Effects of Ca(II) ions on Cu(II) ion-phytic acid interactions

In vitro interactions among phytic acid (PA), Cu(II) ions, and Ca(II) ions were examined as functions of PA:Cu(II):Ca(II) molar ratios and pH. Ca(II) ions competed with Cu(II) ions for binding by the soluble phytate species for PA:Cu(II) molar ratios ranging from 10:1 to 1:6 and pH values in the 2.4-5.9 range. At pH values where precipitation occurred, Ca(II) ions potentiated Cu(II) ion binding by the precipitated phytate species for PA:Cu(II) molar ratios of 10:1 to 1:3. At lower PA:Cu(II) molar ratios, Ca(II) ions competed with Cu(II) ions for binding by the precipitated phytate species. Compositions of the precipitated copper-calcium phytates are reported.     

Some aspects of the copper(II)-DNA interaction

The Cu(II) ion interaction with calf-thymus DNA was studied by means of differential pulse polarography and sweep voltammetry as well as chromatography and viscosimetry. Most of the complexes formed at high ionic strength (0.2 M) and lower Cu(II) concentrations are of a nondenaturing nature. Their formation has but a minor effect on unwinding process of the DNA double helix. The excess of Cu(II) (P = 5) leads, however, to distinct denaturation of the DNA structure. Metal ions have little effect on the denaturation induced by the polarographic reduction of DNA on the mercury electrode. This conclusion is consistent with the character of the polarographic process and with the fact that Cu(II) ions are not very effective in the interaction with AT pairs. Cupric ions have no renaturing ability towards thermally denatured DNA at 0.2 M ionic strength but distinct renaturation was observed at low ionic strength (0.05 M).     

Copper(II)-DNA denaturation. II. The model of DNA denaturation

In a continuing study of the denaturation of DNA as brought abought about by Cu(II) ions, results are presented for the dependence of T_m and τ (the terminal relaxation time) on ionic strength, pH, reactant concentrations, and temperature. Maximum stability of the double helix, as reflected by the longest relaxation times and highest T_m values, was observed between pH 5.3 and 6.2. Outside this range both T_m and τ decreased sharply. A second, faster relaxation time was deduced from the kinetic cureves. The apparent activation energies of the rapid and slow (“terminal”) relaxations were found to be 12 and 55 kcal/mole, respectively. Several lines of evidence led to the conclusions that (1) the rate-determining step in DNA denaturation, when occurring in the transition region, is determined by chemical events and (2) the interactions which are disrupted kinetically in the rate-determining step are those which account for the major portion of the thermal (T_m) stability of helical DNA.     

The dynamics of 57Fe nuclei in Fe(II)-DNA and [Fe(II)(1-methyl-2-mercaptoimidazole)2]-DNA condensates

Alcoholic solutions of FeCl(2) and Fe(II)(Hmmi)(2)Cl(2) (Hmmi=1-methyl-2-mercaptoimidazole) induce calf thymus DNA condensation from aqueous solutions buffered at pH 7.4. A 1:1 Fe(II)-(DNA monomer) stoichiometry is assumed. The (57)Fe M?ssbauer hyperfine parameters suggest an octahedral coordination environment, severely distorted, in both Fe(II)-(DNA monomer) and [Fe(II)(Hmmi)(2)]-(DNA monomer) condensates. The dynamic properties of iron nuclei in freeze-dried samples were investigated by means of variable temperature (57)Fe M?ssbauer spectroscopy. Mean square displacements, (T), were calculated, such as the effective vibrating mass and the M?ssbauer lattice temperature of the solids. increases linearly with the temperature in the whole temperature range explored; the absolute values are typical for lattice or solid-state vibrations. Very similar values for the effective vibrating masses were extracted, suggesting comparable covalency of the bonding interaction between the metal atom and its ligands, while the M?ssbauer lattice temperatures show a softening of the lattice for [Fe(II)(Hmmi)(2)]-(DNA monomer) with respect to Fe(II)-(DNA monomer) condensate.     

Phytic acid-metal ion interactions. II. The effect of pH on Ca(II) binding

The interaction of phytic acid with Ca(II) has been studied by potentiometric titration and by measurement of free Ca(II) concentrations using an ion Selective electrode. With increasing Ca(II) concentration, the titration curve of phytic acid is displaced to regions of lower pH. In the binding of calcium ions to phytic acid, there is no evidence that significant binding occurs below approximately pH 5. Above this pH, the extent of binding is dependent upon both pH and the calcium to phytic acid ratios. Maximum binding obtains at a Ca(II):phytate ratio of 6 with 4.8 mol of Ca(II) bound per mol of phytate above pH ca. 8. Binding constants are apparently very large since binding isotherms at any Ca(II):phytate ratio are a linear function of the total calcium ion concentration. In all cases, binding occurs only when one or more phosphate groups have been converted to the oxo dianion form. The apparent pK' values (curve-fit parameters) that describe the potentiometric titration data are in good agreement with the constants evaluated from the binding of Ca(II) to phytate as a function of pH. Using CPK space-filling models, structures containing six metal ions in coordinate linkage to pairs of oxo dianions have been constructed and discussed within the framework of the axial conformation of phytic acid and the order of proton removal with an increase in pH based upon NMR studies.     

