, most likely dihydrokalafungin, as the final steps in its formation. Mutations in the actVB gene block these last steps, and the mutants secrete kalafungin as a shunt product. To investigate the role of the actVB gene in these transformation, we have overexpressed the gene in Escherichia coli and purified and characterized the recombinant protein. ActVB was shown to catalyze the reduction of FMN by NADH to give NAD and FMNH2, which, unusually, is released into solution. The protein contains no chromogenic cofactors and exhibits no requirements for added metal ions. The reaction obeys simple kinetics and proceeds through the formation of a ternary complex; Km values for FMN and NADH are 1.5 and 7.3 microM, respectively, and kcat is about 5 s-1. FAD and riboflavin are also substrates for the enzyme, although they have much higher Km values. The subunit structure of the enzyme was investigated by analytical ultracentrifugation, which showed the protein to exist in rapid equilibrium between monomer and dimer forms. The possible role of this oxidoreductase in the oxidative chemistry of actinorhodin biosynthesis is discussed.

Construction of Zn2+/Cd(2+)-tolerant cyanobacteria with a modified metallothionein divergon: further analysis of the function and regulation of smt.

This paper reports the (de novo) construction of mutants of Synechococcus PCC 7942 lacking the repressor (SmtB) of the metallothionein gene, smtA. These smtA+/B- cells are more tolerant to elevated [Zn2+] and [Cd2+] than cells containing an intact metallothionein divergon (smt). Previously selected (by step-wise adaptation) Cd(2+)-tolerant mutants contain additional copies of smtA and possibly other undetected mutations. It is now confirmed that these cells also contain a deletion within 'all' copies of smtB and hence fail to revert to wild type following subculture in medium which has not been supplemented with Cd2+ or Zn2+. Northern analysis showed enhanced accumulation of smtA transcripts, even in the absence of added metal ions in these mutants. An increase in the accumulation of Zn2+ is reported in cells containing an intact metallothionein divergon compared to cells deficient in both smtA and smtB. This supports the assumption that SmtA binds Zn2+ within cyanobacterial cells. We also describe the use of the above mentioned mutants to identify additional factors involved in the regulation of transcription from the smtA operator-promoter.

Effects of added metal ions on the interaction of chitin and partially deacetylated chitin with an azo dye carrying hydroxyl groups

AbstractThe binding of chrome violet, which is a monoazo dye and involves two hydroxyl groups in the o and o′ positions to an azo group, to chintin and partially decetylated chitin, was examined in the presence of metal ions. Zn2+ and Cu2+ ions do not perceptively influence the binding affinity of chrome voilet to chitin. In contrast, Co2+ ion enhances the binding and Ni2+ ion suppresses it. In the lower free dye concentrations the dye uptake by partially deceteylated chitin was tremendously enhanced by adding. Co2+ ion in the buffer solution of pH 5. The dye uptake by the polymer was considerably increased by the addition of Cu2+ ion at pH 5 and became much larger at pH 6. The amount corresponded to that in the presence of Co2+ ion. To investigate further the action of added metal ions, a cobalt‐ or a chrome–complex dye was prepared, and the binding properties for the polymers were compared with those of chrome violet in the presence of Co2+ and Cu2+ ions. Some possible mechanisms for the enhancement of chrome violet binding by the addition of Co2+ and Cu2+ ions are described. © 1995 John Wiley & Sons, Inc.

Transport of adsorbing metals from stream water to a stationary sand-bed in a laboratory flume

The transport of zinc and lithium ions between the overlying water column and the stationary sand-bed in a laboratory flume with bottom bedforms is investigated. Experiments have been performed under simplified conditions in a recirculating laboratory flume with straight impermeable walls and a sand-bed. The sand is well sorted and acid-washed to provide reproducible experimental conditions. The chemical composition of the recirculating water is controlled and steady flow conditions are maintained in the experiments. The concentrations of initially added metal ions are monitored both in the circulating overlying water and in the pore water of the sediment bed. Batch experiments were performed to investigate the chemical partitioning of the metal ions to the sand grain surfaces, and the data were compared with adsorption values obtained from the flume experiments. A model based on pressure-driven advective flow and linear partitioning of the pollutant to the sediment has been developed and accurately predicts the rate of transfer of the metal ions (zinc and lithium) into the bed in the case of stationary bedforms.

Influence of metal ions on hydroxymethylfurfural formation in honey

The effects of added magnesium, manganese, zinc and iron(II) ions on the production of hydroxymethylfurfural (HMF) in honey were studied using a spectrophotometric method using p-toluidine and barbituric acid for the formation of a coloured complex. All the metal ions used significantly accelerated HMF formation in honey, especially at high temperatures. As the concentration of the added metal ions was increased, there was a corresponding increase in the amount of HMF formed. Manganese ions caused the highest increase in the level of HMF, followed by zinc, magnesiumn and iron (II) ions in that order.

