Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.     

Arsenazo I and tetramethylmurexide as optical calcium indicators

Calcium-binding stoichiometry, dissociation equilibrium constants at zero ionic strength (K⁰), and molar extinction difference coefficients (Δ?_λ) at the wavelength λ of the metallochromic indicators arsenazo I (ArsI) and tetramethylmurexide (TMX) were reevaluated with a computerized method based on mass conservation and thermodynamic consistency checks. This new method is shown to provide a more critical assessment of the assumed calcium-dye complexing model than is afforded by the commonly used reciprocal-plot method. The analyses of spectrophotometric Ca titrations confirm that both dyes form only 1:1 complexes in aqueous solution. For TMX, K⁰ = 1.3 × 10^?3m and Δ?₄₈₀ = 1.5 × 10⁴m^?1 cm^?1; for ArsI, K⁰ = 5.8 × 10^?3m and Δ?₅₆₂ = 1.8 × 10⁴m^?1 cm^?1 at pH 7.0 and T = 293°K. The discriminatory power of the analytical method is demonstrated by comparison of these results with those found for a different dye, arsenazo III, which complexes Ca in 1:1, 1:2, and 2:1 forms.     

Computer simulation for effect of Pr(III) on Ca(II) speciation in human interstitial fluid

A multiphase model of metal ion speciation in human interstitial fluid was constructed and the effect of Pr(III) on Ca(II) speciation was studied. Results show that free Ca²⁺, [Ca(HCO₃)], and [Ca(Lac)] are the main species of Ca(II). Because of the competition of Pr(III) for ligands with Ca(II), the percentages of free Ca²⁺, [Ca(Lac)], and [Ca(His)(Thr)H₃] increase gradually and the percentages of CaHPO₄(aq) and [Ca(Cit)(His)H₂] decrease gradually with the increase in the total concentration of Pr(III). However, the percentages of [Ca(HCO₃)] and CaCO₃(aq) first increase and then begin to decrease when the total concentration of Pr(III) exceeds 6.070×10⁻⁴ M.     

Computer simulation for effect of Pr(III) on Ca(II) speciation in human interstitial fluid

A mutiphase model of metal ion speciation in human interstitial fluid was constructed and the effect of Pr(III) on Ca(II) speciation was studied. Results show that Ca(II) mainly distributes in free Ca²⁺, [Ca(HCO₃)], and [Ca(Lac)]. Because of the competition of Pr(III) for ligands with Ca(II), with the total concentration of Pr(III) rising, the percentages of free Ca²⁺, [Ca(Lac)] and [Ca(His)(Thr)H₃], gradually increase and the percentages of CaHPO₄(aq) and [Ca(Cit)(His)H₂] gradually decrease. However, the percentages of [Ca(HCO₃)] and CaCO₃(aq) first increase, and then begin to decrease when the total concentration of Pr(III) exceeds 6.070×10⁻⁴ M.     

Binding studies of Mn+2 to isolated acetylcholine receptor as a probe for cation-receptor interaction.

The paramagnetic cation Mn⁺² binds to $\text{Torpedo californica}$ acetylcholine receptor (AcChR) at sites with at least two different affinity constants. For each α-Bungarotoxin (α-Bgt) binding site AcChR has between 3 to 4 Mn⁺² sites with Kd values of 1.74 ± 1.0 × 10^?4 M. An additional 10–12 sites/α-Bgt site have a weaker affinity for Mn⁺² (Kd ? 1 mM). The α-Bgt does not displace bound Mn⁺², however Ca⁺² displaces all bound Mn⁺² in a competitive fashion with Kd of 0.90 × 10^?3 M and Mg⁺² is as effective as Ca⁺² in the displacement. Decamethonium, carbamylcholine and NaCl at high concentrations are also effective in displacing Mn⁺². A constant enhancement value (?_b) for the binary metal · AcChR complexes was obtained when simultaneous EPR measurements and the water proton relaxation rates were made. Similarity of the AcChR environment and/or coordination number for the Mn⁺² sites in AcChR is inferred. It appears that Mn⁺² binds to many AcChR sites, different from those responsible for binding cholinergic ligands. The Mn⁺² site seem to be the same as those responsible for binding the electrophysiologically significant Ca⁺².     

