Equilibrium Association Constant Research Articles

Moment Analysis Method for Measurement of Reaction Equilibrium and Rate Constants by Using High-Performance Liquid Chromatography.

The moment analysis method was developed for the determination of association equilibrium constant (KA) and association (ka) and dissociation (kd) rate constants of intermolecular interactions between solute and ligand molecules. They are accurately determined by using moment equations from elution peak profiles because they are measured by using high-performance liquid chromatography (HPLC) under preferable conditions that neither immobilization nor chemical modification (i.e., fluorescence labeling) of solute and ligand molecules is required. To demonstrate the effectiveness of the method, it was applied to the inclusion complex formation system between dibenzo-18-crown-6 (DB18C6) and alkaline earth metal cations, i.e., Mg2+, Ca2+, and Sr2+, as a concrete example. Because the diameter of the three metal cations is smaller than that of the inner cavity of DB18C6, the values of KA, ka, and kd were analytically determined by assuming the stoichiometry of 1:1 between DB18C6 and the metal cation. They reflected the influence of the difference in the size between the inner cavity of DB18C6 and the metal cations on the inclusion complex formation. It seems that the moment analysis method based on HPLC separation is effective for the multifaceted analysis of chemical reactions because some characteristics of the method are different from those of other conventional methods. It must contribute to the dissemination of an opportunity for the study of chemical reactions to many researchers because of the versatility of HPLC.

Generation of affinity maps for thiazolidinediones with human serum albumin using affinity microcolumns. I. Studies of effects by glycation on multisite drug binding

Human serum albumin (HSA) is known to undergo modifications by glucose during diabetes. This process produces glycated HSA that can have altered binding to some drugs. In this study, high-performance affinity microcolumns and competition studies were used to see how glycation affects the binding by two thiazolidinedione-class drugs (i.e., pioglitazone and rosiglitazone) at specific regions of HSA. These regions included Sudlow sites I and II, the tamoxifen and digitoxin sites, and a drug-binding site located in subdomain IB. At Sudlow site II, the association equilibrium constants (or binding constants) for pioglitazone and rosiglitazone with normal HSA were 1.7 × 105 M−1 and 2.0 × 105 M−1 at pH 7.4 and 37 °C, with values that changed by up to 5.7-fold for glycated HSA. Sudlow site I of normal HSA had binding constants for pioglitazone and rosiglitazone of 3.4 × 105 M−1 and 4.6 × 105 M−1, with these values changing by up to 1.5-fold for glycated HSA. Rosiglitazone was found to also bind a second region that had a positive allosteric effect on Sudlow site I for all the tested preparations of HSA (binding affinity, 1.1–3.2 × 105 M−1; coupling constant for Sudlow site I, 1.20–1.34). Both drugs had a strong positive allosteric effect on the tamoxifen site of HSA (coupling constants, 13.7–19.9 for pioglitazone and 3.7–11.5 for rosiglitazone). Rosiglitazone also had weak interactions at a site in subdomain IB, with a binding constant of 1.4 × 103 M−1 for normal HSA and a value that was altered by up to 6.8-fold with glycated HSA. Neither of the tested drugs had any significant binding at the digitoxin site. The results were used to produce affinity maps that described binding by these thiazolidinediones with HSA and the effects of glycation on these interactions during diabetes.

Unraveling the Dynamics of SARS-CoV-2 Mutations: Insights from Surface Plasmon Resonance Biosensor Kinetics.

Surface Plasmon Resonance (SPR) technology is known to be a powerful tool for studying biomolecular interactions because it offers real-time and label-free multiparameter analysis with high sensitivity. This article summarizes the results that have been obtained from the use of SPR technology in studying the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations. This paper will begin by introducing the working principle of SPR and the kinetic parameters of the sensorgram, which include the association rate constant (ka), dissociation rate constant (kd), equilibrium association constant (KA), and equilibrium dissociation constant (KD). At the end of the paper, we will summarize the kinetic data on the interaction between angiotensin-converting enzyme 2 (ACE2) and SARS-CoV-2 obtained from the results of SPR signal analysis. ACE2 is a material that mediates virus entry. Therefore, understanding the kinetic changes between ACE2 and SARS-CoV-2 caused by the mutation will provide beneficial information for drug discovery, vaccine development, and other therapeutic purposes.

Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid

A new method for the determination of cadherin 12 (CDH12)—an adhesive protein that has a significant impact on the development, growth, and movement of cancer cells—was developed and validated. The method is based on a biosensor using surface plasmon resonance imaging (SPRi) detection. A quartz crystal microbalance was used to analyze the characteristics of the formation of successive layers of the biosensor, from the linker monolayer to the final capture of CDH12 from solution. The association equilibrium constant (KA = 1.66 × 1011 dm3 mol−1) and the dissociation equilibrium constant (KD = 7.52 × 10−12 mol dm−3) of the anti-CDH12 antibody–CDH12 protein complex were determined. The determined analytical parameters, namely the values determining the accuracy, precision, and repeatability of the method, do not exceed the permissible 20% deviations specified by the aforementioned institutions. The proposed method is also selective with respect to possible potential interferents, occurring in up to 100-fold excess concentration relative to the CDH12 concentration. The determined Limit of Quantification (LOQ = 4.92 pg mL−1) indicates the possibility of performing quantitative analysis in human plasma or peritoneal fluid without the need to concentrate the samples; however, particular attention should be paid to their storage conditions, as the analyte does not exhibit high stability. The Passing–Bablok regression model revealed good agreement between the reference method and the SPRi biosensor, with ρSpearman values of 0.961 and 0.925.

Development of Visible Spectrophotometric Methods for the Determination of Tricyclic Antidepressants Based on Formation of Molecular Complexes with p-Benzoquinones

Tricyclic antidepressants are commonly employed in the management of major depressive disorders. The present work describes two visible (VIS) spectrophotometric techniques that utilize the formation of charge transfer complexes between four antidepressant compounds, namely, amitriptyline hydrochloride (AMI), imipramine hydrochloride (IMI), clomipramine hydrochloride (CLO), and trimipramine maleate (TRI) acting as electron donors and two p-benzoquinones, namely, p-chloranilic acid (pCA) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), serving as electron acceptors. The stoichiometry of the compounds produced exhibited a consistent 1:1 ratio in all instances, as established by Job’s method. Molar absorptivities, equilibrium association constants, and several other spectroscopic properties were determined for all complexes. The developed spectrophotometric techniques were validated intra-laboratory and successfully applied for quantitative assessment of the four antidepressant active ingredients in several commercial pharmaceutical formulations. The methods are relatively simple, fast, and use readily available laboratory instrumentation, making them easily applicable by most quality control laboratories worldwide.

Sterically attenuated electronic communication in cobalt complexes of meridional isoquinoline-derived ligands for applications in electrocatalysis.

The ability to synthetically tune the ligand frameworks of redox-active molecules is of critical importance to the economy of solar fuels because manipulating their redox properties can afford control over the operating potentials of sustained electrocatalytic or photoelectrocatalytic processes. The electronic and steric properties of 2,2':6',2″-terpyridine (Terpy) ligand frameworks can be tuned by functional group substitution on ligand backbones, and these correlate strongly to their Hammett parameters. The synthesis of a new series of tridentate meridional ligands of 2,4,6-trisubstituted pyridines that engineers the ability to finely tune the redox potentials of cobalt complexes to more positive potentials than that of their Terpy analogs is achieved by aryl-functionalizing at the four-position and by including isoquinoline at the two- and six-positions of pyridine (Aryl-DiQ). Their cobalt complex syntheses, their electronic properties, and their catalytic activity for carbon dioxide (CO2) reduction are reported and compared to their Terpy analogs. The cobalt derivatives generally experience a positive shift in their redox features relative to the Terpy-based analogs, covering a complementary potential range. Although those evaluated fail to produce any quantifiable products for the reduction of CO2 and suffer from long-term instability, these results suggest possible alternate strategies for stabilizing these compounds during catalysis. We speculate that lower equilibrium association constants to the cobalt center are intrinsic to these ligands, which originate from a steric interaction between protons on the pyridine and isoquinoline moieties. Nevertheless, the new Aryl-DiQ ligand framework has been engineered to selectively tune homoleptic cobalt complexes' redox potentials.

