Determination Of Binding Constants Research Articles

Evaluation of the effect of background electrolyte composition and independence of parameters in determining binding constants of betulin derivatives to β- and dimethyl-β-cyclodextrins by affinity capillary electrophoresis.

The values of the apparent binding constants for β-cyclodextrin complexes of betulin derivatives determined by mobility shift affinity capillary electrophoresis were found to be independent of the composition of the two background electrolytes used (tetraborate buffer, pH 9.18, and phosphate buffer, pH 8.00, both of them with 20 mM ionic strength). It has been found that if there is not a constant plateau on the binding curve then four independent parameters can be determined: binding constants (also referred to as stability, association, or formation constants) and ionic mobilities of 1:1 and 1:2 complexes. However, at least 10-12 data points in the binding curve should be used to reliably estimate the parameters. For the first time, the apparent binding constants for complexes of ester betulin derivatives with dimethyl-β-cyclodextrin have been determined by mobility shift affinity capillary electrophoresis. The logarithms of the constants for 1:1 and 1:2 complexes at 25°C for betulin 3,28-diphthalate with a 95% confidence interval are 4.98 (4.95-5.01) and 7.52 (7.26-7.68); for betulin 3,28-disulfate, the values are 4.97 (4.89-5.03) and 8.24 (6.82-8.52). It has been found that betulin 3,28-disuccinate forms only a 1:1 complex and the binding constant logarithm is 5.25 ± 0.02.

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs.

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

Shedding light on the binding mechanism of kinase inhibitors BI-2536, Volasetib and Ro-3280 with their pharmacological target PLK1.

In the present work, the interactions of the novel kinase inhibitors BI-2536, Volasetib (BI-6727) and Ro-3280 with the pharmacological target PLK1 have been studied by fluorescence spectroscopy and molecular dynamics calculations. High Stern-Volmer constants were found in fluorescence experiments suggesting the formation of stable protein-ligand complexes. In addition, it was observed that the binding constant between BI-2536 and PLK1 increases about 100-fold in presence of the phosphopeptide Cdc25C-p that docks to the polo box domain of the protein and releases the kinase domain. All the determined binding constants are higher for the kinase inhibitors than for their competitor for the active center (ATP) being BI-2536 and Volasertib the inhibitors that showed more affinity for PLK1. Calculated binding free energies confirmed the higher affinity of PLK1 for BI-2536 and Volasertib than for ATP. The higher affinity of the inhibitors to PLK1 compared to ATP was mainly attributed to stronger van der Waals interactions. Results may help with the challenge of designing and developing new kinase inhibitors more effective in clinical cancer therapy.

Food additive dye–lysozyme complexation: Determination of binding constants and binding sites by fluorescence spectroscopy and modeling methods

The interaction mechanisms of QY with lysozyme were investigated using multispectral and molecular docking methods in the current study. Quinoline yellow (QY) is a synthetic dye that is widely used in the food industry. The interaction of QY with this proteinase was studied using fluorescence spectroscopy. The fluorescence quenching results demonstrated that QY could quench this proteinase's intrinsic fluorescence using a static quenching procedure. Through hydrogen bonding and van der Waals interactions, QY binds to lysozyme. The binding constant (Ka) and the number of binding sites (n) for this binding interaction were determined using associated fluorescence quenching data. The presence of QY dye reduced enzyme activity, according to kinetic technique analysis. The CD results confirmed the protein's conformational changes during the complexation process. Aside from the experimental studies, molecular docking revealed the precise binding location of QY within the lysozyme enzyme structure. Molecular dynamics simulation was also used to assess the stability of the lysozyme-QY complex.

Selective Detection and Ultrasensitive Quantification of SARS-CoV-2 IgG Antibodies in Clinical Plasma Samples Using Epitope-Modified Nanoplasmonic Biosensing Platforms.