Effect of concanavalin A on Ca2+ binding, uptake and the Ca2+ ATPase of lymphocyte plasma membranes

Lymphocyte plasma membranes bind ⁴⁵Ca²⁺ with three affinity sites: K_Al = 4.0 . 10⁶ M^?1, K_A2 = 8.5 . 10⁴ M^?1 and K_A3 = 4.2 . 10² M^?1, and Ca²⁺ binding capacities are 0.10, 1.2 and 85 nmoles Ca²⁺/mg protein. In the presence of 15 μg/ml ConA the Ca²⁺ binding constants were K_A1 = 4.6 . 10⁶ M^?1, K_A2 = 4.4 . 10⁴ M^?1 and K_A3 = 4.2 . 10² M^?1. The Ca²⁺ binding capacity was increased by ConA, to 0.13, 2.4 and 91 nmoles/mg protein. The Ca²⁺ ATPase activity of lymphocyte membranes was increased by ConA from 1 to 2 μmol P/protein × h. The ⁴⁵Ca²⁺ uptake was stimulated by ConA and PHA to about 16 %.     

Heparin binding to antithrombin III: Variation in binding sites and affinity

Heparin fractions of different molecular weights and anticoagulant activities were prepared by chromatography on protamine-Sepharose, and the association constants and stoichiometry for binding to antithrombin III were determined by measurement of enhancement of tryptophan fluorescence. A 7,900 molecular weight heparin preparation bound to antithrombin III with a stoichiometry of close to 2:1, whereas 14,300 and 21,600 molecular weight fractions bound at approximately 1:1 with the protein. Apparent association constants were 0.66 × 10⁶ M^?1 for the low molecular weight preparation and 2.89 × 10⁶ M^?1 for the high molecular weight material. Maximal fluorescence enhancement was greater with the higher molecular weight heparin. These results suggest a model of heparin-antithrombin III binding in which two sites on antithrombin III can accommodate one large heparin molecule with high affinity or two smaller molecules with low affinity.     

Excitatory neuromuscular transmission in crayfish: Calcium dependence is unaffected by picrotoxin

Picrotoxin, 1 × 10^?5M to 1.6 × 10^?3M, had little or no effect on the amplitude of intracellularly recorded excitatory junctional potentials (EJPs) at extracellular calcium concentrations [Ca²⁺]₀ ranging from 0.5 to 15 mM. The slope of the log EJP vs. log[Ca²⁺]₀ relationship was approximately 1 with or without picrotoxin. The reduction of EJP amplitude resulting from the addition of 5 × 10^?5M GABA was largely reversed by 10^?5M picrotoxin.     

Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein

Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be K_Umb-1=3.1 ± .01 × 10⁴ M^?1, K_Umb-2 = 7 ± .01 × 10⁴ M^?1, which corresponds to ?6.1 and ?6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of K_Umb-1-AGP=3.1 ± .01 × 10³ M^?1 and K_Umb-2-AGP = 4.6 ± .01 × 10³ M^?1. Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster’s theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1–HSA and Umb-2–HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.     

The non-specific role of Mg2+ in ribosomal subunit association: kinetics and equilibrium in the presence of other divalent metal ions.

The effects of Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ on the kinetics and equilibrium of the association of vacant “tight” ribosomal subunits from Escherichia coli were studied. Increments of Mg²⁺, Ca²⁺, Sr²⁺ and, by and large, Ba²⁺, to ribosomes dissociated to 30 S and 50 S particles at 1.2 mm-Mg²⁺ (60 mm-M²⁺, pH 7.5, 25°C) produce nearly indistinguishable association curves, with midpoints at 1.8 mm total M²⁺ and complete association to 70 S particles at 4 to 5 mm total M²⁺ . The association rate constants at 1 mm-Mg²⁺, 2 mM-M²⁺ are similar (0.5 × 10⁶ to 0.9 × 10⁶m^?1s^?1), as are the dissociation rate constants at 1 mm-(Mg²⁺ + M²⁺) (0.2 to 0.4 s^?1). Mn²⁺ and Zn²⁺ increase the degree of association, as well as further aggregation (Zn²⁺ especially), at lower concentrations than the alkaline earth ions. Co²⁺ and Ni²⁺ produce lower degrees of association, by promoting dissociation of the 70 S particle : the association rate constants at 1 mm-Mg²⁺, 2 mm-M²⁺ for the transition metal ions are all grouped at 2 × 10⁶ to 3 × 10⁶m^?1s^?1. Ni²⁺ also causes a slower inactivation of one or both subunits.The results are compatible with the view that the effects on the rate and equilibrium constants arise from decreases in the electrostatic free energies of the 30 S, 50 S and 70 S particles produced by large-scale, relatively indiscriminate, charge-neutralization “binding” of M²⁺ , and are difficult if not impossible to reconcile with a specific-sites mode of action of M²⁺.     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^? to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^?1s^?1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^?1. It has also been demonstrated that in the absence of DNA O₂^?1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^?1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

