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.     

The electrochemical investigation of the manganese complexes of lactobionic acid

Interaction of manganese in different oxidation states with lactobionate ions in alkaline media was studied by polarographic, pH-metric and spectrophotometric methods. The results demonstrated that the lactobionate ligand forms stable parent and hydroxo mixed complexes with manganese(II) and manganese(IV), even in alkaline media. The composition of the complexes, and in the case of the manganese(II) system the corresponding conditional stability constants, were determined. The central atom of the manganese(III) complex is reduced to manganese(II) by the coordinated ligand. For comparison some analogous gluconate complexes were also studied.     

Interactions between different selenium compounds and zinc, cadmium and mercury

In this paper, we present a polarographic study of systems containing different inorganic and organic selenium compounds (sodium selenite, sodium selenate, seleno-methionine and seleno-urea) and metal ions (Zn2+, Cd2+ Hg2+) of the 12th group of elements in the periodic table. While zinc is a trace element known to be essential for plants and animals, cadmium and mercury are exogenous elements and are harmful pollutants that accumulate during aging; selenium is also recognized as an important micronutrient and is sometimes added to the diet. Experiments investigating the interactions were carried out using polarographic techniques in unbuffered systems. The three metal cations originated complexes with different strength and solubility in the presence of selenite anions; in the presence of selenate, polarography was not able to detect formation of complexes with these metal ions, at least under the experimental conditions used: a decrease of Hg2+ ion concentration was observed. Seleno-methionine did not react with Cd2+; in the presence of Zn2+, a soluble complex with a co-ordination number 1 was formed, while, again, the concentration of Hg2+ decreased in the presence of increasing concentrations of the selenium derivative. Seleno-urea did not react with Zn2+, but formed a complex with Cd2+ with limited solubility. Finally, this ligand could not be studied with Hg2+ because of the overlapping of the reduction potentials of both the ligand and the metal cation. Overall equilibrium constants for complex formation (Kf) and the solubility product (Ksp) for poorly soluble species are also reported.     

Thallium (I) interactions in biological fluids: a potentiometric investigation of thallium(I) complex equilibria with some sulphur-containing amino acids

Formation constants for thallium(I) complexes of L-cysteine (CysH2), DL-penicillamine (PenH2), N-acetyl-L-cysteine (AcyH2), and N-acetyl-DL-penicillamine (ApeH2) in aqueous solution have been determined in 150 mmol dm-3 NaCl medium at 37 degrees C by potentiometric titrations using a glass electrode. Glycine has been used as a model for simple amino acids. The experimental data may be explained by the formation of the complexes T1(Cys)-, T1(Cys)H, T1(Pen)-, T1(Pen)H, T1(Acy)-, and T1(Ape)- with log formation constants 3.26, 11.28, 3.60, 12.05, 2.27, and 2.45, respectively. Analysis of the results obtained and comparison of thallium(I) complexing ability with that of dimethyl-thallium(III) seem to indicate that thallium(I) toxicity does not directly stem from its interference with the metabolism of sulphur-containing compounds.     

Kinetic studies and thermodynamic results for complex formation and solvation of thallium(I) cryptates in acetonitrile and in acetonitrile-water mixtures

Stability constants and dissociation rate constants of a range of thallium(I) cryptates in acetonitrile and of the cryptate Tl(2,2,2)⁺ in water-acetonitrile mixtures have been measured at 25°C. Solvation free energies of transfer for Tl⁺ from water to acetonitrile and to water-acetonitrile mixtures have been estimated from polarographic measurements using the ferrocene assumption. The results allow the calculation of transfer free energy data for the stable cryptate, TlCry⁺, and for the transition state (Tl⁺…Cry)^≠.In mixtures of water and acetonitrile the stability constant of Tl(2,2,2)⁺ increases substantially with increasing acetonitrile content. This variation arises almost equally from an increase in the formation rate constant and a decrease in dissociation rate constant. Alternatively, the increase of stability constants for Tl(2,2,2)⁺ with increasing mole fraction of acetonitrile results from a strongly decreasing transfer free energy of the cryptate which surpasses in magnitude the increasing transfer free energies of the reactants.     

