Ligand Dissociation Constants Research Articles

The effect of ligand heterogeneity on the Scatchard plot. Particular relevance to lipoprotein binding analysis.

Computer simulations of equilibrium binding studies of a mixture of two labeled ligands binding competitively to a single class of identical and independent sites (receptors) were performed to investigate how ligand heterogeneity affects the observed data in such studies. The simulated data are presented in Scatchard plots. Ligand heterogeneity was generally found to be indistinguishable from the case of a homogeneous ligand when usual experimental conditions applied (that is, Scatchard plots of the data were straight lines). Some factors that increased the probability of recognizing heterogeneity in the system were identified, however. These are 1) a large difference between the dissociation constants of the two ligands, 2) a high concentration of receptors relative to the dissociation constant of the higher-affinity ligand, 3) a high concentration of the lower-affinity ligand relative to that of the higher-affinity ligand, 4) a high specific activity of the lower-affinity ligand relative to that of the higher-affinity ligand, and 5) lack of experimental error. When ligand heterogeneity (under certain conditions) did cause curvilinearity in the Scatchard plot, the curve formed was always concave-downwards. Thus, ligand heterogeneity may occasionally mimic positive cooperativity, but never mimics negative cooperativity or multiple classes of binding sites. Implications of these findings for equilibrium binding studies involving lipoproteins (which are generally isolated as heterogeneous mixtures of particles) are discussed in detail. These findings are also relevant to equilibrium binding studies using ligands which are mixtures of stereoisomers or which contain chemical or radiochemical impurities.

Structural Chemistry Studies of Hydroxyanthraquinone Complexes

Abstract The interaction of iron(II), iron(III) cobalt(II), nickel(II), copper(II), palladium(II) and platinum(IV) salts with some chelating hydroxyanthraquinone ligands was investigated. Nineteen solid complexes were synthesized and were characterized by elemental analysis, infrared and electronic spectra and pH and conductance measurements. These complexes were nonconducting. The iron(III) complexes were of octahedral geometry but are subject to some distortion. The ligand dissociation constants and the stability constants of the complexes were evaluated.

Ontogenic appearance of Ca 2+ channels characterized as binding sites for nitredipine during development of nervous, skeletal and cardiac muscle systems in the rat

The appearance of specific receptors for the Ca 2+ channel antagonist nitrendipine has been followed during the fetal and post-natal development of rat brain without cerebellum, cerebellum, skeletal muscle and cardiac muscle. The number of nitrendipine receptors is low at the fetal stage and increases drastically during post-natal development of brain, cerebellum, skeletal muscle and cardiac muscle. The time course of this increase is different for each type of tissue studied. No significant change in receptor ligand dissociation constant ( K d) can be detected over the development period studied. The results are discussed in relation with the known properties of the differentiation process in the four types of excitable tissues studied.

6-(p-toluidinyl)naphthalene-2-sulfonic acid as a fluorescent probe of yeast hexokinase: conformational states induced by sugar and nucleotide ligands.

The fluorescent dye 6-(p-toluidinyl)naphthalene-2-sulfonic acid (2,6-TNS) has been shown to be a sensitive and nonperturbing probe of conformational states of yeast hexokinase. The binding of sugar ligands to hexokinase induced conformational states of the enzyme which could be distinguished by monitoring 2,6-TNS fluorescence and correlated well with their behavior during the catalytic reaction. The binding of five-carbon sugar inhibitors such as lyxose induced a conformational state of hexokinase that demonstrated a small quenching of 2,6-TNS fluorescence but an increased ability to bind metal-ligands when compared to free enzyme. The binding of good sugar substrates such as glucose produced a conformational state of hexokinase which demonstrated a large enhancement (37%) of bound 2,6-TNS fluorescence. This glucose-induced conformational state had an increased ability to bind metal-ATP ligands; however, the relative changes in the dissociation constants for the various metal-ATP ligands differ from those observed with hexokinase in the presence of lyxose. Hence, the lyxose-induced conformational state of hexokinase was concluded to be significantly different from the glucose-induced conformational state. The binding of poor sugar substrates such as 5-thioglucose induced a conformational state of hexokinase similar to the conformational state induced by glucose, but with a smaller enhancement of 2,6-TNS fluorescence (15%) and a lesser ability to increase the affinity for metal-ATP ligands. The six-carbon inhibitor with a bulky group on the 2-position, N-acetylglucosamine, gave minimal changes in 2,6-TNS fluorescence and effects on metal-nucleotide binding. These conformational states are interpreted in terms of the closure of the cleft between the two domains observed by X-ray crystallography. The binding of A1ATP to free hexokinase was not observed at concentrations up to 100 microM, which is consistent with the kinetic properties reported for this metal-ATP ligand. Although both CrATP and A1ATP have been reported to produce a slow burst-type transient in the progress curve of hexokinase, only CrATP demonstrated slow changes in 2,6-TNS fluorescence, indicating that the conformational state of hexokinase induced by A1ATP is different from the conformational state induced by CrATP.

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K L, values over 20,000-fold lower than the K m for l-phenylalanine. From these and other K L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.

