Effective Association Constant Research Articles

Steric Effects on the Binding of Phosphate and Polyphosphate Anions by Zinc(II) and Copper(II) Dinuclear Complexes of m-Xylyl-bis-cyclen.

The triethylbenzene-bis-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) compound (tbmce) was designed with an imposed structural rigidity at the m-xylyl spacer to be compared to a less restrained and known parent compound (bmce). The framework of both compounds differs only in the substituents of the m-xylyl spacer. The study was centered in the differences observed in the acid-base reactions of both compounds, their copper(II) and zinc(II) complexation behaviors, as well as in the uptake of phosphate and polyphosphate anions (HPPi3-, ATP4-, ADP3-, AMP2-, PhPO42-, and HPO42-). On the one hand, the acid-base reactions showed lower values for the third and fourth protonation constants of tbmce than for bmce, suggesting that the ethyl groups of the spacer in tbmce force the two cyclen units to more conformational restricted positions. On the other hand, the stability constant values for copper(II) and zinc(II) complexes revealed that bmce is a better chelator than tbmce pointing out to additional conformational restraints imposed by the triethylbenzene spacer. The binding studies of phosphates by the dinuclear copper(II) and zinc(II) complexes showed much smaller effective association constants for the dicopper complexes. Single-crystal X-ray and computational (density functional theory) studies suggest that anion binding promotes the formation of tetranuclear entities in which anions are bridging the metal centers. Our studies also revealed the dinuclear zinc(II) complex of bmce as a promising receptor for phosphate anions, with the largest effective association constant of 5.94 log units being observed for the formation of [Zn2bmce(HPPi)]+. Accordingly, a colorimetric study via an indicator displacement assay to detect phosphates in aqueous solution found that the [Zn2bmce]4+ complex acts as the best receptor for pyrophosphate displaying a detection limit of 2.5 nM by changes visible to naked eye.

Cellular Adhesion: Evaluating the Effect of Receptor-Ligand Chemistries, Distribution of Receptors, and Spread Versus Spherical Cell Geometry

The initial stage of cellular adhesion is similar to a functionalized nanocarrier binding to a cell membrane through specific receptor-ligand interactions. Therefore, the computational models developed for nanocarrier binding can be used to investigate cell-substrate interactions. In this work, we use a Monte Carlo based free energy model to determine the binding affinities of a cell interacting with a functionalized substrate. In particular, we focus on how the chemical variations of the cell surface receptors (e.g., integrins) mediate the adhesive interactions and thereby affect the binding affinity. Our multiscale model explicitly accounts for (i) a mixture of specific and non-specific receptor-ligand interactions, (ii) flexural motion of the ligands, (iii) thermal motion of the cell, and (iv) feedback between molecular adhesion and membrane dynamics. The receptor-ligand interactions are first characterized using conventional docking studies and the interaction energy landscape is determined from all atom umbrella sampling simulations. For simplicity, we model the dynamics of the cell membrane using a harmonic potential that mimics the undulations of the membrane. We first study how the underlying molecular interactions, composition of receptor, and size and shape of the cell manifest as effective association constants. We also show that binding affinities, computed for cells whose dimensions are less than 1 micron, follow an asymptotic scaling behavior that can be used to predict the binding affinities of typical mammalian cells with larger dimension (> 1 micron) with minimal computational costs.

The evaluation of the association constant between R-SN and Hg(2+).

The effective determination of heavy metals from environmental media is among the most important issues for many industrialized countries. The interaction between RS-N, as novel heavy metal probe, and metal ions was studied. RS-N shows selective color change from colorless to pink in the presence of Hg(2+) in methanol/water solvent and the UV-Vis study shows peak at 560nm. Fluorescence data revealed that the fluorescence enhance of RS-N by Hg(2+) dramatically was the result of the formation of [Hg(2+)]RS-N complex. The effective association constants (K a ) were 3.97 × 10(5) and 0.204 × 10(5)M(-1) for Hg(2+) and Cu(2+) to RS-N, respectively. The thermodynamic parameters, enthalpy change (ΔH (0)) and entropy change (ΔS (0)), were calculated to be -6.431 ± 0.226kJ/mol and -0.129 ± 0.008J/K/mol, respectively, according to van't Hoff equation on the basis of Gibbs free energy (ΔG (0)) ranged from -33.8326 to -28.5389kJ/mol.

