Free Metal Ion Concentrations Research Articles

The effects of sulfite or nitrate on turnover-dependent inhibition in the ATPase from Halobacterium saccharovorum are related to the binding of the second metal ion.

The turnover-dependent inhibition of the Halobacterium saccharovorum ATPase is dependent on two parameters: pH and the concentration of the divalent cation present. At pH 6 and 1 mM Mn2+ the inhibition is small, but increases steeply with 6 mM Mn2+. In contrast, at pH 8.5 the inhibition is more than 90% at 1 mM Mn2+, and higher concentrations have little additional effect. A relationship between the occupation of a second metal ion binding site and turnover-dependent inhibition was postulated previously [Schobert, B. (1992) J. Biol. Chem. 267, 10252-10257]. The results lead to a model where this site (X-) can alternatively bind protons (XH), depending on the pH and the free metal ion concentration. The pKa of XH is estimated to be 9. The turnover-dependent inhibition is diminished by bisulfite, whereas sulfite is ineffective. The kinetics show that bisulfite and metal ion compete for the same site. In the proposed model, bisulfite binds via its negative charge to the site from which Pi was released and is arranged such as to interact with X- via its protonated group (X-HSO3-). In this way, formation of the inhibited enzyme species XMe is prevented. Inhibitory anions like nitrate, which carry a permanent dipole as a common feature, show uncompetitive inhibition vs metal ions. The data are compatible with a model in which these inhibitors bind to the vacant Pi site and position their positive charges near XH. As a consequence, the pKa of XH is decreased and X- is stabilized, which in turn favors the formation of XMe. The downshift in pKa was calculated to be 0.7 pH unit.

Copper and Cadmium Binding to Fish Gills: Estimates of Metal–Gill Stability Constants and Modelling of Metal Accumulation

Fathead minnows (Pimephales promelas) were exposed to 17 μg Cu∙L−1or 6 μg Cd∙L−1in synthetic soft water in the presence of competing ligands. Measured gill metal concentrations correlated with free metal ion concentrations, not with total metal. Langmuir isotherms were used to calculate conditional metal–gill equilibrium constants and the number of binding sites for each metal. Log KCu-gillwas estimated to be 7.4 and the number of Cu binding sites on a set of gills (70 mg, wet weight) was ~2 × 10−10mol (~30 nmol∙g wet weight−1). Log KCd-gillwas ~8.6, and the number of Cd binding sites on minnow gills was ~2 × 10−10mol (~2 nmol∙g wet weight−1). Stability constants for H+and Ca interactions at metal–gill binding sites and for metal interactions with dissolved organic carbon (DOC) were estimated using these metal–gill constants. All stability constants were entered into the MSNEQL+aquatic chemistry program, to predict metal accumulation on fish gills using metal, DOC, and Ca concentrations, and water pH. Calculated metal accumulation on gills correlated well with measured gill metal concentrations and with LC50values. Our approach of inserting biological data into an aquatic chemistry program is useful for modelling and predicting metal accumulation on gills and therefore toxicity to fish.

Calorimetric determination of the standard transformed enthalpy of a biochemical reaction at specified pH and pMg

In a biochemical reaction there is generally a change in the binding of hydrogen ions and metal ions. Therefore, calorimetric measurements of enthalpies of reaction have to be adjusted for the enthalpies of reaction of the hydrogen ions and metal ions produced or consumed with the buffer. It can be shown that this yields the standard transformed enthalpy of reaction that determines the change in the apparent equilibrium constant K′ (written in terms of sums of concentrations of species of a reactant) with temperature at the chosen pH and concentration of free metal ion. The derivations are based on the assumption that the changes in pH and free metal ion concentrations in the calorimetric experiment are small. This assumption is experimentally realized if a solution is well buffered for hydrogen and metal ions. The derived equations are discussed in terms of the implications they have for the performance and interpretation of calorimetric measurements.

Physicochemical and physiological effects on the uptake of dissolved zinc and cadmium by the amphipod crustacean Orchestia gammarellus

An artificial medium of NaCl in distilled water, of predictable dissolved metal speciation equilibria, has been used to define the effects of physicochemical changes on the uptake of zinc (Zn) and cadmium (Cd) by the euryhaline littoral amphipod crustacean Orchestia gammarellus (Pallas). At salinities between 36.5 and 25‰, changes in uptake rate of either metal correlated with predicted changes in free metal ion concentrations upon change in salinity, indicating the dominant role of medium physicochemistry in defining metal uptake rates. At 15‰ salinity, however, the change in uptake rate of each metal was no longer proportional to the change in free metal ion concentration, indicating a physiological response on the part of the amphipod to low salinity. It is proposed that such a response might be a reduction in apparent water permeability, characteristic of some euryhaline crustaceans. It is concluded that the use of a simple medium of defined physicochemistry has permitted the resolution of physicochemical and physiological effects on trace metal uptake rates, and that physiological adaptations of euryhaline organisms are significant in determining rates of trace metal uptake.