Modulation of mitochondrial succinate dehydrogenase activity, mechanism and function

The mitochondrial succinate dehydrogenase (E.C. 1.3.3.99) is subjected to apparently complicated regulatory mechanism. Yet, systematic analysis of the mechanism reveals the simplicity of the control. There are two stable forms of the enzyme; the non-active form stabilized as 1:1 complex with oxaloacetate and the active form stabilized by binding of activating ligands. This model quantitatively describes either the equilibrium level of active enzyme or the kinetics of activation-deactivation, in the presence of various concentrations of opposing effectors. The site where the regulatory ligands interact with the enzyme is not the substrate bonding site. The marked differences of dissociation constants of the same ligand from the two sites clearly distinguish between them. This model is fully developed for simple cases where the activating ligands are dicarboxylic acids or monovalent anions. On the other hand with activators such as ATP or CoQH2, quantitation is still not at hand. This stems from the difficulties in maintaining determined, measurable, concentrations of the ligand in equilibrium with the membranal enzyme. While in active form the histidyl flavin moity of the enzyme is reduced by physiological substrate (succinate; CoQH2). The non-active form is not reduced by these compounds, only strong reductants with low redox potential reduce the non-active enzyme. It is suggested that deactivation is a simple modulation of the redox potential of the flavin form E′ ≃ 0 mV in the active enzyme to E′ < −190 mV. The switch from one state to another might be achieved by distortion of the planar form of oxidized flavin to the bend configuration of the reduced flavin. Thus, in the active enzyme such distortion will destabilize the oxidized state of the flavin, shifting the redox potential to the higher value. The binding of oxaloacetate to the regulatory sites releases the distorting forces by relaxing the conformation of the enzyme. Consequently, the flavin assumes its planar form with the low redox potential. This assumption is supported by the spectral shifts of the flavin associated with the activation deactivation transition. The suicidal oxidation of malate to oxaloacetate, carried by the succinate dehydrogenase, plays an important role in modulating the enzyme activity in the mitochondria. This mechanism might supply oxaloacetate for deactivation in spite of the negligible concentration of free oxaloacetate in the matrix. The oxidation of malate by the enzyme is controlled by the redox potential at the immediate vicinity of the enzyme, and is imposed by the redox level of the membranal quinone. Finally, the modulation of succinate dehydrogenase activity is closely associated with regulation of NADH oxidation through the mutual inhibition between oxidases (Gutman, M. in Bioenergetics of Membranes, L. Packer et al., ed. Elsevier 1977, p. 165). The consequence of these interactions is the selection for the main electron donnor for the respiratory chain, during mixed substrate respiration, according to the metabolic demands from the mitochondria.

Ternary complexes: Equilibrium studies of mixed ligand complexes of vanadyl ion with some carboxylic and phenolic acids

The interactions of VO 2+ with bidentate ligands such as carboxylic and phenolic acids were studied in an aqueous medium by pH metric titrations at a temperature of 30 ± 0.1°C and ionic strength μ ∼ 0.1M (NaClO 4). Vanadyl forms only 1:1 complexes with carboxylic and phenolic acids, except 3,5-dinitro salicylic acid with which it forms both 1:1 and 1:2 chelates. The possibility of π-bonding between vanadium and ligands is examined from the plots of log K vs p K. Ternary complexes of VO 2+ with (i) two carboxylic acids, (ii) one carboxylic and one phenolic acid and (iii) two phenolic acids were investigated. The equilibrium constants for the various ternary complexes have been calculated. The values log K 2 YX are directly related to the product of the acid dissociation constants of secondary ligand. The overall stability is found to depend on the binary stabilities.

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ : II. The permeability response of the receptor-rich membrane fragments to cholinergic agonists in vitro

Purified receptor-rich membrane fragments (spec. act. 1000 ± 400 nmol 3H-labelled α-toxin sites/g protein) from Torpedo marmorata make closed vesicles which retain 22Na + in free and membrane-bound compartments. Acetylcholine (after blocking acetylcholinesterase with 0.01 m m-Tetram † † Abbreviations used: Tetram, O,O′-diethyl S-(β-diethylamino)ethyl phosphorothiolate; SKF-525A, diethylamino ester of diphenylpropylacetic acid; methyldilvasene, 2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide; EGTA, ethyleneglycol bis(β-aminoethylether)- N,N′-tetraacetic acid. ), carbamylcholine, phenyltrimethylammonium, suberyldicholine and methyldilvasene enhance the release of the free 22Na + from these microsacs and the effect of carbamylcholine is blocked by Naja nigricollis α-toxin, d-tubocurarine, flaxedil and hexamethonium. Decamethonium, an agonist on T. marmorata electroplaque, acts as a competitive antagonist on this in vitro system. Two local anaesthetics, dimethisoquin and SKF-525A block the response in vitro. Apparent dissociation constants for cholinergic ligands are determined from the dose-response curves. The values of the constants are, for most of them, significantly larger than those measured in vivo with the electroplaque by following steady-state depolarization in the presence of agonist added in the bath (on the other hand, they coincide rather well with the equilibrium dissociation constants given by direct binding studies with the cholate-solubilized receptor). The real dissociation constants measured with the membrane-bound receptor are close to the apparent ones for all the antagonists tested. However, the agonists bind at concentrations two orders of magnitude smaller than those necessary to trigger the response.

