Determination Of Binding Constants Research Articles

Painkiller Isoxicam and Its Copper Complex Can Form Inclusion Complexes with Different Cyclodextrins: A Fluorescence, Fourier Transform Infrared Spectroscopy, and Nuclear Magnetic Resonance Study

The interaction of a painkiller Isoxicam, belonging to the oxicam group of nonsteroidal anti-inflammatory drugs (NSAIDs) and its copper complex with different cyclodextrins (β-CD, γ-CD, HPβCD, and HPγCD), has been investigated in both solution and the solid state. Steady state and time-resolved fluorescence spectroscopy, fluorescence anisotropy, 1H NMR, and FTIR spectroscopy are used. Both the drug and its copper complex form a host-guest inclusion complex with all CDs. Fluorescence spectroscopy is used to determine binding constants and stoichiometries of the host-guest complex. The strongest binding is seen for γ-CD. 1H NMR study showed that Isoxicam penetrates into the CD cavity from the more accessible wider side. For β- and γ-CD, Isoxicam showed one type of binding, i.e., formation of an inclusion complex, whereas, for HPβCD and HPγCD, it showed two types of binding, i.e., inclusion in the CD cavities and interaction with the outer surface of the CD molecules mainly near the hydroxy propyl group. Deeper penetration occurred into the larger diameter cavity of γ-CD and HPγCD compared to β-CD and HPβCD. From FTIR and 1H NMR study, it is seen that predominantly the π-electron-rich benzene part of the drug and its complex penetrate into the host cavity.

Specific Binding Constant and Stoichiometry Determination in Free Solution by Mass Spectrometry and Capillary Electrophoresis Frontal Analysis.

A free solution method was developed for evaluating the specific binding affinity and stoichiometry of small molecules with oligo DNA subsequent to cation-induced G-quadruplex formation. A nonlinear curve fitting equation capable of extracting specific binding constants in the presence of nonspecific binding without the need for reference compounds was proposed and tested. Electrospray ionization mass spectrometry was first used to rapidly screen the small molecule candidates; then, the stoichiometry and affinity constants of the native state binding pair in solution were obtained with capillary electrophoresis frontal analysis (CE-FA). The B cell lymphoma 2 (Bcl-2) oncogene is directly responsible for the expression of Bcl-2 protein, which plays a significant role in cell apoptosis. The binding of a G-quadruplex formed in the promoter region of the Bcl-2 oncogene with a small molecule could stabilize the quadruplex structure and potentially regulate the transcription of Bcl-2. Four natural product drug candidates were tested for their ability to bind the Bcl-2 promoter G-quadruplex. Using this reference-free method based on CE-FA data, jatrorrhizine and palmatine were found to bind specifically to the Bcl-2 promoter G-quadruplex with stoichiometries of 4:1 and 3:1, respectively.

Evaluation of the binding of four anti-tumor Casiopeínas® to human serum albumin

The metal complexes designated by Casiopeínas® are mixed-ligand CuII-compounds some of them having promising antineoplastic properties. We report studies of binding of Cu(glycinato)(4,7-dimethyl-1,10-phenanthroline) (Cas-II-Gly (1)), Cu(acetylacetonato)(4,7-dimethyl-1,10-phenanthroline) (Cas-III-Ea (2)), Cu(glycinato)(4,4′-dimethyl-2,2′-bipyridine) (Cas-IV-Gly (3)) and Cu(acetylacetonato)(4,4′-dimethyl-2,2′-bipyridine) (Cas-III-ia (4)) to human serum albumin (HSA) by circular dichroism (CD), Electron paramagnetic resonance (EPR) and fluorescence spectroscopy. The results indicate that HSA may bind up to three molecules of the tested Casiopeínas. This is confirmed by inductively coupled plasma – atomic absorption spectroscopy measurements of samples of HSA-Casiopeínas after passing by adequate size-exclusion columns. The binding of Cas-II-Gly to HSA was also confirmed by MALDI-TOF mass spectrometric experiments. In the physiological range of concentrations the Casiopeínas form 1:1 adducts with HSA, with conditional binding constants of ca. 1×109 (1), 4×107 (2), 1×106 (3) and 2×105 (4), values determined from the CD spectra measured, and the fluorescence emission spectra indicates that the binding takes place close to the Trp214 residue. Overall, the data confirm that these Casiopeínas may bind to HSA and may be transported in blood serum by this protein; this might allow some selective tumor targeting, particularly in the case of Cas-II-Gly. In this work we also discuss aspects associated to the reliability of the frequently used methodologies to determine binding constants based on the measurement of fluorescence emission spectra of solutions containing low concentrations of proteins such as HSA and BSA, by titrations with solutions of metal complexes.