Preparation of β-cyclodextrin-modified multi-walled CNTs and its application in capturing β-naphthol from wastewater

Multi-walled carbon nanotubes (MWCNTs) were covalently modified with water-soluble β-cyclodextrin (β-CD) through ester bond. The products cyclodextrin-modified multi-walled carbon nanotubes (MWCNTs-COO-CD) were characterised by 1H-NMR, Fourier transform infrared spectroscopy and thermogravimetric analysis. The solubility of MWCNTs-COO-CD in water was 357.3 mg/l and no precipitates were observed after 18 months. The fluorescence spectra proved that the MWCNTs-COO-CD as a spectral probe could be used in the molecular recognition of β-naphthol (β-NOH), and the effective association constant (Ka) was calculated 2.22×104 from fluorescence spectra. By centrifugation at a speed of 16 000 rpm/min, the β-NOH with MWCNTs-COO-CD could be separated from water, collected and reused repeatedly. The results showed that the MWCNTs-COO-CD could be used to capture the aromatic pollutant such as β-NOH from wastewater. This study will widen the application of nanosupramolecule materials.

NMR spectroscopic characterization of β-cyclodextrin inclusion complex with vanillin

The inclusion of vanillin by β-cyclodextrin was investigated by 1H NMR. The continuous variation technique was used to evidence the formation of soluble 1:1 complex in aqueous solution. The association constant of vanillin with β-cyclodextrin has been obtained at 298 K by fitting the experimental chemical shifts differences, Δδobs = δfree – δobs of the observed guest and host protons, with a non-linear regression method. Besides the effective association constant, the fitting procedure allows a precise determination of all chemical shift parameters characterizing the pure complex. They can by used for an analysis of the geometry of the molecular complex in solution.

Curvature-Induced Base Pair “Slipping” Effects in DNA-Nanoparticle Hybridization

Experiments are presented that suggest DNA strands chemically immobilized on gold nanoparticle surfaces can engage in two types of hybridization: one that involves complementary strands and normal base pairing interactions and a second one assigned as a "slipping" interaction, which can additionally stabilize the aggregate structures through non-Watson-Crick type base pairing or interactions less complementary than the primary interaction. The curvature of the particles appears to be a major factor that contributes to the formation of these slipping interactions as evidenced by the observation that flat gold triangular nanoprism conjugates of the same sequence do not support them. Finally, these slipping interactions significantly stabilize nanoparticle aggregate structures, leading to large increases in T(m)'s and effective association constants as compared with free DNA and particles that do not have the appropriate sequence to maximize their contribution.

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.

The Effects of Buffers on the Thermodynamics and Kinetics of Binding between Positively-Charged Cyclodextrins and Phosphate Ester Guests

Cyclodextrin derivatives in which the primary hydroxyl groups at the 6-positions of the sugar units have been replaced with 2-hydroxethylamino groups are known to bind phosphate esters with significant affinity. Association between the host and guest is mediated by a combination of hydrophobic and electrostatic interactions. Association constants for two cyclodextrin hosts with three phosphate ester guests are measured in a variety of buffer solutions, and it is found that the buffer has a large impact on the electrostatic component of the binding interaction. Negatively charged buffers such as phosphate and ADA (N-(2-acetamido)iminodiacetic acid) compete with the phosphate ester guests for binding to the positively charged cyclodextrins. This competition lowers the effective association constant between host and guest. Positively charged buffers such as Tris-HCl do not strongly compete and are thus more conducive to binding. The kinetics of dissociation of the host−guest complexes were measured in several buffers using 31P EXSY experiments. These measurements demonstrate that negatively charged buffers decrease the equilibrium constant by lowering the rate of association of these complexes but in most cases do not effect the dissociation rates.

Interaction of dipyridamole with micelles of lysophosphatidylcholine and with bovine serum albumin: fluorescence studies

The interaction of the coronary vasodilator dipyridamole with biological systems, protein and membranes has been studied through optical absorption and fluorescence spectroscopies. Using the analysis of the spectra and fluorescence intensity of dipyridamole (DIP) in solution, the interaction of this compound with the transport protein albumin (BSA) and with a model of cell membranes, namely micelles of lysophosphatidylcholine (L-PC), was investigated. Measurements were performed at pH 5.0 and pH 7.0 where the molecule of DIP is fully protonated and partially protonated, respectively. The quenching of fluorescence with nitroxide-stable radicals 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) as well as with acrylamide and iodide allowed the localization of the drug in the polar interface of micelles. Quenching by acrylamide and iodide in L-PC micelles demonstrated the effect of micelle protonation which increased the accessibility of iodide to the chromophore. An effective association constant was obtained both at pH 7.0 (7.5 · 10 3 M −1) and pH 5.0 (2.5 · 10 3 M −1) and a very good agreement with the proposed binding model was observed. The quantum yields of fluorescence data agree very well with the fluorescence lifetimes. The measurement of lifetimes was important to understand the kinetic data obtained from Stern-Volmer plots both of radical, acryamide acrylamide and iodide quenching of fluorescence. It was observed that, in the presence of micelles, the k q value increased for TEMPO while decreased for TEMPOL. This result, together with the vanishing solubility of DIP in saturated hydrocarbons and the preferential partition of TEMPO in micelles, suggested the localization of DIP in the polar micellar interface. This is also supported by the enhanced iodide quenching at pH 5.0, constancy of acrylamide quenching in the range of pH 7.0−5.0 and the partition of TEMPO and TEMPOL in SDS micelles. The association constant of DIP to BSA was also estimated both at ppH 7.0 (2 · 10 4 M −1) and pH 5.0 (4 · 10 3 M −1). Quenching studies with nitroxide radicals, acrylamide and iodide also suggested the binding of the drug to a hydrophobic region of the protein. At pH 5.0, the protein undergo a conformational change which leads to a loosening of the overall structure so that the accessibility of the nitroxide radicals for DIP is increased at this pH. The differences in k q values at pH 7.0 and pH 5.0 suggested that at pH 7.0 the chromophore is protected in the protein site. Our results with model systems demonstrate that the drug interacts with these systems and suggest that the interaction of DIP with biological systems could be mediated by the cell membrane, although the possible interaction with the protein moiety cannot be discounted.