Monitoring the human immune response by assaying (detection and quantification) the antibody level against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important in conducting epidemiological surveillance and immunization studies at a population level. Herein, we present the design and fabrication of a solid-state nanoplasmonic biosensing platform that is capable of quantifying SARS-CoV-2 neutralizing antibody IgG with a limit of detection as low as 30.0 attomolar (aM) and a wide dynamic range spanning seven orders of magnitude. Based on IgG binding constant determination for different biological motifs, we show that the covalent attachment of highly specific SARS-CoV-2 linear epitopes with an appropriate ratio, in contrast to using SARS-CoV-2 spike protein subunits as receptor molecules, to gold triangular nanoprisms (Au TNPs) results in a construction of a highly selective and more sensitive, label-free IgG biosensor. The biosensing platform displays specificity against other human antibodies and no cross reactivity against MERS-CoV antibodies. Furthermore, the nanoplasmonic biosensing platform can be assembled in a multi-well plate format to translate to a high-throughput assay that allowed us to conduct SARS-CoV-2 IgG assays of COVID-19 positive patient (n = 121) and healthy individual (n = 65) plasma samples. Most importantly, performing a blind test in an additional cohort of 30 patient plasma samples, our nanoplasmonic biosensing platform successfully identified COVID-19 positive samples with 90% specificity and 100% sensitivity. Very recent studies show that our selected epitopes are conserved in the highly mutated SARS-CoV-2 variant “Omicron”; therefore, the demonstrated high-throughput nanoplasmonic biosensing platform holds great promise for a highly specific serological assay for conducting large-scale COVID-19 testing and epidemiological studies and monitoring the immune response and durability of immunity as part of the global immunization programs.

Metal(II) Complexes of the Fluoroquinolone Fleroxacin: Synthesis, Characterization and Biological Profile.

A series of complexes of divalent transition metals (Cu(II), Mn(II), Zn(II), Co(II) and Ni(II)) with the quinolone antibacterial agent fleroxacin, in the absence or presence of an α-diimine such as 2,2′-bipyridine, 1,10-phenanthroline or 2,2′-bipyridylamine, were prepared and characterized. The complexes were characterized by various physicochemical and spectroscopic techniques and by single-crystal X-ray crystallography. The in vitro antibacterial activity of the complexes was studied against the bacterial strains Staphylococcus aureus, Bacillus subtilis and Xanthomonas campestris and was higher than that of free quinolone. The affinity of the complexes for bovine and human serum albumin was studied by fluorescence emission spectroscopy and the determined binding constants showed tight and reversible binding to the albumins. The interaction of the complexes with calf-thymus DNA was studied by various techniques, which showed that intercalation was the most plausible mode of interaction.

Cupric Ions Selectively Modulate TRAAK-Phosphatidylserine Interactions.

TRAAK and TREK2 are two-pore domain K+ (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca2+, Mg2+, and Zn2+) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu2+ and Zn2+ than TREK2. In the presence of Cu2+, TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu2+. High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu2+-bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK-PS interactions.

Molecular structure-affinity relationship of selected phenolic compounds for lupin seed γ-conglutin

The molecular basis underlying the interaction between proteins and phenolic compounds are still not fully understood. The specific structural properties of proteins, as well as phenolics, strongly determine the complex formation. In this work, the interactions between γ-conglutin, a unique lupin seed protein, and twenty-one different phenolic compounds representing six different classes of phenolics that differ in their structures were investigated. The interactions were studied based on a fluorescence quenching experiment, and the determined binding constant (Ka) ranged from 6.88 × 102 (protocatechuic acid) to 5.06 × 106 (hesperidin), while the number of binding sites of phenolic compound molecules to the protein (n) was on average 1.14 ± 0.16. Within the analysed compounds, phenolic acids interacted the weakest with the protein, while flavonoids showed considerable higher affinity strength to γ-conglutin. Notably, flavanones and flavones were the phenolics that formed the complex with markedly higher values of Ka (1-3 orders of magnitude) and n. Additionally, principal component analysis allowed to formulate the general regularities of phenolic compounds preferences for γ-conglutin. Finally, the obtained results also indicate that phenolic compounds' binding preferences for γ-conglutin can result from their native occurrence in lupin seeds.

Standard errors or asymmetric confidence intervals of the protein-ligand binding constant

The determination of equilibrium binding constants (Kb) between chemical compounds (ligands) and target proteins in vitro is important for the understanding of biological interaction mechanisms. Accuracy of the data is critical for decision-making in many scientific fields. The Kb is commonly assumed to follow a symmetric normal, Gaussian, distribution, and is often reported as experimental value plus/minus standard deviation. However, in general, the distributions of the measured Kb values (and the equivalent dissociation constant, Kd=1/Kb) and the corresponding Gibbs energy change (ΔGb=-RTlnKb) are all asymmetric to varying degrees.