Differential pulse polarography: a method for the direct study of biosorption of metal ions by live bacteria from mixed metal solutions

The technique of differential pulse polarography is shown here to be applicable to the monitoring directly the biosorption of metal ions from solution by live bacteria from mixed metal solutions. Biosorption of Cd(II), Zn(II) and Ni(II) by P. cepacia was followed using data obtained at the potential which is characteristic of the metal ion in the absence and presence of cells. Hepes buffer (pH 7.4, 50 mM) was used as a supporting electrolyte in the polarographic chamber and metal ion peaks in the presence of cells of lower amplitude were obtained due to metal-binding by the cells. Well defined polarographic peaks were obtained in experiments involving mixtures of metal ions of Cd(II)-Zn(II), Cu(II)-Zn(II), Cu(II)-Cd(II) and Cd(II)-Ni(II). Biosorption of Cd(II), Zn(II) increased with solution pH. The method was also tested as a rapid technique for assessing removal of metal ions by live bacteria and the ability of the polarographic technique in measuring biosorption of metal ions from mixed metal solutions is demonstrated. Cu(II) was preferentially bound and removal of metals was in the order Cu(II) > Ni(II) > Zn(II), Cd(II) by intact cells of P. cepacia. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Electrochemistry of 2-thio- and 2-oxo-pyrimidines in dimethyl formamide in the presence of dioxygen

Electrocatalysis of dioxygen reduction to superoxide ion in dimethylformamide is afforded by a reversible interaction with thiol or hydroxo forms of pyrimidine which takes place only on the mercury surface electrode even in the absence of transition metal ions. The interaction is revealed by a polarographic wave and by a voltammetric peak which can be recorded only in the presence of dioxygen in the pyrimidines solution. The polarographic wave and voltammetric peak are attributed to the reduction of the oxygenated pyrimidine. In the range of concentration where the polarographic limiting currents due to the reduction of the oxygenated and unoxygenated pyrimidine are of the same order of magnitude, the stoichiometric ratio dioxygen/pyrimidine is determined to be 1. The electrode process probably involves the formation of a complex of mercury with the thiol or the hydroxo form of the pyrimidine on the electrode surface. Adsorption or coadsorption of the reactants seems to be involved.     

Kinetics of formation and dissociation of metallocarboxypeptidases.

The rates of formation of a number of metallocarboxypeptidases from metal ions and bovine apocarboxypeptidase A (CPA) have been measured directly and by a competitive method. Rates were determined with pH = 6-8 by utilising the pH change attending metal-ion incorporation, employing indicator and stopped-flow. Second-order rate constants Kf, M-1 s-1 at 25 degrees C, I = 1 M NaCl, pH = 7, Tris = 25 micrometer) were 1.7 X 10(5) (Mn2+), 3 X 10(4) (Co2+), 5 X 10(3) (Ni2+), 7 X 10(5) Zn2+), and 9 X 10(5) (Cd2+). Relative incorporation rate constants were determined at 25 degrees, pH = 7.0, Tris = 0.1 M, by competing two metal ions for a deficiency of apoprotein and analyzing the products by differential enzyme activity. Agreement between the two methods was reasonable. Rate constants for dissociation of CoCPA, NiCPA, and ZnCPA were measured by loss of enzyme activity on addition of the metal ion scavenger EDTA. Values of kd at 25 degrees, I = 1.0 M NaCl, pH = 7.0 were 8 X 10(-3), 3 X 10(-5), and 4 X 10(-4) s(-1), respectively. Values of K obtained kinetically (kf/kd) were in good agreement with those determined by activity measurements of equilibrated solutions. Results are compared with those of bovine apocarbonic anhydrase, where generally significantly slower rates are encountered.     