Impact of bound ssRNA length on allostery in the Dengue Virus NS3 helicase.

The presence of ATP is known to stimulate helicase activity of the Dengue Virus Non-structural protein 3 helicase (NS3h), and the presence of RNA stimulates NS3h ATPase activity, however this coupling is still mechanistically unclear. Here we use atomistic models and molecular dynamics simulations to evaluate the single-stranded RNA(ssRNA)-length dependence of the NS3h-ssRNA binding affinity and its modulation by bound ATP. Considering complexes with 7, 11, 16and 26 nucleotides (nts), we observe that both the binding affinity and its modulation by bound ATP are augmented with increased ssRNA lengths. In models with at least 11 nts bound, the binding of ATP results in a shift from a tightly bound to a weakly bound state. We find that the weakly bound state persists during both the ADP-Pi- and ADP-bound stages of the catalytic cycle. We obtain the equilibrium association constants for NS3h binding to an ssRNA 10-mer in vitro, both in the absence and presence of ADP, which further support the alternation between tightlyand weaklybound states during the catalytic cycle. The length of bound ssRNA is critical for understanding the NS3h-RNA interaction as well as how it is modulated during the catalytic cycle.

The role of structural heterogeneity in the homodimerization of transmembrane proteins.

The equilibrium association of transmembrane proteins plays a fundamental role in membrane protein function and cellular signaling. While the study of the equilibrium binding of single pass transmembrane proteins has received significant attention in experiment and simulation, the accurate assessment of equilibrium association constants remains a challenge to experiment and simulation. In experiment, there remain wide variations in association constants derived from experimental studies of the most widely studied transmembrane proteins. In simulation, state-of-the art methods have failed to adequately sample the thermodynamically relevant structures of the dimer state ensembles using coarse-grained models. In addition, all-atom force fields often fail to accurately assess the relative free energies of the dimer and monomer states. Given the importance of this fundamental biophysical process, it is essential to address these shortcomings. In this work, we establish an effective computational protocol for the calculation of equilibrium association constants for transmembrane homodimer formation. A set of transmembrane protein homodimers, used in the parameterization of the MARTINI v3 force field, are simulated using metadynamics, based on three collective variables. The method is found to be accurate and computationally efficient, providing a standard to be used in the future simulation studies using coarse-grained or all-atom models.

A fluorescent probe for detection of Hg2+ ions constructed by tetramethyl cucurbit[6]uril and 1,2-bis(4-pyridyl)ethene.

In this paper, tetramethyl cucurbit[6]uril (TMeQ[6]) and 1,2-bis(4-pyridyl)ethene (G) were used to construct a supramolecular fluorescent probe G@TMeQ[6]. The host-guest interaction between TMeQ[6] and G was investigated using 1H NMR spectroscopy, single-crystal X-ray diffraction and various experimental techniques. The results show that TMeQ[6] and G form an inclusion complex with a host-guest ratio of 1:1 and the equilibrium association constant (Ka) was 2.494 × 104 M-1. The G@TMeQ[6] fluorescent probe can sensitively recognize Hg2+ ions by fluorescence enhancement. The linear range is 0.33 × 10-5-1.65 × 10-5 mol·L-1, R2 = 0.9926, and the limit of detection is 4.12 × 10-8 mol·L-1. The fluorescent probe can be used to detect the concentration of Hg2+ ions in aqueous solution, and provides a theoretical basis for the development of new fluorescent probes for detecting heavy metal ions.