A new FRET-based functional chemosensor for fluorometric detection of Fe3+and its validation through in silico studies

The present study aims to examine fluorescence resonance energy transfer (FRET) between coumarin 151 (C151) and bovine serum albumin (BSA) in different pH mediums beside determining binding constants and energy transfer parameters using different methods considering pH levels. Once the basis of the BSA-C151 FRET system had been established, this system was designed as a new FRET-based functional chemosensory system for detection of Fe3+ ion. Fe3+ ion was observed to have specifically interacted with the system in question, thus affecting the fluorescence energy transfer between two molecules. The limit of detection (LOD) value of the system for Fe3+ ion was determined as 1.42 × 10−9 M (1.42 nM) and Fe3+concentration ranging from 10 nM to 1 μM. The molecular docking results showed that BSA had interacted with C151, which we defined as a new system. It was also revealed that BSA had interacted with two amino acid residues (Trp214 and Ser202) as a result of FeCl3 being added to the system. Beside, the in-silico studies demonstrated that the FRET occurring in the BSA-C151 system had taken place via the Trp214 amino acid residue of BSA, and that the fluorometric determination of the Fe3+ ion was based on the fluorescence quenching that occurred due to the interaction between the Trp214 amino acid residue of BSA and Fe3+ present in the BSA-C151-FeCl3 system. The BSA–C151 FRET system has good potential for Fe3+ detection in analytical systems as it shows high selectivity for Fe3+. The low LOD value in the present study also contributed to this potential.

Linking molecular composition to proton and copper binding ability of fulvic acid: A theoretical modeling approach based on FT-ICR-MS analysis

Dissolved organic matter (DOM) is one of the most important ligands governing the geochemical cycling of metals in the environment, but recent studies with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) have shown enormous complexity and diversity of DOM composition. How the diverse molecular composition of DOM affects the reactivity of DOM with metals is still largely unknown, which precludes us from developing accurate geochemical models for the fate of metals in the environment. In this study, we combined FT-ICR-MS analysis and theoretical modeling approaches and specifically elucidated the link between molecular composition and the proton and Cu binding ability of DOM, using the Suwannee River fulvic acid (FA) as a model humic substance. Batch adsorption experiments were conducted to generate different extents of molecular fractionation of FA samples by ferrihydrite. FT-ICR-MS analyses were employed to investigate the changes of molecular composition while Cu titration and the Windermere Humic Aqueous Model (WHAM) were used to quantify the variations on the Cu binding capacities of FA samples. We developed a general theoretical modeling approach, which integrated a suite of theoretical modeling methods, including the Vienna Soil-Organic-Matter-Modeler (VSOMM), SPARC Performs Automated Reasoning in Chemistry (SPARC), and the linear free energy relationships (LFER), for molecular modeling based on FT-ICR-MS data. Based on the FT-ICR-MS results, we found that, despite of the complex molecular composition of FA, FA molecules can be divided into three representative groups and each group of molecules had distinct chemical properties. Interestingly, molecules within the same group had similar distributions of molecular properties. Based on the chemical properties of the three groups of FA molecules, we successfully constructed three molecular models of FA using VSOMM, and quantified the distributions of proton and Cu binding constants with SPARC and LFER. Those independently determined binding constants were comparable to the WHAM default proton and Cu binding constants, supporting the validity of our modeling approach. Our modeling results suggested that the molecular complexity of DOM may be simplified with representative groups of molecules based on their binding ability with metals in theoretical modeling. Our modeling approach based on FT-ICR-MS data shed light on developing mechanistical models for metal reactions with DOM based on the molecular data, which is helpful for predicting the geochemical cycling of carbon and metals in the environment.

Miscalculating Equilibrium Constants for Multivalent Protein Complexes: Site-Binding Concentration or Protein Molecular Concentration?

Because the authors continue to note instances in the scientific literature of failure to use the correct receptor binding site concentration for determining binding constants, herein we discuss the fundamental concepts that need to be considered to determine correct binding constants or conversely calculate accurate reactant concentrations with known equilibrium constants. We also show the derivation and analytical solutions of the cubic and quartic equations that give the exact free ligand concentration in bivalent and trivalent receptor systems at equilibrium as a function of the macroscopic equilibrium dissociation constants and the total concentrations of ligand and multivalent protein. These equations and solutions strongly reemphasize the critical dependency of deriving the correct concentrations of bound or free ligand and multivalent protein on the choice of the correct concentration basis for the multivalent protein, which is in turn dependent upon the type of equilibrium constant used. These results demonstrate the importance of choosing the proper multivalent protein concentration for the determination of either valid microscopic or valid macroscopic equilibrium dissociation constants from binding isotherms of ligand-multivalent protein complexes.