Uptake and binding of β-ecdysone in imaginal discs of Drosophila melanogaster

The kinetics of uptake and retention of β-ecdysone by imaginal discs from late third instar larvae of Drosophila melanogaster correspond well with those of the first synthetic response of discs to hormone, an increase in RNA synthesis.Competition studies indicate the presence of two types of hormone binding sites, specific and non-specific. The specific sites are saturated at hormone concentrations which fully induce morphogenesis. Results are consistent with the hypothesis that analogs which induce morphogenesis at differing concentrations bind to the same sites. Experiments with the inhibitors N-ethylmaleimide, actinomycin d, and cycloheximide suggest that the binding sites are pre-existing in the cell and require functional sulfhydryl groups for binding.Specific binding, binding that is competed by excess unlabeled β-ecdysone, is saturable (70–80 nM). Kinetic rate constants for this specific binding were estimated to be k_a = 1.5 × 10⁵M^?1 min^?1, k_d = 3 × 10^?2 min^?1. The equilibrium dissociation constant calculated from the kinetic rate constants was K_eq = 2 × 10^?7M compared to 1.7 × 10^?7M β-ecdysone required to induce morphogenesis in vitro and 2.5 × 10^?7M determined to be the in vivo concentration at the time of induction of morphogenesis.     

Revised model of calcium and magnesium binding to the bacterial cell wall

Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane as lipoteichoic acid or covalently bound to the cell wall as wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, which contradicts previous reports that calcium binding is 100 % dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, which means that the binding affinity decreases as more ions become bound to the sample. For Ca²⁺, Region I has a K_A = (1.0 ± 0.2) × 10⁶ M^?1 and Region II has a K_A = (0.075 ± 0.058) × 10⁶ M^?1. For Mg²⁺, K_A1 = (1.5 ± 0.1) × 10⁶ and K_A2 = (0.17 ± 0.10) × 10⁶. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η₂, which are 0.70 ± 0.04 and 0.67 ± 0.03 µmol/mg for Ca²⁺ and Mg²⁺ respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.     

An investigation into the altered binding mode of green tea polyphenols with human serum albumin on complexation with copper

Green tea is rich in several polyphenols, such as (?)-epicatechin-3-gallate (ECG), (?)-epigallocatechin (EGC), and (?)-epigallocatechin-3-gallate (EGCG). The biological importance of these polyphenols led us to study the major polyphenol EGCG with human serum albumin (HSA) in an earlier study. In this report, we have compared the binding of ECG, EGC, and EGCG and the Cu(II) complexes of EGCG and ECG with HSA. We observe that the gallate moiety of the polyphenols plays a crucial role in determining the mode of interaction with HSA. The binding constants obtained for the different systems are 5.86?±?0.72?×?10⁴ M^?1 (K _ECG-HSA), 4.22?±?0.15?×?10⁴ M^?1 (K _{ECG-Cu(II)-HSA}), and 9.51?±?0.31?×?10⁴ M^?1 (K _{EGCG-Cu(II)-HSA}) at 293?K. Thermodynamic parameters thus obtained suggest that apart from an initial hydrophobic association, van der Waals interactions and hydrogen bonding are the major interactions which held together the polyphenols and HSA. However, thermodynamic parameters obtained from the interactions of the copper complexes with HSA are indicative of the involvement of the hydrophobic forces. Circular dichroism and the Fourier transform infrared spectroscopic measurements reveal changes in α-helical content of HSA after binding with the ligands. Data obtained by fluorescence spectroscopy, displacement experiments along with the docking studies suggested that the ligands bind to the residues located in site 1 (subdomains IIA), whereas EGC, that lacks the gallate moiety, binds to the other hydrophobic site 2 (subdomain IIIA) of the protein.     

Interaction of AMP with cytosolic apo-aspartate aminotransferase

Interaction of cytosolic apo-aspartate aminotransferase with AMP has been studied under equilibrium conditions: e.g., equilibrium dialysis and spectrophotometric titration. Results show that a 1:1 stoichiometric complex AMP—apo-aspartate aminotransferase monomer is formed. The calculated dissociation constants with the two different experimental techniques are 40.4 × 10^?6 M^?1 and 31.4 × 10^?6 M^?1, respectively. These findings substantiate a previous hypothesis of control of the reconstitution of cytosolic apo-aspartate aminotransferases exerted by AMP.     