An efficient fluorescence reversible regulation strategy with single labelled oligonucleotide HEX-OND successively triggered by Hg(II) and Cysteine: The application and mechanism

An efficient fluorescence reversible regulation system with HEX-OND was developed. Then the application potential was explored in probing Hg(II) & Cysteine (Cys) in real samples and the thermodynamic mechanism was further investigated by precise theory analysis combining multiple spectroscopic methods. The results showed that only mere disturbances were observed among 15 and 11 kinds of other substances for the optimal system in detecting Hg(II) & Cys, respectively; The linear ranges of quantification were identified as 1.0 ∼ 14.0 and 2.0 ∼ 20.0 (×10-8 mol/L) with LODs of 8.75 and 14.09 (×10-9 mol/L) for Hg(II) and Cys, respectively; no significant deviations were found in the quantification results of Hg(II) in three traditional Chinese herbs and Cys in two samples between the well-understood methods with ours respectively, showing excellent selectivity, sensitivity, and tremendous application feasibility. The detailed mechanism was further verified as that the introduced Hg(II) forced HEX-OND to transform into the Hairpin structure with the apparent equilibrium association constant of 6.02 ± 0.62 × 1010 L/mol in the bimolecular ratio, leading to the equimolar quencher, consecutive two guanine bases ((G)2), approaching and spontaneously static-quenching the reporter HEX (hexachlorofluorescein) (equilibrium constant, 8.75 ± 1.97 × 107 L/mol) in the Photo-induced Electron Transfer (PET) way that was driven by the Electrostatic Interaction. The additional Cys destructed the equimolar Hairpin structure with the apparent equilibrium constant of 8.87 ± 2.47 × 105 L/mol through breaking one of the formed T-Hg(II)-T mismatches by association with the involved Hg(II), occasioning (G)2 apart from HEX and consequently the fluorescence recovery.

Critical Analysis of Association Constants between Calixarenes and Nitroaromatic Compounds Obtained by Fluorescence. Implications for Explosives Sensing.

The binding behaviour of two ureido-hexahomotrioxacalix[3]arene derivatives bearing naphthyl (1) and pyrenyl (2) fluorogenic units at the lower rim towards selected nitroaromatic compounds (NACs) was evaluated. Their affinity, or lack of it, was determined by UV-Vis absorption, fluorescence and NMR spectroscopy. Different computational methods were also used to further investigate any possible complexation between the calixarenes and the NACs. All the results show no significant interaction between calixarenes 1 and 2 and the NACs in either dichloromethane or acetonitrile solutions. Moreover, the fluorescence quenching observed is only apparent and merely results from the absorption of the NACs at the excitation wavelength (inner filter effect). This evidence is in stark contrast with reports in the literature for similar calixarenes. A naphthyl urea dihomooxacalix[4]arene (3) is also subject to the inner filter effect and is shown to form a stable complex with trinitrophenol; however, the equilibrium association constant is greatly overestimated if no correction is applied (9400 M-1 vs 3000 M-1), again stressing the importance of taking into account the inner filter effect in these systems.

Building a molecular model for ion channel and transporter stability in membranes through a partnership of experiments and computation.

Ion channels and transporters are the molecular gatekeepers of biology, governing the passage of atoms and molecules in and out of cells. They provide essential nutrients for metabolism, eliminate waste, enable cell-to-cell communication and store the potential energy that fuels life. Yet, despite their overwhelming importance, we still lack a physical and molecular understanding of why membrane transport proteins form stable structures that enable their specific functions within the oil-filled environment of the lipid bilayer. Our lack of understanding here is well rationalized by the inherent challenge of measuring equilibrium reactions of membrane protein assembly in membranes, compounded by the complexity of the reaction solvent as a structured lipid bilayer. However, through a combination of novel experimental single-molecule microscopy approaches coupled with coarse-grained and all-atom computational modeling, we can now measure equilibrium constants of protein association in membranes and dissect out the important molecular contributions from the protein and surrounding membrane. Here, I present our results on two systems where reversible binding within membranes can be quantitatively assessed - the homodimeric CLC-ec1 chloride/proton antiporter and the dual-topology homodimeric Fluc fluoride ion channel. By combining experimental and computational studies, we identify a driving force for protein association that arises from state-dependent perturbation of the membrane structure and a mechanism for tuning dimerization stability by altering solvation energetics through preferential solvation. These results provide generalizable mechanisms that drive membrane protein assembly in membranes that are expected to apply to all ion channels and transporters.