Misinterpretations in Evaluating Interactions of Vanadium Complexes with Proteins and Other Biological Targets

In aqueous media, VIV- and VV-ions and compounds undergo chemical changes such as hydrolysis, ligand exchange and redox reactions that depend on pH and concentration of the vanadium species, and on the nature of the several components present. In particular, the behaviour of vanadium compounds in biological fluids depends on their environment and on concentration of the many potential ligands present. However, when reporting the biological action of a particular complex, often the possibility of chemical changes occurring has been neglected, and the modifications of the complex added are not taken into account. In this work, we highlight that as soon as most vanadium(IV) and vanadium(V) compounds are dissolved in a biological media, they undergo several types of chemical transformations, and these changes are particularly extensive at the low concentrations normally used in biological experiments. We also emphasize that in case of a biochemical interaction or effect, to determine binding constants or the active species and/or propose mechanisms of action, it is essential to evaluate its speciation in the media where it is acting. This is because the vanadium complex no longer exists in its initial form.

Binding Constants Determination of Ofloxacin and Ornidazole Enantiomers with Sulfated β-Cyclodextrin as Single Ligand by Capillary Electrophoresis using Three Different Linear Plotting Methods

The aim of this research is to use capillary electrophoresis to establish/determine the binding constants for ofloxacin and ornidazole enantiomers with the negatively charged chiral selector sulfated-β-cyclodextrin (S-β-CD). Using electrophoretic mobility values of ofloxacin and ornidazole enantiomers at various concentrations of S-β-CD used in the context of background electrolyte (BGE), binding constants were calculated using three separate linearization plots, namely double-reciprocal, X-reciprocal and Y-reciprocal. The R-ofloxacin enantiomer-S-β-CD complex had the highest inclusion affinity of the ofloxacin and ornidazole enantiomers, which matched with previously reported estimation. Every enantiomer-S-β-CD complex’s binding constants, as well as thermodynamic binding parameters, were calculated at different temperatures. The host-guest binding constants using double reciprocal fit showed greater linearity (R2 > 0.99) at all temperature ranges measured (15-30 ºC) as compared to the other two fit approaches. The thermodynamic complexation parameters were found to be dependent on the temperature of the enantiomers, as seen by the linear van’t Hoff (15-30 ºC) plot.

Mass transfer kinetics on modern Whelk-O1 chiral stationary phases made on fully- and superficially-porous particles

In this work, a detailed study of mass transfer properties of trans-stilbene oxide (TSO) enantiomers on two Whelk-O1 chiral stationary phases (CSPs) has been performed. The CSPs were prepared by using both fully-porous silica particles of 2.5 μm particle diameter and superficially-porous ones of 2.6 μm particle diameter as base materials. By combining stop-flow and dynamic measurements in normal-phase conditions, the different contributions to mass transfer have been estimated. The study of intraparticle diffusion has revealed that the adsorption of both enantiomers is localized (i.e., characterized by absence of surface diffusion). The determination of thermodynamic binding constants (measured through adsorption isotherms) supports this finding.

Determining binding constants for 1:1 and 1:2 inclusion complexes of ester betulin derivatives with (2-hydroxypropyl)-β-cyclodextrin by affinity capillary electrophoresis.

The complexation of ester betulin derivatives with (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) was studied by mobility shift affinity CE. Electrophoretic mobility for triangular peaks was calculated using the parameter a1 of the Haarhoff-Van der Linde function instead of the peak top time. Dependences of the viscosity corrected electrophoretic mobility on HP-β-CD concentration were not described on the basis of only complexes with 1:1 stoichiometry due to the fact that these binding curves did not reach a plateau. However, the dependences were well described taking into account both 1:1 and 1:2 complexes. The presence of higher order equilibria was also revealed by x-reciprocal plots. The values of apparent binding constant logarithm, obtained for the first time, for 1:1 and 1:2 HP-β-CD complexes of betulin 3,28-diphthalate and betulin 3,28-disuccinate with 95% confidence interval limits in brackets are the same within error and are equal to 4.85 (4.73-4.95), 8.56 (7.75-8.82), 4.92 (4.86-4.97), and 8.54 (8.23-8.72) at 25°C, respectively. These values for 1:1 and 1:2 HP-β-CD complexes of betulin 3,28-disulfate at 25°C are 4.61 (4.57-4.64) and 7.11 (6.57-7.34), respectively. The binding constants for betulin 3,28-disulfate agree with the previously obtained results from the separation in the thermostatted capillary segment.