強酸性ＭｎＳＯ４溶液のオゾン酸化による二酸化マンガン析出反応の速さと反応に及ぼす共存金属イオンの影響

Ozone oxidation-precipitation reaction was investigated in strongly acidified solutions of MnSO4 with/without added sulfate.The rate of the oveall reaction was determined by chemical analysis on manganese ion species in the solution and the variation of oxidation-reduction potential (ORP) was continuously measured to follow the ozonation process in MnSO4 solutions of initial Mn concentration of 0.10 mol/l and sulfuric acid concentration of 1.0 to 5.0mol/l over a temperature range between 25 and 80°.The behavior of 15 elements (18 ionic species of metal ions and oxonium complex ions) added to the MnSO4 solution (0.1mol/l in Mn and 1.0mol/l in acid) during ozone oxidation at 25 and 70° was examined by ORP measurement and chemical analysis of the precipitates.The results are summarised as follows: 1) The rate of ozone oxidation-precipitation is strongly affected by acid concentration. An increase in acid concentration resulted in elongated induction period for the precipitation of MnO2, the reduced overall reaction rate and also higher minimum concentration of manganese remaining in the solution.2) A rise in reaction temperature favors the ozonation reaction. At 70°, precipitation of MnO2 took place even at sulfuric acid concentration of 4 or 5.0mol/l.3) Li, Na, K, Al and Mg ions added to the solutions caused only a little change in ORP during ozonation and the content of these ions of the precipitates was fairly low.4) Co, Ni and Cu in the solution were incorporated at about 2% in the precipitate without a significant change in ORP.5) ORP measurement showed that As (III), V (IV) and Fe (II) were oxidized prior to the oxidation of Mn (II). These ions and Ti (IV) are incorporated in the precipitates to a considerable extent of 7 to 10 wt % of the dried samples. None of the added metal ions is precipitated alone from their single acidic solution.6) Zn and Cd were incorporated in the precipitate at about 1 wt %. More than 70% of the added mercury was eliminated with manganese from the solution of an initial mercury concentration of 0.001mol/l.

A comparison of oil stability based on the metrohm rancimat with storage at 20°C

AbstractThe induction time for oxidative stability by the Rancimat method has been compared with peroxide development during storage at 20°C for six edible oils and rapeseed oil samples with added metal ions, antioxidants or phosphatidylethanolamine. The Rancimat method correlated highly (r=0.966;P=0.000) with oil stability measured by peroxide development for all samples except the oils containing added phosphatidylethanolamine or added butylated hydroxytoluene.

Ability of Aspergillus niger to tolerate metal ions and minerals in a solid-state fermentation system for the production of citric acid

Biosynthesis of citric acid by Aspergillus niger in molasses medium in submerged and liquid surface fermentations is highly sensitive to the concentration of trace elements present in the medium. Studies on citric acid production in wheatbran medium in a solid-state fermentation system, however, showed no such influence despite high concentrations of metal ions and total ash content in the wehatbran. In contrast, supplementation of wheatbran with additional amounts of mineral ions such as iron, copper, zinc, manganese, magnesium and phosphorous was found to be necessary for increased production of citric acid. All the mineral ions when used individually at an optimum level increased citric acid production by 1·4–1·9 times. However, only iron, copper and zinc were effective in combination. Citric acid production with this combination was 49·21 g/kg commercial wheatbran at 48 h as against 22·49 g formed in the absence of any added metal ions at the same fermentation time. These results indicate that the advantage of solid-state fermentations in tolerating high concentrations of metal ions in the production of citric acid is probably due to concentration gradients and impaired transport of the metal ions in the conditions of limited water availability.

The Cytochrome c Reductase Integrated Processing Peptidase from Potato Mitochondria Belongs to a New Class of Metalloendoproteases.

The general mitochondrial processing peptidase that removes the N-terminal targeting signals from proteins imported into mitochondria forms part of a respiratory protein complex in potato (Solanum tuberosum L.). We have termed this complex the "cytochrome c reductase/processing peptidase complex" and show that it acts on a variety of precursor proteins from different intramitochondrial locations. In potato, biochemical methods fail to separate the ubiquinol cytochrome c oxidoreductase function from the function of the processing protease. On the other hand, inhibition of electron flow with antimycin A or myxothiazol does not affect processing activity. The integration into an oligomeric protein complex causes the unique properties of the processing enzyme. It is fully active at high pH and in the presence of high salt. It does not need externally added metal ions, but it is inhibited by EDTA and 1,10-phenanthroline. Other protease inhibitors have no effect on the processing activity. Taken together, the molecular genetic and physiological results indicate that the mitochondrial processing protease does not belong to the thermolysin superfamily of metalloproteinases but may be a member of a new class of metalloendoproteases.

Potentiation of 45Ca uptake and acute toxicity mediated by the N-methyl-D-aspartate receptor: the effect of metal binding agents and transition metal ions.