Handling Detection Limits of Multiplex Lateral Flow Immunoassay by Choosing the Order of Binding Zones.

Changes in the limits of detection (LODs) for a multiplex lateral flow immunoassay (LFIA) caused by different locations of the binding zone on the test strips were studied. Due to the non-equilibrium conditions of the immune reactions in LFIAs, their analytical parameters are susceptible to the binding constants of antigen-antibody reactions and assay duration. Consequently, the integration of several tests into one multiplex assay can cause a significant worsening of the sensitivity. In this study, we propose a simple methodology for the determination of the best arrangement of binding zones, which takes into account the binding constants for immunoreagents. LFIAs of four mycotoxins, namely, aflatoxin B1, deoxynivalenol, T-2 toxin, and ochratoxin A, were integrated into a multiplex test strip. An enzyme-linked immunosorbent assay was applied to determine the equilibrium and kinetic constants of the immunoreactants for each analyte. It was found that the arrangement of binding zones with a descending order of the equilibrium association constants was optimal and provided both lower detection limits and a more uniform coloration. The selected position of the binding zones allowed decreasing the LODs down to 2 and 27 times for ochratoxin A and deoxynivalenol, respectively. The proposed approach can be applied to multiplex LFIAs for different analytes.

Single -and Multi-Walled Carbon Nanotubes as Nanocarriers for the Delivery of 7-Hydroxyflavone.

The research on flavonoids has exponentially grown since their first therapeutic evidence, in 1937. They are effective in vitro in a wide range of human diseases, particularly those mediated by free radicals, such as cancer, atherosclerosis, AIDS, or neuronal diseases. However, their applications have been reduced due to their low solubility, poor absorption, and rapid metabolism. Flavonoid encapsulation in nanocarriers significantly improves their oral absorption, protects the drug against degradation, decreases the first-pass hepatic effect, and makes absorption through the lymphatic system easier. In this work, carbon nanotubes were used as nanocarriers of 7-hydroxyflavone, 7-HF. The encapsulation of 7-HF into pristine single- and multi-walled carbon nanotubes, and into -COOH functionalized single-walled carbon nanotubes has been investigated. The equilibrium association constants were estimated. The structural backbone of 7-HF, two benzene rings linked through three carbon atoms that form a pyran heterocyclic ring containing a keto group, seems to play a key role in the 7-HF/CNT interactions, although other types of interactions are also at work. The in vitro release of 7-HF was studied at three pHs, 2.0, 7.4, and 9.2, mimicking the different biological barriers of the human organism.

Analysis of the binding of warfarin to glyoxal- and methylglyoxal-modified human serum albumin by ultrafast affinity extraction

Ultrafast affinity extraction (UAE) and affinity microcolumns containing immobilized human serum albumin (HSA) were employed to evaluate the effect of advanced stage glycation on HSA and its binding to warfarin, a common site-specific probe for Sudlow site I of this protein. The modification of HSA by glyoxal (GO) and methylglyoxal (MGO) was considered, where GO and MGO are known to be important in the formation of many types of advanced glycation end products. Free drug fractions were measured by UAE for warfarin in solutions containing normal HSA or HSA that had been modified by GO or MGO at levels seen in serum during diabetes. The free fractions measured with the GO-modified HSA gave association equilibrium constants that ranged from 2.42–2.63 × 105 M−1 at pH 7.4 and 37 °C. These values were not significantly different from a value of 2.33 (±0.15) × 105 M−1 that was determined by the same method for warfarin with normal HSA. Similar studies using MGO-modified HSA gave association equilibrium constants for warfarin in the range of 3.07–3.31 × 105 M−1, which were 1.32- to 1.42-fold higher than the value seen for normal HSA (differences that were significant at the 95% confidence level). These results will be valuable in future binding studies based on affinity chromatography or other methods that employ warfarin as a probe to examine drug interactions at Sudlow site I of HSA and modified forms of this protein. This work also illustrates how UAE can be used, with analysis times of only minutes, to detect and measure small changes in the binding by drugs with unmodified or modified forms of a soluble binding agent or protein.