An ion-exchange/atomic absorption method for the measurement of ionized calcium and magnesium at micromolar concentrations

A quantitative ion-exchange/atomic absorption method is described for measuring the concentration of free (hydrated) calcium and magnesium in solution at micromolar levels. Sample solutions are pumped through a micro-column of strong acid-type cation-exchange resin until equilibrium has been achieved between resin and solution. After removal of interstitial solution by first air, then water, the sorbed metal ion is eluted from the resin with nitric acid directly into an atomic absorption spectrophotometer. In a 0.1 M 1:1 electrolyte, here KNO3, the amount of metal ion sorbed on the resin is directly proportional to the free metal ion concentration in solution over a concentration range of 1.25 to 5 × 10−5 mol/L (12–50 μmol/L). Selectivities for free calcium and magnesium in the presence of complexing ligands such as citrate and phosphate compare well with calculated values.

Bound and determined: A computer program for making buffers of defined ion concentrations

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40°C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i–iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.

The effect of pH, aluminum, and chelator manipulations on the growth of acidic and circumneutral species of Asterionella

The growth rates of two diatoms, acidophilic Asterionella ralfsii and circumneutral A. formosa, were differentially affected by varying pH, Al, and EDTA in chemically defined media. Free Al ion concentration increased as pH and EDTA concentration decreased. Free trace metal ion concentration decreased as EDTA levels increased but increased by orders of magnitude upon addition of Al. pH had an overriding species specific effect on growth rate; at low pH A. ralfsii had higher growth rates than A. formosa and vice versa at high pH. For both species higher EDTA levels depressed growth rates. Moderate additions of Al generally resulted in growth stimulation. The growth rate stimulations, especially at 200 and 400 μg L−1 Al additions, correlate to increases in free trace metal ion concentrations. The EDTA-AI interaction effects on growth rate were both pH and concentration dependent: at pH 7 both species were stimulated by addition of Al at all EDTA levels (except A. ralfsii at 5.0 mM EDTA and A. formosa at 0.5 mNM EDTA); at pH 6 Al addition either stimulated or had no effect on the growth rates of both species (except at low EDTA and high Al levels); at pH 5 A. formosa did not grow and additions of 200 μg L−1 Al stimulated growth of A. ralfsii. It is likely that the effect of pH, Al, and EDTA on speciation of essential or toxic trace metals affects growth rates of these diatoms in a species specific manner.

Influence of metals and metal speciation on the growth of filamentous bacteria

Calcium, copper, nickel and zinc were examined for their effect on the growth of axenic filamentous bacteria which included strains of Thiothrix, type 021N and type 1701. All organisms grew best at the upper range of calcium concentrations tested. Heavy metals were also less toxic to Thiothrix strain A1 at higher calcium concentrations. Copper was more inhibitory than either nickel or zinc and copper-nickel and copper-zinc mixtures appeared to act synergistically in suppressing the development of Thiothrix strain A1. In contrast, nickel toxicity could be reduced by the addition of zinc to the medium. Metal toxicity could also be diminished by the chelating agent, EDTA; therefore, free metal ions were most likely responsible for the observed toxic effects. A computer model, MINEQL, was used to predict metal speciation and to determine the potential for various media components to control speciation of the metals. The suitability of the computer model was determined by comparison of predicted and analyzed free metal ion concentrations.