PREPARATION, CHARACTERIZATION, ANTIOXIDANT ACTIVITY OF FUNCTIONALIZED 2-(FERROCENYLMETHYLAMINO) BENZONITRILE BY ZnO NANOPARTICLE USING 3 AMINOPROPYLTRIETHOXYSILANE(APTES)

The aim of the present study was study the interaction between 2,2-diphenyl-1- picrylhydrazyl radical (DPPH•) and ZNO@APTES@2- (ferrocenylmethylamino)benzonitrile and Galic acide Then determination of binding parameters like binding constant and binding free energy The determination of binding constant and binding free energy is based upon the decrease in absorbance of the electronic absorption spectrum .The ZNs was synthesise according to chemical method. The characterizations of ZnO NPs were measured by FTIR and X-ray diffraction.

Time-Dependent Fluorescence Spectroscopy to Quantify Complex Binding Interactions.

Measuring the binding affinity for proteins that can aggregate or undergo complex binding motifs presents a variety of challenges. In this study, fluorescence lifetime measurements using intrinsic tryptophan fluorescence were performed to address these challenges and to quantify the binding of a series of carbohydrates and carbohydrate-functionalized dendrimers to recombinant human galectin-3. Collectively, galectins represent an important target for study; in particular, galectin-3 plays a variety of roles in cancer biology. Galectin-3 binding dissociation constants (KD) were quantified: lactoside (73 ± 4 μM), methyllactoside (54 ± 10 μM), and lactoside-functionalized G(2), G(4), and G(6)-PAMAM dendrimers (120 ± 58 μM, 100 ± 45 μM, and 130 ± 25 μM, respectively). The chosen examples showcase the widespread utility of time-dependent fluorescence spectroscopy for determining binding constants, including interactions for which standard methods have significant limitations.

Investigation of the enantioselective interaction between selected drug enantiomers and human serum albumin by mobility shift-affinity capillary electrophoresis.

Mobility shift-affinity capillary electrophoresis was employed for enantioseparation and simultaneous binding constant determination. Human serum albumin was used as a chiral selector in the background electrolyte composed of 20mM phosphate buffer, pH 7.4. The applied setup supports a high mobility shift since albumin and the drug-albumin complex hold negative net charges, while model compounds of amlodipine and verapamil are positively charged. In order to have an accurate effective mobility determination, the Haarhoff-van der Linde function was utilized. Subsequently, the association constant was determined by nonlinear regression analysis of the dependence of effective mobilities on the total protein concentration. Differences in the apparent binding status between the enantiomers lead to mobility shifts of different extends (α). This resulted in enantioresolutions of Rs=1.05-3.63 for both drug models. R-(+)-Verapamil (KA 1844M-1 ) proved to bind stronger to human serum albumin compared to S-(-)-verapamil (KA 6.6M-1 ). The association constant of S-(-)-amlodipine (KA 25073M-1 ) was found to be slightly higher compared to its antipode (KA 22620M-1 ) when applying the racemic mixture. The low measurement uncertainty of this approach was demonstrated by the close agreement of the association constant of the enantiopure S-(-)-form (KA 25101M-1 ).

Determination of binding constants for strong complexation by affinity capillary electrophoresis: the example of complexes of ester betulin derivatives with (2-hydroxypropyl)-γ-cyclodextrin.

Complexation plays an important role in many biological phenomena, the analysis of different samples, optimization of separation processes, and increasing the pharmacological activity of drugs. This paper discusses the features of using mobility shift affinity capillary electrophoresis for studying strong complexation. Electrophoretic peaks for this case are often triangular. It was shown that the use of electrophoretic mobility obtained from the peak apex time to calculate binding constants leads to significant systematic and random errors, and the parameter a1 of the Haarhoff-Van der Linde function should be used instead of the apex time. Distorted triangular peaks with dips were shown to be observed at too high a ratio of analyte concentration in the sample to ligand concentration in the background electrolyte, and the peaks and parameter a1 significantly shifted. It was found that the permissible excess of analyte concentration over ligand concentration was approximately 10-35, provided that the parameter a1 was used, but the peak shape should be used as a landmark, and only triangular peaks without dips should be fitted with the function. The lowest possible analyte concentration should be utilized, which allows the use of a wider range of ligand concentration leading to higher precision of determining the binding constants values. Kinetically labile 1:1 complexes between (2-hydroxypropyl)-γ-cyclodextrin (HP-γ-CD) and betulin 3,28-diphthalate (DPhB) and betulin 3,28-disuccinate (DScB) were studied as an example. The binding constant logarithms at 25°C are 7.23 ± 0.03 and 7.13 ± 0.10 for the HP-γ-CD complexes of DPhB and DScB, respectively. Graphical Abstract.