Moment theory of affinity capillary electrophoresis for analysis of reaction kinetics of intermolecular interactions

A theoretical basis for affinity capillary electrophoresis (ACE) was systematically developed by applying the moment theory to the study on intermolecular interactions between solute (S) and ligand (L) molecules to form complex (X). Moment equations were developed for both complete filling and partial filling ACE systems, in which SLm or SnL forms as X. They are effective for the determination of equilibrium and kinetic constants of association between S and L and dissociation of X from the first absolute and second central moments of elution peaks measured by ACE. In order to demonstrate their effectiveness, simulative calculations of partial filling ACE behavior was conducted by assuming the stoichiometry of 1:1 between S and L. Because partial filling ACE systems are classified into five categories based on experimental conditions, calculation results for all the five cases are explained. The combination of ACE and moment theory is effective because there are some advantages concerning the accurate analysis of intermolecular interactions. The results of this study provide an opportunity for the study on intermolecular interactions to many researchers because CE is already versatile.

Characterization of binding by repaglinide and nateglinide with glycated human serum albumin using high-performance affinity microcolumns.

High-performance affinity microcolumns were used to characterize binding by the anti-diabetic drugs repaglinide and nateglinide with normal and glycated forms of human serum albumin. The microcolumns contained only nmol amounts of protein and provided a detailed analysis of these drug interactions with good precision and in a matter of minutes per experiment. The overall binding by repaglinide to normal and glycated albumin fits a model with two types of binding sites: a set of one or two moderate-to-high affinity regions and a larger set of weaker regions with association equilibrium constants of ∼105 and 103 M-1 , respectively, at pH 7.4 and 37°C. Competition studies gave site-specific association constants for repaglinide and nateglinide at Sudlow site I of 4.2 × 104 and 5.0 × 104 M-1 for normal albumin, with a decrease of 26%-30% being seen for nateglinide with glycated albumin and no significant change being noted for repaglinide. At Sudlow site II, repaglinide and nateglinide had association constants for normal albumin of 6.1 × 104 and 7.1 × 105 M-1 , with glycated albumin giving an increase in the association constant at this site for repaglinide of 1.6- to 1.8-fold and a decrease for nateglinide of 51%-58%.

Real-time kinetics and affinity analysis of the interaction between protein A and immunoglobulins G derived from different species on silica colloidal crystal films

Kinetic and affinity analysis of protein interactions reveals information on their related activities in biological processes. Herein, we established a system for evaluating the kinetics and affinity of the interaction between protein A and various IgG species on the surface of silica spheres of silica colloidal crystal (SCC) films by the extraordinary optical interference capabilities of 190 nm silica spheres after self-assembly. The equilibrium association constant (KA) was calculated by the equilibrium Langmuir model and nonlinear least-squares analysis of time-dependent data. The relative protein A/IgG binding affinity is human > rabbit >cow >goat. In addition, the competitive interaction of distinct species of IgG with protein A at the interface of SCC films was studied and performed. These findings may help with the use of protein A and other recognition components in a number of sensor types. Furthermore, this research might offer a novel approach to determining the kinetics and affinity of proteins on the surface of spheres particles, which may contribute to the development of the application of spheres particles in pharmaceutical science, biomedical engineering, and other techniques.

Sulfate Protonation in Sodium Chloride and Sodium Perchlorate Media

The equilibrium (association) constant, KA, for the first protonation of the sulfate ion in aqueous solution: [H+(aq) + {text{SO}}_{4}^{2 - }(aq) ⇌ {text{HSO}}_{4}^{ - }(aq)] has been measured at 298.15 K in NaCl and NaClO4 media using glass-electrode potentiometry at five ionic strengths in the range 0.5 ≤ I/(mol·dm−3) ≤ 5.0. The present KA values vary smoothly with I and are in good agreement with reliable literature data but indicate that the previously reported value of KA at I = 5 mol·dm−3 in NaClO4 media is in error.

Determination of association equilibrium constant from single molecule fluorescence localization microscopy.

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.