Horseradish peroxidase. XXXVII. Compound I formation from reconstituted enzyme lacking free carboxyl groups as heme side chains

Recombination of apo horseradish peroxidase with 2,4 dimethyldeutero hemin and its mono- and dimethyl esters was performed. The number of free carboxyl side chains in these three hemins is 2, 1 and 0 respectively. Despite such a difference, all of these three reconstituted enzymes can react with H₂O₂ to produce compound I. The second order rate constants for compound I formation are 1.3 × 10⁷ M^?1s^?1, 8.5 × 10⁶ M^?1s^?1 and 5.9 × 10⁶ M^?1s^?1. Therefore the propionate side chain of hemin has no direct role in compound I formation.     

The inhibition of activated bovine coagulation factors X and VII by antithrombin III

The inhibition of activated bovine Factors VII and X by antithrombin III has been studied by kinetic methods. The reaction between Factor X_a and antithrombin III is characterized by second-order kinetics, with a rate constant of 3.9 × 10³m^?1s^?1 at pH 7.5 at 37 °C. Inhibition in the presence of excess antithrombin III does not proceed to completion: The decay of Factor X_a deviates from pseudo-first-order kinetics and a final equilibrium is reached, suggesting reversibility. The apparent association constant, at pH 7.5, 37 °C, is 2.3 × 10⁹m^?1. The interaction of three forms of bovine Factor VII with antithrombin III has been studied by the same methods. Factor VII and the two-chain activated form, α-Factor VII_a, and the tissue factor-Factor VII_a complex are not significantly inhibited by plasma levels of antithrombin III, in the either the presence or absence of heparin.     

Reaction of imidazole and hydroquinone with oxymyoglobin

Oxymyoglobin reacts with imidazole, substituted imidazoles, and hydroquinone to give metmyoglobin. The kinetics of these reactions have been studied. The rates are first order in both reactants, and second-order rate constants are reported. At pH 8.2, k₁ for imidazole is 2.5 ± 0.3 × 10^?3 M^?1 sec^?1 and for hydroquinone is 4 ± 0.4 × 10^?1 M^?1 sec^?1. The rates are independent of pH for imidazole but increase rapidly with pH for hydroquinone. The mechanism for all these reactions is thought to involve the two-electron reduction of molecular oxygen to peroxide with concurrent oxidation of both the protein and the reactant. An analogous mechanism has been suggested previously [1] for the reaction of oxyhemoglobin with hydroquinone. It has previously been shown [6] that imidazole can mediate the transfer of electrons to heme proteins by forming a transient reduced radical. The present results indicate that it can also form a transient oxidized radical under mild conditions. This dual capability may be important in biological electron-transfer processes.     

Free radicals and singlet oxygen scavengers: Reaction of a peroxy-radical with β-carotene,diphenyl furan and 1,4-diazobicyclo(2,2,2)-octane

The ‘singlet oxygen scavengers’. 1,4-diazobicyclo(2,2,2)-octane (DABCO), diphenyl furan and β-carotene react rapidly with the organic peroxyradical CCl₃O₂^?. The absolute reaction rate constants k = 1.2 ± 0.2 × 10⁷, 6 ± 2 × 10⁷ at 1.5 ± 0.2 × 10⁹ M^?1s^?1 respectively have been determined by pulse radiolysis. Comparison with other data suggest that other free radicals are also likely to react with these compounds; in the case of the hydroxyl radical and DABCO k = 1.25 × 10⁹ M^?1s^?1 has been determined.     

Synthesis,characterization, DNA interaction,and cytotoxicity of novel Pd(II) and Pt(II) complexes

Four complexes [Pd(L)(bipy)Cl]·4H₂O (1), [Pd(L)(phen)Cl]·4H₂O (2), [Pt(L)(bipy)Cl]·4H₂O (3), and [Pt(L)(phen)Cl]·4H₂O (4), where L = quinolinic acid, bipy = 2,2’-bipyridyl, and phen = 1,10-phenanthroline, have been synthesized and characterized using IR, ¹H NMR, elemental analysis, and single-crystal X-ray diffractometry. The binding of the complexes to FS-DNA was investigated by electronic absorption titration and fluorescence spectroscopy. The results indicate that the complexes bind to FS-DNA in an intercalative mode and the intrinsic binding constants K of the title complexes with FS-DNA are about 3.5?×?10⁴ M^?1, 3.9?×?10⁴ M^?1, 6.1?×?10⁴ M^?1, and 1.4?×?10⁵ M^?1, respectively. Also, the four complexes bind to DNA with different binding affinities, in descending order: complex 4, complex 3, complex 2, complex 1. Gel electrophoresis assay demonstrated the ability of the Pt(II) complexes to cleave pBR322 plasmid DNA.