Development and Characterization of a Fluorescent Tracer for the Free Fatty Acid Receptor 2 (FFA2/GPR43)

The free fatty acid receptor 2 (FFA2/GPR43) is considered a potential target for treatment of metabolic and inflammatory diseases. Here we describe the development of the first fluorescent tracer for FFA2 intended as a tool for assessment of thermodynamic and kinetic binding parameters of unlabeled ligands. Starting with a known azetidine FFA2 antagonist, we used a carboxylic acid moiety known not to be critical for receptor interaction as attachment point for a nitrobenzoxadiazole (NBD) fluorophore. This led to the development of 4 (TUG-1609), a fluorescent tracer for FFA2 with favorable spectroscopic properties and high affinity, as determined by bioluminescence resonance energy transfer (BRET)-based saturation and kinetic binding experiments, as well as a high specific to nonspecific BRET binding signal. A BRET-based competition binding assay with 4 was also established and used to determine binding constants and kinetics of unlabeled ligands.

Mapping the Binding Motifs of Deprotonated Monounsaturated Fatty Acids and Their Corresponding Methyl Esters within Supramolecular Capsules.

A suite of NMR techniques revealed that a cavitand (1) formed 2:1 host-guest complexes with a range of monounsaturated fatty carboxylates and their corresponding methyl esters. All of the carboxylates bound to the capsule in a J-shaped motif with the carboxylate at the equatorial region of the dimeric capsule, and the reverse turn of the chain and the methyl terminal in each polar region of the host. Guest exchange was slow on the NMR time scale, while tumbling was slow or close to the NMR time scale depending on the position and stereochemistry of the double bond. In contrast, the methyl esters were found to bind in three motifs depending on the position and stereochemistry of the double bond. Thus, the esters were observed to bind in a J-shaped, U-shaped (the turn in the guest occupying a polar region and the two termini competing for occupancy of the other pole), or a reverse J-shaped motif (ester moiety and turn each occupying a pole and the methyl terminal located near the equator). Relative binding constant (Krel) determinations revealed that the affinity for the capsule was dependent on the position and stereochemistry of the double bond.

Label-free quantification of calcium-sensor targeting to photoreceptor guanylate cyclase and rhodopsin kinase by backscattering interferometry

Quantification of protein binding to membrane proteins is challenging and a limited set of methods is available to study such systems. Here we employed backscattering interferometry (BSI), a free-solution label-free method with high sensitivity, to quantify the interaction of neuronal Ca2+-Sensor proteins with their targets operating in phototransduction. We tested direct binding of guanylate cyclase–activating proteins (GCAP1 and GCAP2) to their membrane target guanylate cyclase 1. The regulatory mechanism of GCAPs including their binding interface in the target is unresolved. Here we used a label-free, free-solution assay method based on BSI to determine binding constants of GCAP1 and GCAP2 to the full-length membrane-bound guanylate cyclase type 1. GCAP1 and GCAP2 bound to different regions on the target guanylate cyclase with submicromolar affinity (apparent KD-values of 663 ± 121 nM and 231 ± 63 nM for Ca2+-free GCAP1 and GCAP2, respectively). A guanylate cyclase construct containing the juxta-membrane and kinase homology domain harbored an exclusive binding site for GCAP1 with similar affinities as the full-length protein, whereas GCAP2 did not bind to this region. We provide a model in which GCAP1 and GCAP2 do not share a single binding site to the target, thus cannot exchange upon fluctuating Ca2+ levels.

Interaction of mycotoxin zearalenone with human serum albumin

Zearalenone (ZEN) is a mycotoxin produced mainly by Fusarium species. Fungal contamination of cereals and plants can result in the formation of ZEN, leading to its presence in different foods, animal feeds, and drinks. Because ZEN is an endocrine disruptor, it causes reproductive disorders in farm animals and hyperoestrogenic syndromes in humans. Despite toxicokinetic properties of ZEN were studied in more species, we have no information regarding the interaction of ZEN with serum albumin. Since albumin commonly plays an important role in the toxicokinetics of different toxins, interaction of ZEN with albumin has of high biological importance. Therefore the interaction of ZEN with human serum albumin (HSA) was investigated using spectroscopic methods, ultrafiltration, and molecular modeling studies. Fluorescence spectroscopic studies demonstrate that ZEN forms complex with HSA. Binding constant (K) of ZEN-HSA complex was quantified with fluorescence quenching technique. The determined binding constant (logK=5.1) reflects the strong interaction of ZEN with albumin suggesting the potential biological importance of ZEN-HSA complex formation. Based on the results of the investigations with site markers as well as docking studies, ZEN occupies a non-conventional binding site on HSA. Considering the above listed observations, we should keep in mind this interaction if we would like to precisely understand the toxicokinetic behavior of ZEN.