Ternary Complexs: Equilibrium Studies of Mixed-ligand Complexes of Neodymium Ion with Carboxylic and Phenolic Acids in Aqueous Medium

Abstract The stability constants of simple and mixed ligand systems of neodymium ion with bidentate ligands such as carboxylic and phenolic acids are studied in an aqueous medium by pH metric titrations at 30±0.1 °C and at an ionic strength of 0.1 M (NaClO4). Neodymium forms 1 : 1 complexes with succinic, adipic, itaconic, malonic, and 3,5-dinitrosalicylic acids and 1 : 1 and 1 : 2 complexes with phthalic, 5-sulfo-, and 4-hydroxysalicylic acids. Ternary complexes of neodymium with these acids when (i) both the ligands are carboxylic acids, (ii) one is carboxylic and the other a phenolic acid, (iii) both ligands are phenolic acids, are studied by potentiometry. The stability constants of these complexes are compared with those of binary complexes. The results have been discussed on the basis of the magnitudes of ΔlogK, KDXY, and logβXY−1⁄2[logβxx+logβyy], and also from the comparison of K2YX and KMX1 or K2XY and KMY1. The values of K2YX are directly related to the acid dissociation constant of the secondary ligand. The overall stability (βXY) is found to be depending on the binary stabilities and the basicities of the ligands.

Conformational Changes in Glycogen Phosphorylase Studied with a Spin-Label Probe

Phosphorylase b and a were covalently modified on essentially one -- SH group per subunit by a spin label 4-(2-iodoacetamido)2,2,6,6-tetramethyl piperidinyloxyl. The labelled enzyme is fully active and exhibits all the characteristics of the native molecule. The electron spin resonance spectrum of the label depends on the nature of the ligand that is bound to the enzyme. This property of the spin label is used to study the interaction between the enzyme (both in the b and a forms) and activators (AMP, IMP, CMP), inhibitors (ADP, ATP, UDPG, glucose 6-phosphate), substrates (phosphate and glucose 1-phosphate) and other ligands (adenosine, beta-glycerol-2-phosphate). The interactions are analysed in terms of the apparent ligand dissociation constants and the multiplicity of conformations that this regulatory enzyme exhibits.

An extended scatchard analysis for competitively bound ligands and its application to cyclic adenosine-3′,5′-monophosphate and cyclic 5′-amido-5′-deoxyadenosine-3′,5′-monophosphate binding to beef skeletal muscle protein

A method for determining the dissociation constants of ligands and ligand analogs is described. It is based on competition binding studies in the presence of an isotope-labeled, or otherwise measurable, ligand and suitable analog concentrations. The steps used are determination of (1) the maximal amount of radioactive ligand that can be bound, (2) the slopes and intercepts from Scatchard plots at different analog concentrations and (3) the values for the dissociation constants of radioactive ligand and ligand analog from replots of the reciprocals of the slopes and intercepts obtained from the Scatchard plots. Application of the method to a cyclic AMP-binding protein from beef muscle is demonstrated, yielding dissociation constants of 2 · 10 −9 M for cyclic [ 3H]AMP and cyclic AMP, and 3 · 10 −5 M for cyclic 5′-amido-5′-deoxyadenosine-3′,5′-monophosphate.

Protein fluorescence of nicotinamide nucleotide-dependent dehydrogenases

1. The decrease in the protein fluorescence (F) of Neurospora crassa glutamate dehydrogenase is linearly related to the increase in the fraction of the coenzyme sites occupied by NADPH (alpha) at pH6.35. Under these conditions NADPH causes this enzyme to dissociate to monomers. 2. There is a non-linear relationship of F to alpha for NADH binding to give the alcohol dehydrogenase-NADH-isobutyramide complex, the l-glycerol 3-phosphate dehydrogenase-NADH complex and the bovine glutamate dehydrogenase-NADH-glutamate complex. The non-linearity is accurately represented by F=[1-alpha(1-x)](n) where n is the number of NADH-binding sites per protein molecule. 3. The co-operative binding of GTP to bovine glutamate dehydrogenase in the presence of NADH gives a linear relationship between F and alpha. 4. The prediction from the equation F=[1-alpha(1-x)](n) that initial tangents to non-linear protein-fluorescence-quenching curves will intercept the fluorescence when alpha=1 at a value of total ligand concentration less than the sum of the concentration of binding sites in the solution plus the dissociation constant of ligand is quantitatively fulfilled. 5. Non-linear protein-fluorescence titrations may be used to obtain information about the distribution of ligand among the protein molecules in solution.

Rapid graphic methods for the estimation of enzyme normalities and their ligand dissociation constants

Methods are presented for the rapid graphic evaluation of spectrophotometric titrations by which both the active site normalities and reactant dissociation constants may be estimated.