A theoretical study of zinc(II) interactions with amino acid models and peptide fragments

Density functional theory calculations have been employed to study the interaction between the Zn²⁺ ion and some standard amino acid models. The highest affinities towards the Zn²⁺ ion are predicted for serine, cysteine, and histidine. Relatively high affinities are reported also for proline and glutamate/aspartate residues. It was found that the zinc complexes with cysteine adopt a tetrahedral conformation. Conversely, complexes with one or two histidine moieties remain in an octahedral geometry, while those with three or more histidine groups adopt a square-planar geometry. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A comparative study of DNA complexation with Mg(II) and Ca(II) in aqueous solution: major and minor grooves bindings

Although structural differences for the Mg-DNA and Ca-DNA complexes are provided in the solid state, such comparative study in aqueous solution has been less investigated. The aim of this study was to examine the bindings of Mg and Ca cations with calf thymus DNA in aqueous solution at physiological pH, using constant concentration of DNA (1.25 or 12.5 mM) and various concentrations of metal ions (2 microM-650 microM). Capillary electrophoresis, UV-visible, and Fourier transform infrared spectroscopic methods were used to determine the cation-binding modes, the binding constants, and DNA structural variations in aqueous solution. Direct Ca-PO(2) binding was evident by major spectral changes (shifting and splitting) of the backbone PO(2) asymmetric stretching at 1222 cm(-1) with K = 4.80 x 10(5) M(-1), whereas an indirect Mg-phosphate interaction occurred (due to the lack of shifting and splitting of the phosphate band at 1222 cm(-1)) with K = 5.6 x 10(4) M(-1). The metal-base bindings were directly for the Mg with K = 3.20 x 10(5) M(-1) and indirectly for the Ca cation with K = 3.0 x 10(4) M(-1). Both major and minor groove bindings were observed with no alteration of the B-DNA conformation.     

Energetics of Ca(2+)-EDTA interactions: calorimetric study

The interaction between Ca(2+) and EDTA has been studied using isothermal titration calorimetry to elucidate the detailed mechanism of complex formation and to relate the apparent thermodynamic parameters of calcium binding to the intrinsic effects of ionization. It has been shown that Ca(2+) binding to EDTA is an exothermic process in the temperature range 5-48 degrees C and is highly dependent on the buffer in which the reaction occurs. Calorimetric measurements along with pH-titration of EDTA under different solvent conditions shows that the apparent enthalpy effect of the binding is predominantly from the protonation of buffer. Subtraction of the ionization effect of buffer from the total enthalpy shows that the enthalpy of binding Ca(2+) to EDTA is -0.56 kcal mol(-1) at pH 7.5. The DeltaH value strongly depends on solvent conditions as a result of the degree of ionization of the two amino groups in the EDTA molecule, but depends little on temperature, indicating that the heat capacity increment for metal binding is close to zero. At physiological pH values where the amino groups of EDTA with pK(a)=6.16 and pK(a)=10.26 are differently ionized, the coordination of the Ca(2+) ion into the complex leads to release of one proton due to deprotonation of the amino group having pK(a)=10.26. Increasing the pH up to 11.2, where little or no ionization occurs, leads to elimination of the enthalpy component due to ionization, while its decrease to pH 2 leads to its increase, due to protonation of the two amino groups. The heat effect of Ca(2+)/EDTA interactions, excluding the deprotonation enthalpy of the amino groups, i.e. that associated with the intrinsic enthalpy of binding, is higher in value (Delta(b)H(o)=-5.4 kcal mol(-1)) than the apparent enthalpy of binding. Thus, the large DeltaG value for Ca(2+) binding to EDTA arises not only from favorable entropic but also enthalpic changes, depending on the ionization state of the amino groups involved in coordination of the calcium. This explains the great variability in DeltaH obtained in previous studies. The ionization enthalpy is always unfavorable, and therefore dramatically decreases Ca(2+) affinity by reduction of the enthalpy term of the stability function. The origin of the enthalpy and entropy terms in the stability of the Ca(2+)-EDTA complex is discussed.     