High-Field Nuclear Magnetic Resonance Techniques for the Investigation of a β-Cyclodextrin:Indomethacin Inclusion Complex

The inclusion complex of indomethacin sodium salt in β-cyclodextrin has been studied by proton NMR at high magnetic field. The continuous variation technique was used to evidence the formation of a soluble 1:1 complex in aqueous solution at physiological pH. The effective association constant was determined by the Benesi–Hildebrand procedure to be 760M−1 at 303K. This technique requires NMR measurements in the presence of a very large excess of one of the complex components and, since both β-cyclodextrin and the sodium salt of indomethacin are sparingly soluble in water, NMR spectrometers operating at very high magnetic fields were used. Besides the effective association constant, the Benesi–Hildebrand approach allows a precise determination of all NMR parameters of the pure inclusion complex which may be used for a complete analysis of the geometry of this complex in solution.

Phosphatidylcholine-fatty acid membranes. I. Effects of protonation, salt concentration, temperature and chain-length on the colloidal and phase properties of mixed vesicles, bilayers and nonlamellar structures

The phase and colloidal properties of phosphatidylcholine/fatty acid (PC/FA) mixed vesicles have been investigated by optical methods, acid-base titration, and theoretically as a function of temperature (5–80°C), molar lipid ratio (0–1), lipid chain length (C 14–C 18), headgroup ionization ( 1.5 ⩽ pH ⩽ 10 ), vesicle concentration (0.05–32 μmol vesicle · dm −3, and ionic strength ( 0.005 ⩽ J ⩽ 0.25). Increasing the fatty acid concentration in PC bilayers causes the phase transition temperatures (at 4 ⩽ pH ⩽ 5 ) to rise until, for more than 2 FA molecules per PC molecule, the sample turbidity exhibits only two transitions corresponding to the chain-melting of the 1:2 stoichiometric complexes of PC/FA, and pure fatty acid. The former transition is into a nonlamellar phase and is accompanied by extremely rapid vesicle aggregation (with association rates on the order of C a ≈ 10 7 dm 3 · mol − · s −1 ) and massive lipid precipitation. Fluid-phase vesicles with less than 2 FA per PC associate much more slowly ( C a ≈ 10 3 dm 3 · mol −1 · s −1 ), their aggregation being comparable to that of the ordered-phase liposomes. Under no conditions was the relation between the fatty acid concentration and the vesicle association rate for the fluid-phase vesicles linear. In contrast to the X-ray diffraction data, optical measurements reveal a ‘pretransitional region’ between the chain-melting temperature of the PC component and the temperature at which the gross transformation into a nonlamellar phase sets in. This is seen for all lipid mixtures investigated. On the relative temperature scale, lipids with different chain lengths behave qualitatively similarly; however, the effective association constants determined for samples of constant lipid concentration seem to decrease somewhat with the number of CH 2 groups per chain. Fatty acid protonation, which yields electrically neutral bilayers, invariably increases the rate of vesicle association; we have measured, for example, C a ≈ 10 2 at pH ≈ 7 and C a ≈ 10 7 dm 3 · mol −1 · s −1 at pH ≈ 4 ). Pronation of the phosphatidylcholine phosphate groups, which causes a net positive charge to accumulate on the lipid vesicles, initially increases ( C a ≈ 10 8 dm 3 · mol −1 · s −1 ) but ultimately decreases ( C a ≈ 10 7 dm 3 · mol −1 · s −1 ) the rate of association between PC/FA (1:2) mixed vesicles. The presence of ions also facilitates the association between PC/FA mixed vesicles, not only for charged but also for electrically neutral vesicles. This shows that the nonelectrostatic effects of protonation and ion screening, which probably arise from changes in the interfacial polarity (hydrophilicity), significantly affect the colloidal and phase properties of such mixed PC/FA membranes. Concomitantly, surface electrostatics and interfacial hydration together determine the titration properties of PC/FA (1:2) membranes, the latter effect in acidic suspensions being at least as strong as the former. From our C a vs. pH data the pK values at ionic strength J = 0.005 (0.1) for FA and PC in the fluid phase are found to be 7.5 ( ≈ 7) and 1.5, respectively. This suggests the electrostatic and hydrational contributions to the pK shifts are 1.5 and 1 pH units, respectively, again indicative of the importance of interfacial hydration. Direct acid-base titrations yield similar results and provide evidence, moreover, for the occurrence of an isothermal, pH-induced phase transition at 65° C around pH 6 for the C 14 mixture.