The cryptonephridial system in the mealworm Tenebrio molitor: Transport of radioactive potassium,thallium and sodium; a functional and structural study

Larvae of the mealworm Tenebrio molitor were injected with radioactive potassium, sodium or thallium solution. It was found that the rectal complex of the animal was labelled with potassium and thallium, but not with sodium. Potassium and thallium labelled the complex to the same level as if the two ions were tracers for each other. Ramsay has found that potassium is actively transported to the complex from the hemocoel and there are reasons to believe that T1+ follows the same pathway. Therefore animals injected with thallium were investigated both by light and electron microscopy. The results suggest that thallium spreads from the hemocoel through the leptophragma to the neighbouring ordinary tubular cells, and in moist mealworms thallium is further found in the perirectal space. Due to diffusion and washing out of thallium during fixation it can not be determined whether T1+ and K+ follow identical pathways, but it is possible to determine how far thallium has penetrated during the experiments.     

The binding of nucleotides and metal ions to elongation factor Tu from Bacillus stearothermophilus as studied by equilibrium dialysis

EF-Tu from B. stearothermophilus binds divalent metal ions even in the absence of guanine nucleotides. The association constants necessary for characterizing the multiple equilibria between EF-Tu, GDP and the divalent ions magnesium and manganese were determined by equilibrium dialysis. The constants are 4.6 X 10(4) M-1 and 5.4 X 10(5) M-1 for the binding of Mg2 and 1.0 X 10(5) M-1 and 1.1 X 10(6) M-1 for the binding of Mn2 to EF-Tu and EF-Tu . GDP, respectively. In the absence of divalent ions EF-Tu binds GMP, GDP and GTP with association constants of 3 x 10(3) M-1, 1.7 x 10(7) M-1 and 1.3 x 10(6) M-1, respectively. The binding of GDP in the presence of metal ions is an order of magnitude stronger than in the absence of metal ions.     

Silver and thallium(I) complexation with dibenzo-16-crown-4.

Dibenzo-16-crown-4 (1) indicates high silver and thallium(I) ion selectivity over sodium, potassium, and rubidium ion evaluated from the solvent extraction of metal picrates, while its cation-binding ability is lower than those of dibenzo-18-crown-6 (2) and dibenzo-22-crown-6 (3). Taking account of the highest thallium(I) ion selectivity for 1 obtained from extraction experiments, PVC membrane thallium(I)-selective electrodes based on 1 are prepared. The electrode shows the best potentiometric selectivity coefficients for thallium(I) over potassium and rubidium than those of 2 and 3, and commercially available bis(crown ether)s (4).     

Phospholipid-deacylating enzymes of rabbit platelets.

The inhibition of selenium-glutathione peroxidase by metal ions was studied by means of a direct spectrophotometric assay that monitors at 237 nm the decrease of GS^? concentration with time. Cadmium (II) and zinc (II) ions were the most potent inhibitors, while silver (I), mercury (II), cobalt (II), and lead (II) inhibited to a lesser extent. Inhibition by these metal ions was competitive with respect to the donor substrate, GSH. Competitive inhibition was verified for cadmium (II) ion by means of an assay employing Ellman's reagent, 5,5′-dithiobis-2-nitrobenzoic acid. Inhibition by cadmium (II) ion was noncompetitive with respect to the acceptor substrate, t-butyl hydroperoxide. Inhibitor constants obtained from Lineweaver-Burk plots and binding constants obtained from Scatchard plots were comparable. Correlation of inhibitor constants with chemical and physical properties showed a dependence on the softness of the metal ion as an acid and also a dependence on ionic size.     

Polyphasic character of ATP hydrolysis in actomyosin system.

The interaction of 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 0.1 or 1.0 M, KNO3, 25 degrees C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for delta log KM, where delta log KM = log KM(ATP)M(ATP)Tris--log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Tris-hydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident.     