Critical role of chromium (Cr)-DNA interactions in the formation of Cr-induced polymerase arresting lesions

The genotoxicity associated with the metabolic reduction of hexavalent chromium [Cr(VI)] is complex and can impede DNA polymerase-mediated replication in vitro. The exact biochemical nature of Cr-induced polymerase arresting lesions (PALs) is not understood, but is believed to involve the formation of Cr-DNA interstrand cross-links (ICLs). The aim of this investigation was to determine the dependence of direct Cr-DNA interactions on the development of PALs in DNA treated with trivalent Cr [Cr(III)] or with Cr(VI) in the presence of ascorbic acid (Asc), a major intracellular reductant, using an in vitro, acellular system. The formation of Cr-DNA adducts, ICLs, and PALs was maximal at Asc:Cr(VI) molar ratios of 0.5-2, but gradually decreased at higher ratios. EDTA, a Cr(III) chelator, significantly decreased Cr-DNA binding and ICL and PAL formation. Co-treatment of DNA with Cr(VI)/Asc and mannitol, a Cr(V) chelator, selectively inhibited the formation of mono/bifunctional DNA adducts and PALs produced by Cr(VI) reduction, but had no effect on Cr(III)-DNA binding or Cr(III)-induced polymerase arrest. Blocking Cr-DNA phosphate interaction by preincubation of DNA with MgCl(2) abrogated DNA binding and ICL and PAL production. DNA strand breaks and abasic sites may lead to the in vitro arrest of DNA polymerases; however, we failed to detect significant increases in the frequency of these lesions following Cr(VI)/Asc treatment. These data indicate that the bifunctional adduction of Cr to DNA phosphates (ICLs) constitutes a major PAL. Furthermore, the generation of DNA strand breaks and abasic sites by Cr(VI) reduction is insufficient to explain PALs observed in vitro.     

Nickel(II) binding to Cap43 protein fragments

Cap43 protein has been tested for metal binding domains. The protein, specifically induced by nickel compounds in cultured human cells, had a new mono-histidinic motif consisting of 10 amino acids repeated three times in the C-terminus. The 20-Ac-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly(360) - peptide 1) and the 30-Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(366)-Thr-Ser-Glu-Gly(370) - peptide 2) amino acids sequence has been analyzed as a site for Ni(II) binding. A combined pH-metric and spectroscopic (UV-visible, CD, NMR) studies of Ni(II) binding to both fragments were performed. The 20-amino acid peptide can bind one and two metal ions while the 30-amino acid fragment one, two and three metal ions. At physiological pH, depending on the metal to ligand molar ratio, peptide 1 forms the Ni(2)L species while peptide 2 the NiL, Ni(2)L and Ni(3)L complexes where each metal ion is coordinated to the imidazole nitrogen atom of the histidine residue of the 10-amino acid fragment. Octahedral complexes at pH 8-9 and planar 4N complexes with (N(Im), 3N(-)) bonding mode at pH above 9, are formed. This work supports the existence of an interesting binding site at the COOH-terminal domain of the Cap43 protein.     

Substrate interactions with brain(Ca+Mg)-ATPase

ATP hydrolysis by a partially purified (Ca+Mg)-ATPase preparation from rat brain increased with substrate concentration in a biphasic fashion, with apparentK _m values of 3 M and 0.1 mM. Ca-dependent phosphorylation, however, had only a singleK _m value, 3 M. KCl increased ATPase activity in both concentration ranges, but theK _0.5 for KCl decreased from 7 mM to 0.3 mM as the ATP concentration was reduced from 1 mM to 10 M. TheK _0.5 for MgCl₂ decreased somewhat less, from 3 mM to 0.6 mM with ATP concentrations from 1 mM to 1 M, but was far lower for steady-state phosphorylation, 0.03 mM. (Ca+Mg)-dependent hydrolysis was not demonstrable with other nucleotide triphosphates or p-nitrophenyl phosphate, and these substances, as well as a reaction product, P_i, were also inhibitors. On the other hand, ADP inhibited at both ATP concentration ranges, and also stimulated dephosphorylation. This pattern of responses to substrate and cations is reminiscent of that of well-characterized transport ATPases, suggesting similar roles and mechanisms.     

Platinum determination in cis-dichlorodiammineplatinum(II)-DNA complexes by differential pulse polarography

A simple polarographic assay for platinum determination in cis-dichlorodiammineplatinum(II)-DNA complexes is described. The method makes it possible to determine the free (unbound) drug in the presence of DNA or platinum-DNA complex, i.e., without a separation of free drug and macromolecular components of the solution to be analyzed. This method is based on the polarographic activity of intact cis-dichlorodiammineplatinum(II) at ?1.5 V, which can be measured by differential pulse polarography even in the presence of DNA or platinum-DNA complex. The lower level of analytical utility of this method is ca. 1 × 10^?6m (195 ng of platinum/ml).     