Binding of cadmium by cyanobacterial growth media: Free ion concentration as a toxicity index to the cyanobacteriumNostoc UAM 208

A quantitative study of cadmium binding to three different growth media for nitrogen-fixing cyanobacteria was done with the aid of a solid state ion-specific electrode. Kratz and Myers modified medium and Arnon medium bound large amounts of Cd2+, BG11o medium had less binding capacity. Of the media components, phosphate ion showed the greatest ability to bind Cd2+. Different pHs, the size of cell inoculum and two buffers (Tricine and HEPES, 25 mM) also changed the availability of free cadmium ion in solution. The effect of free Cd2+ ion towards the cyanobacterium Nostoc UAM 208, isolated from a heavy metal polluted environment, also was tested. The effective concentration affecting 50% of population (EC50), at 120 h of exposure, was less for nitrogenase activity (0.26 microgram/mL) than for growth (0.55 microgram/mL), suggesting that this enzyme activity is more sensitive to cadmium than growth. Furthermore, cadmium toxicity was influenced by the addition of buffers to the growth medium. In the presence of buffer, Tricine (25 mM), growth and nitrogenase activity was reduced by 50% at a total cadmium concentration of about 115 micrograms/mL, although no free ion was detected in this case. These results suggest that although generally cadmium toxicity is a function of free metal ion concentration, this can also vary in the presence of complexing agents.

Surface charge of MnCO 3and FeCO 3

The surface charge of two carbonate minerals (rhodochrosite, and siderite) was investigated as a function of pH (4 to 8) and ionic strength (0.03, 0.1, and 1.0 mol dm{sup {minus}3} NaCl) under two different partial pressures of CO{sub 2} (0.5 and 0.005 atm). The acid/base titrations were performed with a flow-through reactor. This technique minimizes the contribution of dissolution and precipitation and thus allows the measurement of the surface charge to be performed. The point of zero charge (PZC) at the mineral surface is found to be equal to the point of zero salt effect and to the pH at which, in solution, the free metal ion concentration is equal in charge to the bicarbonate concentration. At lower pH values, the positive charge of the carbonate mineral surface increases as pH decreases. Above PZC, the surface of carbonate minerals is negatively charged. Whatever partial pressure of CO{sub 2} and ionic strength, this negative charge was found to be a unique function of the variable {l brace}HCO{sup {minus}}{sub 3}{r brace}/{l brace}HCO{sup {minus}}{sub 3}{r brace}{sub PZC}. This variable appears to control the sorption of bivalent trace metals onto carbonate surfaces. This is shown by taking into account previous studies such as sorptionmore » of Mn(II) onto siderite or sorption of Zn(II) onto calcite. Finally, a control of weathering of carbonate minerals by their surface charge is suggested.« less

Complexometric titrations of protein-bound metal ions: A method for determining binding constants

A method for quantifying the affinity of proteins for specific metal ions has been developed. Both the stoichiometry and the binding constants of the protein-bound metal ion can be determined by titrating protein-bound metal ions with complexometric reagents and observing electrochemically the change in free metal ion concentration. The technique is limited to cases where the affinity of the macromolecule for the metal ion is less than or similar to the affinity of the complexometric reagent for the metal ion. The method has been employed successfully in the study of both Cu(II) and Ag(I) binding to the apoprotein of bovine cuprozinc superoxide dismutase.

Studies of the complexation properties of aquatic humic material by differential pulse polarography

Amperometric titrations with copper and cadmium have been carried out for EDTA and two types of humic material of aquatic origin at pH 5.0-6.7. The free metal ion concentration was measured by differential pulse polarography. Accurate results were obtained for EDTA with both copper and cadmium as titrants and vice versa, the concentration of metal was determined accurately by titration with EDTA. The humic materials studied were a standard fulvic acid that had been isolated through a 12-step seperation procedure, and a natural fresh water sample. The formation of the metal-humic complexes was found to be complete within 3 min. The complexation capacity and stability constants were calculated using five different procedures; both inert and labile metal complexes were taken into consideration. The choice of calculation procedure influenced the results to some extent. The complexation capacities and stability constants were in most cases similar for the two types of humic matter. The complexation capacity, in terms of mmol g −1 TOC, was independent of the concentration of humic matter. The titrations of the two humic materials showed a more pronounced complexation of copper than of cadmium. The complexation capacity for cadmium was found to be less than one-tenth of that for copper. The interpretation of the amperometric titration curves is complicated by the uncertainty regarding the polarographic lability of the metal-humic complexes.