Stopped-flow kinetic studies of metal ion dissociation or exchange in a tryptophan-containing parvalbumin

The rates of dissociation of 2 equiv of various metal ions [Ca(II), Cd(II), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Yb(III), and Lu(III)] from the primary CD and EF metal ion binding sites of parvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) were measured by stopped-flow techniques. The removal or replacement of metal ions was monitored by changes in sensitized Tb(III) luminescence or in intrinsic protein tryptophan fluorescence as quenching ions [Eu(III) or Yb(III)] were bound or removed or as the apoprotein was formed. In experiments wherein the bound metal ions were removed by mixing the parvalbumin with an excess of 1,2-diaminocyclohexanetetraacetic acid (DCTA), the kinetic traces were best fit by a double exponential with koff rate constants of 1.07 and 5.91 s-1 for Ca(II), 1.54 and 10.5 s-1 for Cd(II), and approximately 0.05 and approximately 0.5 s-1 for all of the trivalent lanthanide ions. In experiments wherein the bound metal ions were exchanged with an excess of a different metal ion, pseudo-first-order rate constants were proportional to the concentration of excess attacking metal ion for both the fast and slow processes in most experiments. In these cases, extrapolation of the rate constants to zero concentration of attacking metal ion gave values which agree well with the DCTA scavenging results. This finding demonstrates that the off rate constants do not depend on the occupancy of the neighboring site and therefore implies that there is no significant cooperativity in metal ion binding between the two sites in parvalbumin.     

Structural roles of monovalent cations in the HDV ribozyme

The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl(+)). Two Tl(+) ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl(+) ion forms direct inner-sphere contacts with the ribose 2'-OH nucleophile and the pro-S(p) scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.     

Solution-phase secondary-ion mass spectrometry of protonated amino acids

Although sulfolane proved unexpectedly to be a poor solvent for solution-phase secondary-ion mass spectrometry of underivatized amino acids in the presence of thallium(I) salts, glycerol was somewhat more effective. Also, the addition of trifluoromethanesulfonic acid proved more effective than addition of the metal in generating molecular ion complexes. A convenient and reliable method for rapidly determining amino acid molecular ions is based on these observations.     

The monensin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR

Addition of monesin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine (EPC) in sodium or potassium chloride solution and from dioleoylphosphatidylcholine (DOPC) in sodium chloride solutions gives rise to dynamic 23Na- and 39K-NMR spectra. The dynamic spectra arise from the monensin-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of monensin and metal ion concentrations and are compatible with a model in which one monensin molecule transports one metal ion. Rate constants for the association and dissociation of the monensin-metal complex in the membrane/water interface are extracted and the stability constants for complex formation are evaluated. The rate constants in DOPC are similar to those in EPC, confirming that diffusion is not rate-limiting in the transport process and that dissociation of the complex is the rate-limiting step. Although potassium on its own is transported more rapidly, sodium forms the more stable complex and is therefore transported preferentially in competition with potassium.     

Relaxation studies on complex formation of macrocyclic and open chain antibiotics with monovalent cations

The stability constants for the 1 : 1 complexes of macrocyclic antibiotics (nonactin, monactin, dinactin and trinactin) with Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH⁺₄ and for the Na⁺-complexes with the open chain compounds nigericin and monensin in methanol solution have been determined. The relaxation amplitude method was employed to obtain both the equilibrium constants and the enthalpies of reaction. The kinetics were studied with the help of temperature-jump, electric-field pulse and ultrasonic absorption techniques. Although complex formation of the metal ions with the antibiotics involves multidentate ligand chelation, the formation rates are in general very high, i.e. close to the limits imposed for diffusion controlled processes. The data for the macrotetrolides indicate the existence of conformational transition prior to complexation. A sequential substitution or “redressing” mechanism is proposed which is in accord with the high rates of complex formation. The selectivity patterns, as expressed by the equilibrium constants, are similar to those observed for the transport of metal ions across membranes in presence of the antibiotics. Selectivity results from an optimal balance between the strength of metal ion solvation and the stability of the individual metal complex, which in turn is governed by the conformational flexibility of the antibiotics.