Some aspects of lead(II) DNA interactions

The interaction of Pb(II) ions with calf-thymus DNA was studied by differential pulse polarography, sweep voltammetry, cyclic voltammetry, chromatography on hydroxyapatite and viscosity measurements. Pb(II) ions may interact with nucleic acid via phosphate groups causing some stabilization of the DNA structure. However, the more specific interaction occurs with nucleic bases. The latter interaction destabilizes the nucleic acid structure and leads to inter- and intra-chain binding.     

A Comparative study of Fe(II) and Fe(III) interactions with DNA duplex: major and minor grooves bindings

The involvement of the Fe cations in autoxidation in cells and tissues is well documented. DNA is a major target in such reaction, and can chelate Fe cation in many ways. The present study was designed to examine the interaction of calf-thymus DNA with Fe(II) and Fe(III), in aqueous solution at pH 6.5 with cation/DNA (P) (P = phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the cation binding site, the binding constant, helix stability and DNA conformation in Fe-DNA complexes. Structural analysis showed that at low cation concentration (r = 1/80 and 1/40), Fe(II) binds DNA through guanine N-7 and the backbone PO(2) group with specific binding constants of K(G) = 5.40 x 10(4) M(1) and K(P) = 2.40 x 10(4) M(1). At higher cation content, Fe(II) bindings to adenine N-7 and thymine O-2 are included. The Fe(III) cation shows stronger interaction with DNA bases and the backbone phosphate group. At low cation concentration (r = 1:80), Fe(III) binds mainly to the backbone phosphate group, while at higher metal ion content, cation binding to both guanine N-7 atom and the backbone phosphate group is prevailing with specific binding constants of K(G) = 1.36 x 10(5) M(-1) and K(P) = 5.50 x 10(4) M(-1). At r = 1:10, Fe(II) binding causes a minor helix destabilization, whereas Fe(III) induces DNA condensation. No major DNA conformational changes occurred upon iron complexation and DNA remains in the B-family structure.     

A systematic study of ligand intermolecular interactions in crystals of copper(II) complexes of bidentate guanidino derivatives

The synthesis and X-ray structures of copper(II) complexes of the bidentate ligands, N-(4-oxo-5,5-diphenyl-4,5-dihydro-1H-imidazol-2-yl)-N′-phenylguanidino, 2-uanidinobenzimidazolo and N-(4-oxo-3-phenyl-1,3-diazaspiro[4.4]non-1-en-2-yl)guanidino, are reported. These complexes, which possess potential doublet (DA) or triplet (DAD) hydrogen bonding motifs, can form supramolecular structures based on synthons involving hydrogen bonding or phenyl embraces. The changes in supramolecular structure resulting from small changes in ligand structure, as well as from the use of different solvents for their crystallisation, are examined. The structures adopted are compared with others reported previously for complexes of related ligands.     

EPR study of the electronic properties and weak exchange interactions in bis(L-phenylalaninamidato)Cu(II)

We performed EPR measurements in powder and single crystal samples of bis(L-phenylalaninamidato)Cu(II) (C18H22CuN4O2). We evaluated the crystal and molecular g-factors, and estimate an exchange interaction of 0.15 K< or = J(AB)/k < or =0.32 K between coppers spaced apart 6.24 A, transmitted through an amidate bridge Cu-O-C-N-Cu. An unusual line shape is observed in powder samples arising from the relative orientation of symmetry-related molecules in the structure. The dipolar interaction and the layered structure of the copper ions produce a strong temperature variation of the spectral shape when the copper spins are polarized by the applied magnetic field.     

Effect of nickel(II) on DNA-protein interactions

Alterations in DNA-protein interactions (DPI) may play an important role in carcinogenesis. Although the mechanism of nickel carcinogenesis is unknown, nickel reportedly affects DPI. A microfiltration, nitrocellulose filter assay was utilized to study DPI in intact Chinese hamster ovary (CHO) cells and in isolated nuclei. Prior to exposure of CHO cells or isolated CHO cell nuclei, DNA and proteins were radiolabeled using³H-thymidine and³⁵S-methionine, respectively. Nuclei were exposed to NiCl₂ in 10 mM HEPES buffer (pH 6.8). CHO cells were exposed in either complete or a salts-glucose medium. Following exposure, nuclei or cells were incubated at 37°C for 20 min in a high salt lysis solution; aliquots were loaded onto nitrocellulose filters and washed with a low salt solution. DNA (³H) retained on each filter was normalized to protein (³⁵S) bound on the filter. Exposure of either whole cells or isolated nuclei to increasing, noncytotoxic concentrations of NiCl₂ resulted in a dose dependent decrease in DPI. The effect of nickel on specific DNA-protein interactions was examined using a band shift assay and a cloned satellite DNA sequence. Nickel inhibited specific protein binding to the satellite DNA probe. The results of these two independent assays, which were conducted at physiological pH, indicate that NiCl₂ inhibits specific DNA-protein interactions.