Mobility-based correction for accurate determination of binding constants by capillary electrophoresis-frontal analysis.

Capillary electrophoresis frontal analysis (CE-FA) can be used to determine binding affinity of molecular interactions. However, its current data processing method mandate specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. This work shows that significant errors are resulted when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real word applications becomes questionable. An electrophoretic mobility-based correction method is developed in this work based on the flux of each species. A simulation program and a pair of model compounds are used to verify the new equations and evaluate the effectiveness of this method. Ibuprofen and hydroxypropyl-β-cyclodextrinare used to demonstrate the differences in the obtained binding constant by CE-FA when different calculation methods are used, and the results are compared with those obtained by affinity capillary electrophoresis (ACE). The results suggest that CE-FA, with the mobility-based correction method, can be a generally applicable method for a much wider range of applications.

Characterization of complexes between phenethylamine enantiomers and β-cyclodextrin derivatives by capillary electrophoresis-Determination of binding constants and complex mobilities.

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte-cyclodextrin-complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β-cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x-reciprocal, y-reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, methyl-β-cyclodextrin and 6-O-α-maltosyl-β-cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer-cyclodextrin-complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β-cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes.

The Effect of Molar Mass and Charge Density on the Formation of Complexes between Oppositely Charged Polyelectrolytes.

The interactions between model polyanions and polycations have been studied using frontal continuous capillary electrophoresis (FACCE) which allows the determination of binding stoichiometry and binding constant of the formed polyelectrolyte complex (PEC). In this work, the effect of the poly(l-lysine) (PLL) molar mass on the interaction with statistical copolymers of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonate (PAMAMPS) has been systematically investigated for different PAMAMPS chemical charge densities (15% and 100%) and different ionic strengths. The study of the ionic strength dependence of the binding constant allowed the determination of the total number of released counter-ions during the formation of the PEC, which can be compared to the total number of counter-ions initially condensed on the individual polyelectrolyte partners before the association. Interestingly, this fraction of released counter-ions, which was strongly dependent on the PLL molar mass, was almost independent of the PAMAMPS charge density. These findings are useful to predict the binding constant according to the molar mass and charge density of the polyelectrolyte partners.

Quantification of nucleobases/gold nanoparticles interactions: energetics of the interactions through apparent binding constants determination.

We explore the possibilities of quantifying the interaction of both adenine and thymine with non-functionalized, anionic citrate gold nanoparticles (AuNPs). Following the plasmon absorbance band's red shifts, dependent of the AuNPs aggregation state, apparent binding free energies of the system are obtained for the sum of the two processes (nucleobase interaction plus nanoparticle aggregation). A deconvolution procedure confirms those results. Those apparent binding free energies are, in both cases, in good agreement with previous studies indicating an increased reactivity for adenine. Moreover, density functional theory (DFT) calculations were carried out both to model the structures and to theoretically support and help understand the observed experimental stability of adenine as compared to thymine over gold nanoparticles. Theoretical results indicate the N atom in the amino group of adenine adopts a pyramidal sp3 hybridization character, stabilizing the interaction with Au as compared to thymine.

Synthesis, structural, thermal characterization and interaction with calf-thymus DNA and albumins of cationic Ni(II) complexes with 2,2′-dipyridylamine and salicylaldehydes

Five cationic mixed-ligand Ni(II) complexes with 2,2′-dipyridylamine (dpamH) and substituted salicylaldehydes (X-saloH), having the general formula [Ni(dpamH)2(X-salo)](NO3), were prepared and characterized by physicochemical and spectroscopic techniques (FT-IR and UV–vis) and their interaction with calf-thymus (CT) DNA and serum albumins was investigated. The complexes are formulated as [Ni(dpamH)2(3-OCH3-salo)](NO3)·CH3OH, 2, [Ni(dpamH)2(5-CH3-salo)](NO3)·CH3OH, 3, [Ni(dpamH)2(5-NO2-salo)](NO3)·CH3OH, 4, [Ni(dpamH)2(5-Cl-salo)](NO3), 5 and [Ni(dpamH)2(5-Br-salo)](NO3), 6. Moreover, in the absence of dpamH, one neutral nickel-salicylaldehydato complex was also prepared and characterized as [Ni(5-NO2-salo)2(CH3OH)2], 1. The structures of complexes 2 and 4 were verified by X-ray crystallography. The thermal stability of the complexes was examined by the simultaneous thermal analysis technique (TG/DTG-DTA). Spectroscopic (UV–vis), electrochemical (cyclic voltammetry) and physicochemical (viscosity measurements) techniques were employed in order to study the binding mode and binding strength of the complexes to CT DNA, while ethidium bromide (EB) displacement studies (performed by fluorescence emission spectroscopy) revealed the ability of the complexes to displace the DNA-bound EB. Intercalation is the most possible mode of interaction of the complexes with CT DNA. The interaction of the complexes with albumins was studied by fluorescence emission spectroscopy and the determined binding constants present relative high values.