1H nuclear magnetic resonance study of the two calcium-binding sites of porcine intestinal calcium-binding protein.

1H nuclear magnetic resonance has been employed to study the calcium-binding properties of the NH2- and COOH-terminal calcium-binding sites of the porcine intestinal calcium-binding protein. The protein was titrated with calcium in the presence of the chelator EDTA in order to determine the association constants of the protein for calcium relative to the known association constant of EDTA for calcium. The resulting data were compared with various models for the binding of calcium to two sites on the protein. Models were considered for which the two sites in the apoprotein have either intrinsically equal or unequal affinities for calcium. For each of these two cases, positive cooperativity, no cooperativity, and negative cooperativity were considered. The data fit best for the case of random binding to two independent sites with equivalent association constants of 1.0 +/- 0.1 X 10(7) M-1. The case of ordered binding to two sites with intrinsically different affinities, with concomitant positive affinity between the two sites so that the effective association constants were made equal, could not be mathematically excluded when only one protein NMR resonance is considered but can be shown to be implausible when the whole spectrum is considered.

ESR spin-label studies of lipid-protein interactions in membranes

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.

Reduction of antibody affinity in immunized rabbits by ethylenimine and its derivatives

To investigate changes in the functional affinity of antibodies under the influence of immunodepressants, changes in the effective association constant, standard free energy, concentration of hapten-binding sites, heterogeneity index, and concentration equivalents of IgG, Fab, and Fc fragments were studied in 48 rabbits. Under the influence of ethylenimine and its derivatives the affinity indices were reduced and heterogenization of the antibodies took place. Changes in functional affinity of the antibodies were determined by administration of the immunodepressants at different periods of formation of the immune response.

Mathematical model of clonal selection and antibody production

A mathematical model is developed for predicting the cellular proliferation and production of circulating antibodies that takes place when an adult animal is injected with an antigen. A clonal selection theory is used, in which it is assumed that certain target cells are capable of being stimulated by antigen into a proliferating and antibody producing state. The proliferating cells increase in number until the free antigen concentration reaches a low level, whereupon they divide asymmetrically to become plasma cells and memory cells. The plasma cells are terminal antibody producing cells while memory cells become identical to target cells. It is assumed that the target and proliferating cells carry antibody-like surface receptors which are capable of combining reversibly with antigen and that the average number of bound receptors determines the mitotic activity of a cell. The binding of a receptor site to antigen is taken to be characterized by an intrinsic association constant and the state of the binding is calculated, at any time, for univalent antigen interacting with bivalent antibodies and multivalent cells. Heterogeneous antibodies are allowed by having many groups of cells which differ in their affinity for antigen and which produce correspondingly differing antibodies. The rates of change of the various cell populations, antibody and antigen concentrations, are taken to be governed by certain differential equations that embody the biological assumptions of the model. In the model, the level of free antigen selects certain groups of cells, according to their affinity for antigen, for proliferation; the resulting antibody population reflects this proliferation together with the rapid elimination of bound antigen-antibody complexes. A mechanism for inducing high dose tolerance is included in the model and a mechanism for low dose tolerance is suggested, based on depletion of the target cells by repeated production of small clones. The system of equations is solved numerically by a digital computer program and results have been compared with many experiments in which 2,4 dinitrophenyl-bovine γ-globulin was used as antigen in rabbits. Effective association constants and concentrations of antibodies have been compared with experiments and an encouraging degree of agreement is found. It is indicated that in some experiments, the antibody producing system was probably overloaded. Some possible applications of the model to planning and analysis of experiments are discussed . By studying the time dependence of antibody concentration and affinity, following injection of antigen, one can deduce some characteristics of the target cells and their origin. Moreover, if enough is known about the targent cells it may be possible to tailor exposure to antigen so as to achieve special effects, such as high dose tolerance, low dose tolerance, or the production of homogeneous antibodies. Experiments which studied quantitatively the interaction of the receptor sites of target or proliferating cells with antigen would be of great help in confirming or denying this model.