The activities mediated by the N-methyl-D-aspartate (NMDA) receptor were studied in cultured rat cerebellar granule cells. Micromolar concentrations of the metal binding compounds, EDTA, cysteine, and histidine, as well as serum albumin strongly potentiated receptor activity in the presence of millimolar concentrations of Ca2+ and Mg2+. The findings indicated that these agents remove an endogenous metal, probably Zn2+, which attenuates NMDA receptor-mediated 45Ca uptake and toxicity. Several added metal ions were therefore tested at low micromolar concentrations. Zn2+ was found to be the most potent inhibitor of NMDA-induced 45Ca uptake, followed by Cu2+ and Fe2+.Co2+, Cd2+, Fe3+, and Al3+ had no significant effect, whereas Ni2+ potentiated the 45Ca uptake but inhibited at much higher concentrations. The potentiating agents that remove the endogenous metal had a particularly dramatic effect in the presence of Mg2+, the voltage-dependent suppressor of the NMDA receptor. Mg2+ also played an important role in the inhibitory effect of added Zn2+. Much lower concentrations of Zn2+ were needed to achieve inhibition of NMDA-induced 45Ca uptake in the presence of Mg2+. Under a variety of conditions, a very good correlation was found between NMDA receptor-mediated 45Ca uptake and the magnitude of acute neurotoxicity.

Cell growth on immobilized cell growth factor. 7. Protein-free cell culture by using growth-factor-immobilized polymer membrane

A protein-free culture of anchorage-dependent cells, mouse fibroblast cells, STO and 3T3-LI and fibroic sarcoma cells, Swiss albino HSDM 1C 1, grown on a cell-growth protein, insulin, and/or a cell-adhesion protein, collagen, which are immobilized or coimmobilized on surface-hydrolyzed poly(methyl methacrylate) membrane, was investigated. By adding metal ions and lipids to the culture medium, a proteinfree culture medium was composed, which was potent in promoting cell proliferation similarly to serum-containing culture medium. In particular, with insulin/collagen-coimmobilized membrane, a protein-free culture was established without detachment of growing cells over a long period. These protein-immobilized membranes could be used repeatedly.

Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme.

Metal ion requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme have been analyzed. RNA cleavage is observed when Mg2+, Sr2+, or Ca2+ are added to a 40 mM Tris-HCl buffer, indicating that these divalent cations were capable of supporting the reaction. No reaction was observed when other ions (Mn2+, Co2+, Cd2+, Ni2+, Ba2+, Na+, K+, Li+, NH4+, Rb+, and Cs+) were tested. In the absence of added metal ions, spermidine can induce a very slow ribozyme-catalyzed cleavage reaction that is not quenched by chelating agents (EDTA and EGTA) that are capable of quenching the metal-dependent reaction. Addition of Mn2+ to a reaction containing 2 mM spermidine increases the rate of the catalytic step by at least 100-fold. Spermidine also reduces the magnesium requirement for the reaction and strongly stimulates activity at limiting Mg2+ concentrations. There are no special ionic requirements for formation of the initial ribozyme-substrate complex--analysis of complex formation using native gels and kinetic assays shows that the ribozyme can bind substrate in 40 mM Tris-HCl buffer. Complex formation is inhibited by both Mn2+ and Co2+. Ionic requirements for the ribozyme-catalyzed ligation reaction are very similar to those for the cleavage reaction. We propose a model for catalysis by the hairpin ribozyme that is consistent with these findings. Formation of an initial ribozyme-substrate complex occurs without the obligatory involvement of divalent cations. Ions (e.g., Mg2+) can then bind to form a catalytically proficient complex, which reacts and dissociates.(ABSTRACT TRUNCATED AT 250 WORDS)

Structures of bulged three-way DNA junctions.

We have studied a series of three-way DNA junctions containing unpaired bases on one strand at the branch-point of the junctions. The global conformation of the arms of the junctions has been analysed by means of polyacrylamide gel electrophoresis, as a function of conditions. We find that in the absence of added metal ions, all the results for all the junctions can be accounted for by extended structures, with the largest angle being that between the arms defined by the strand containing the extra bases. Upon addition of magnesium (II) or hexamine cobalt (III) ions, the electrophoretic patterns change markedly, indicative of ion-dependent folding transitions for some of the junctions. For the junction lacking the unpaired bases, the three inter-arm angles appear to be quite similar, suggesting an extended structure. However, the addition of unpaired bases permits the three-way junction to adopt a significantly different structure, in which one angle becomes smaller than the other two. These species also exhibit marked protection against osmium addition to thymine bases at the point of strand exchange. These results are consistent with a model in which two of the helical arms undergo coaxial stacking in the presence of magnesium ions, with the third arm defining an angle that depends upon the number of unpaired bases.

Selective removal of some toxic metal ions (HgII,CuII, PbII AND ZnII) by reduction using steel plant granulated slag

Abstract The granulated slag of a steel plant was analysed for major and minor components. It was found to contain iron and manganese (along with some other metals in traces) in a state suitable for causing electrochemical reduction of a number of extraneously added metal ions. The extent of reduction of the metal ions was found to be in the order of their standard electrode potential values. By controlling parameters such as weight of slag used, pH, concentration and flow rate, a scheme for the selective removal of toxic metal ions such as Hg(II), Pb(II), cu(II) and Zn(II) was carried out. The method showed promise of application in the removal of toxic metal ions from solutions and effluents.