Determination of binding modes and binding constants for the complexes of 6H-pyrido[4,3-b]carbazole derivatives with DNA

The binding modes and binding constants for the complexes of forty types of pyridocarbazole derivatives 1–40 with double stranded DNAs (dsDNAs) were reported. The binding modes were determined by a combination of a deflection spectroscopy and orientation of the corresponding molecule in the DNA-based film with chain alignment. All of the compounds exhibited the intercalation-binding mode. Its binding constants Ka for the complexes, determined by quartz crystal microbalance (QCM), varied from 1.7×105 to 4.5×107M−1 according to the substituents on the pyridocarbazole framework and the sequences of dsDNA. The binding constants Ka of pyridocarbazole derivatives possessing the 2-(ω-amino)alkyl group and 5-(ω-amino)alkylcarbamyl group were larger than those of the corresponding ω-ureido derivatives. These ω-amino compounds exhibited strong GC base-pair preference in complexation. The Ka values decreased with the increasing NaCl concentration. It was clarified by a molecular modeling that the framework of the 2-tethered ω-amino derivative was completely overlapped with the stacking GC base-pairs leading to the formation of the stable intercalative-complex, and that the framework of the 5-tethered ureido derivative was half overlapped leading to the formation of the unstable complex. Furthermore, there were good linear relationships between lnKa and the relative stabilities Srel of the complexes. Contrary to our expectation, there was no linear relationship between lnKa and IC50 against Sarcoma-180, NIH3T3, and HeLa S-3 cell lines.

Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia.

LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT. Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance (SPR) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells (HUVEC). The dissociation constant (Kd ) for activated platelet binding was in the 1 μm range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25-50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. LR769 was similar to NovoSevenRT in all kinetic assays. Active site titration demonstrated 0.7 mole of active site/mole of protein. The kcat /Km values for activation of FX and FIX with purified recombinant tissue factor and phospholipids were 10.5 s-1 /0.32 μm and 3.3 s-1 /0.44 μm respectively. The apparent second-order rate constant for inactivation by human plasma AT was 5.9 ± 0.4 × 103 m-1 s-1 . The Kd values for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nm and 0.9 nm, respectively, and the Kd for binding to soluble EPCR was 41 nm. Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.

Determination of rate constant, binding constant, and binding number by fluorescence measurements of Gd3N@C80(OH)20 in D2O

ABSTRACT Today MRI imagining techniques are capable of discerning between abnormal and normal complex tissues by providing contrasting images of these tissues. One drawback of using MRI imagining is its low sensitivity. However, this sensitivity can be greatly enhanced by introducing contrasting agents who can provide a new pathway for water molecules to significantly relax faster and hence generate the desired “contrast” between healthy and unhealthy tissues. We report the first ever recorded fluorescence emission spectrum of Gd3N@C80(OH)20; where -(OH)20 is the average number of hydroxyl groups. Our emission data indicates that the H2O- Gd3N@C80(OH)20 interactions lead to fluorescence quenching via a static quenching mechanism. The binding constant, Kb, on the other hand, was found to be of the same magnitude as interactions between human serum albumin and small organic acid but quite different, several orders of magnitude smaller, than protein nanoparticle complexes. Interestingly, the binding number, n, was found to be approximately 0.5, which in cases like this, is rounded to a whole number of one. The data also indicated an extremely fast rate constant on the order of 1012 L mol-1 s-1 which is outside of the diffusion-control regime. These results are presented within this report.