Effect of physico-chemical heterogeneity of natural complexants : Part II. Buffering action and role of their background sites

The peculiar buffering behaviour of the “background sites” of natural heterogeneous complexants is described. Background sites include most of the minor sites, i.e., a group of sites typically comprising not more than ca. 10% of the total sites. Despite this low proportion, they play an important role in controlling free metal ion concentrations in natural systems, because their complexation energy is higher than that of the major sites and their log K values span a broad range of order of magnitudes, owing to the cooperative effect of the large number of site types that they include, together with such continuous secondary effects as those due to a polyelectrolytic interactions and conformational changes. The main characteristics of these sites are summarized and a mathematical and chemical explanation for their cooperative action in controlling the overall free energy of metal ion complexation and the overall buffering intensity of the system is presented. Comparison is made with a classical system made up of a few simple ligands and devoid of secondary effects. The important parameters useful for describing the properties of background sites as a whole are deduced for complexation data obeying the Freundlich isotherm, which has been shown to fit most of the properties of background sites of natural systems.

The effect of the chelating agent EDTA on the rate of uptake of zinc by Palaemon elegans (Crustacea: Decapoda)

The rate of uptake of zinc by the decapod crustacean Palaemon elegans in the presence of the chelating agent EDTA can be correlated with the free zinc concentration. A simple method of calculating the effective free metal ion concentration, based on conditional stability constants, is presented.

Heavy metal solids association in sewage sludges

In order to study the metal binding characteristics of raw, activated and anaerobically digested sewage sludges, each sludge was passed through a range of filter pore sizes. Each filtrate was then subjected to complexation titration with cadmium, copper and lead using ion selective electrodes to follow the free metal ion concentration. Calibration of the electrodes was extended using metal-ion buffer standards. Conditional stability constants and complexation capacities were determined for each metal in each sludge fraction. The stability of complexes formed in each sludge type followed the general order copper > lead > cadmium. Both copper and lead formed more stable complexes in the treated sludges than in the raw sludge. In general, the capacities of raw and digested sludges to complex all three metals (in terms of unit mass of solids) decreased with increasing particle size. However, the converse was true for activated sludge, particularly for copper, suggesting a lesser contribution from surface area related phenomena. It is concluded that the digestion of sludge plays an important role in controlling the mobility and environmental dispersion of these metals.

The binding of heavy metals to algal surfaces

Biological particles can profoundly influence the distribution of heavy metals in natural waters because the functional groups on the cell surfaces are able to bind metal ions. The interaction of Cu(III) and Cd(II) was evaluated from titration of algal suspensions ( Chlamydomonas rheinhardii) (i) at a constant pH with increments of metal ions and (ii) in the presence of the metal ions with increments of acid. A voltammetric methodology was developed to measure (without prior separation of the solid phase) the metal ions in solution in the presence of algae and to assess the binding of metals to the surfaces of algae. The surfaces of algal cells have a high affinity for Cu(II) and Cd(II), even in the presence of 10 −3 M Ca 2+; their functional group ligands can compete with soluble complex formers typically present in natural waters. The adsorption of metals is readily interpreted in terms of surface complex formation equilibria or—mathematically equivalent—Langmuir type adsorption equilibria. The “average” conditional equilibrium constant extracted from the experimental data permits a generalization valid for a certain range of metal surface loading and can be used in multi-metal multi-ligand speciation calculations. Under-standably, the data cannot be fitted over a large range of free metal ion concentrations or metal loading of the surface because the tendency to form surface complexes decreases with increasing metal loading, because there are a variety of surface ligands, and the metal ions bind first to the surface groups with highest affinity and subsequently to groups with lower affinity. A much better fit of the data is obtained if models are used with more than one adjustable constant such as the constant capacitance or the Fowler Guggenheim Frumkin model or a two-site Langmuir isotherm. The kinetics of adsorption and uptake of Cu(II) to Chlamydomonas is characterized by the establishment of a relatively fast pseudo-adsorption equilibrium with the surface that is followed by slow diffusion-controlled uptake into the inside of the cell.