Characterization of inclusion complexes of organic ions with hydrophilic hosts by ion transfer voltammetry with solvent polymeric membranes

The quantitative characterization of inclusion complexes formed in aqueous phase between organic ions and hydrophilic hosts by ion-transfer voltammetry with solvent polymeric membrane ion sensors is studied, both in a theoretical and experimental way. Simple analytical solutions are presented for the determination of the binding constant of the complex from the variation with the host concentration of the electrochemical signal. These solutions are valid for any voltammetric technique and for solvent polymeric membrane ion sensors comprising one polarisable interface (1PI) and also, for the first time, two polarisable interfaces (2PIs). Suitable experimental conditions and data analysis procedures are discussed and applied to the study of the interactions of a common ionic liquid cation (1-octyl-3-metyl-imidazolium) and an ionisable drug (clomipramine) with two hydrophilic cyclodextrins: α-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin. The experimental study is performed via square wave voltammetry with 2PIs and 1PI solvent polymeric membranes and in both cases the electrochemical experiments enable the detection of inclusion complexes and the determination of the corresponding binding constant.

On the preservation of non-covalent protein complexes during electrospray ionization.

The application range of electrospray ionization mass spectrometry for the quantitative determination of stoichiometries and binding constants for non-covalent protein complexes is broadly discussed. The underlying fundamental question is whether or not the original molecular equilibrium can be preserved during the ionization process and be revealed by subsequent mass spectrometry analysis. Here, we take a new look at this question by discussing recent studies in droplet chemistry.This article is part of the themed issue 'Quantitative mass spectrometry'.

Interactions of helquats with chiral acidic aromatic analytes investigated by partial-filling affinity capillary electrophoresis

Noncovalent molecular interactions between helquats, a new class of dicationic helical extended diquats, and several chiral acidic aromatic drugs and catalysts have been investigated using partial-filling affinity capillary electrophoresis (PF-ACE). Helquats dissolved at 1mM concentration in the aqueous background electrolyte (40mM Tris, 20mM acetic acid, pH 8.1) were introduced as ligand zones of variable length (0–130mm) into the hydroxypropylcellulose coated fused silica capillary whereas 0.1mM solutions of negatively charged chiral drugs or catalysts (warfarin, ibuprofen, mandelic acid, etodolac, binaphthyl phosphate and 11 other acidic aromatic compounds) were applied as a short analyte zone at the injection capillary end. After application of electric field, analyte and ligand migrated against each other and in case of their interactions, migration time of the analyte was increasing with increasing length of the ligand zone. From the tested compounds, only isomers of those exhibiting helical chirality and/or possessing conjugated aromatic systems were enantioselectively separated through their differential interactions with helquats. Some compounds with conjugated aromatic groups interacted with helquats moderately strongly but non-enantiospecifically. Small compounds with single benzene ring exhibited no or very weak non-enantiospecific interactions. PF-ACE method allowed to determine binding constants of the analyte-helquat complexes from the changes of migration times of the analytes. Binding constants of the weakest complexes of the analytes with helquats were less than 50L/mol, whereas binding constants of the strongest complexes were in the range 1 000–1 400L/mol.

Kinetic analysis of IgM monoclonal antibodies for determination of dengue sample concentration using SPR technique

ABSTRACTSurface plasmon resonance (SPR) sensing is recently emerging as a valuable technique for measuring the binding constants, association and dissociation rate constants, and stoichimetry for a binding interaction kinetics in a number of emerging biological areas. This technique can be applied to the study of immune system diseases in order to contribute to improved understanding and evaluation of binding parameters for a variety of interactions between antigens and antibodies biochemically and clinically. Since the binding constants determination of an anti-protein dengue antibody (Ab) to a protein dengue antigen (Ag) is mostly complicated, the SPR technique aids a determination of binding parameters directly for a variety of particular dengue Ag_Ab interactions in the real-time. The study highlights the doctrine of real-time dengue Ag_Ab interaction kinetics as well as to determine the binding parameters that is performed with SPR technique. In addition, this article presents a precise prediction as a reference curve for determination of dengue sample concentration.

Effects of cationic nanoparticles (CNP) on counterion binding constant (RXBr) and catalytic constant (kcat) in micellar system

This paper describes the determination of counterion binding constant, RXBr, and CNP (CTABr/MX/H2O)-catalyzed rate constants, kcat, for the reaction between piperidine and phenyl salicylate ion, PS− (where X=2- and 4-propyl as well as 3, and 4-isopropyl phenolate ions). A plausible reaction mechanism is discussed for CNP catalytic reaction. The calculated mean values of RXBr are 66.3±3.7, 153±8, 60.8±5.5 and 66.6±2.3 for X=2,4-propyl, 3- and 4-isopropyl phenolate ions, respectively. Similarly the mean values of kcat are 0.51±0.09, 1.22±0.07, 0.47±0.07 and 0.53±0.07M−1s−1, respectively.