Etude de la complexation du cadmium par un acide humique de reference

A previous structural investigation of a commercial model of humic acids by Ebenga et al. [ Wat. Res. 20, 1383–1392 (1986)] revealed the presence of phenolic, benzoate, phtalate and salicylate groups close to semiquinonic anion radicals, aromatic units being linked by some alcanoïc chains. These structural features are potential sites of interaction with micropollutants and it is necessary to determine the thermodynamical characteristics of these interactions so as to take them into account in water treatment processes. Cadmium was chosen as a model of micropollutant and Fluka humic acids (FHA) were considered. Complexation phenomenon was first studied by u.v.-visible spectrophotometry and 113Cd NMR. The first failed in the characterization of interaction sites but the second revealed that essentially two kinds of complexes are to be considered (Fig. 1). In order to determine the corresponding stability constants, free metal concentrations were measured by the use of differential pulse anodic stripping voltammetry (DPASV) and uncomplexed metal species were determined with an ion selective electrode (ISE). A preliminary calibration with titrated Cd-solutions revealed that over 100 μgl −1, the DPASV peak corresponding to a medium containing FHA is higher than that observed in a medium without ligand! This observation can surely be matched with the known surfactant character of FHA [Kozarac et al., Wat. Res. 20, 295–300 (1986)], property which is known to disturb the linear relationship between diffusion current intensity and free metal ion concentration. Comparison of DPASV and ISE results shows that over 2 μg Cd mg FHA −1, some complexed species are destroyed during DPASV analytical process (Fig. 2). This comforts the previous NMR hypothesis of different kinds of complexes. Computation of conditional stability constants was then performed taking advantage of the complementarity of DPASV and ISE data. For the first time, Scatchard's method was applied to determine Cd-Fluka humic acids interaction; it fits the best for this kind of investigation since it is a promising method for multisite complexing molecules, already used with benefit by Alberts and Giesy [ Aquatic and Terrestrial Humic Materials (1983)] relatively to isolated natural organic matter. Two kinds of complexation sites were detected, for 1 and 10 Cd ions per FHA molecule, with corresponding stability constants of around 10 7 and 10 5 M −1, respectively. This investigation has thus shown that complexation of Cd by Fluka humic acids leads to very stable structures, from which it is rather difficult to eliminate metallic ions. But fulvic acids are also to be considered since their occurrence in the environment induces the formation of metallic complexes the stability constants of which are smaller [Bhat and Weber, Analytica chim. Acta 141, 95–103 (1982)]. FHA thus seems no longer to be a representative model and one had rather to substitute it by a more convenient model, the determination of which is to be performed. A deeper investigation of the actual structure of the interaction sites and of the complexed form stabilities indeed needs the synthesis of a representative model, considering both the toxicological and the water treatment points of view.

“Light-derepressible” genes are regulated by metal-protein complexes: A hypothesis

The hypothesis is presented that genes whose expression is sensitive to light are regulated by metal-protein complexes. This hypothesis is based on induction of a gene for an Mr 21,000 polypeptide in the prokaryote Arthrobacter sp. by photodynamic conditions, chelating agents, or a decrease in pH. It is proposed that these conditions promote dissociation of a regulatory metal-protein complex by photooxidation of the metal-binding site, lowering the concentration of free metal ion, or protonation of amino acid side-chains in the metal-binding site, respectively. Repression of this gene is restored when cells are returned to the dark, metal ions are added in excess of chelating agents or the pH of the medium is returned to neutral. Restoration of repression is inhibited by chloramphenicol and novobiocin, which suggests that this process requires resynthesis of a regulatory protein and reassociation of the complex with DNA. The hypothesis predicts a number of characteristics of the regulatory system, most of which are found among the effects of phytochrome. The well-documented effects of transition metal ions on the Pfr form of phytochrome suggest that changes in gene expression in plants mediated by photoisomerisation of this chromoprotein are initiated by a chelating activity of Pfr.

6] Metal-buffered systems

The design of a metal ion buffer system useful in a given enzymological application is subject to a number of different requirements. 1. The total concentration of added metal ion, Mt, should be large enough to damp out the effect of any adventitious quantities of the same metal ion and to overwhelm adventitious quantities of other metal ions. 2. The ratio of free to bound ligand should be high enough that the calculated ratio between the concentrations of free metal ion M and Mt will not be unduly sensitive to uncertainties in the values of metal-ligand stability constants. If possible, [Lt]/[Mt] should be large enough that the variation of free metal ion concentration, [M], with [Mt] will be effectively linear in the range of interest. 3. The concentrations of metal ion buffer species, both the free ligand and metal-ligand complexes, should be kept reasonably low in order to minimize the possibility of perturbation of the enzyme/metal ion equilibrium. The best design will be that which most successfully balances these sometimes opposing requirements.

Prediction of Soluble Nickel Removal by Activated Sludge Using Free Metal Ion Concentrations

Batch isotherm studies using spiked sewage samples containing a range of total soluble nickel concentrations typical of municipal sewage strongly suggested that it is the free (i.e. uncomplexed) nickel ion that is sorbed by activated sludge. Equations relating nickel uptake by activated sludge to free nickel ion concentrations and the extent of complexation in untreated sewage were developed and applied. Predicted and measured nickel removals generally agreed to within ± 30%.