Equilibrium Association Constant Research Articles

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.

Investigation and Comparison of Specific Antibodies' Affinity Interaction with SARS-CoV-2 Wild-Type, B.1.1.7, and B.1.351 Spike Protein by Total Internal Reflection Ellipsometry.

SARS-CoV-2 vaccines provide strong protection against COVID-19. However, the emergence of SARS-CoV-2 variants has raised concerns about the efficacy of vaccines. In this study, we investigated the interactions of specific polyclonal human antibodies (pAb-SCoV2-S) produced after vaccination with the Vaxzevria vaccine with the spike proteins of three SARS-CoV-2 variants of concern: wild-type, B.1.1.7, and B.1.351. Highly sensitive, label-free, and real-time monitoring of these interactions was accomplished using the total internal reflection ellipsometry method. Thermodynamic parameters such as association and dissociation rate constants, the stable immune complex formation rate constant (kr), the equilibrium association and dissociation (KD) constants and steric factors (Ps) were calculated using a two-step irreversible binding mathematical model. The results obtained show that the KD values for the specific antibody interactions with all three types of spike protein are in the same nanomolar range. The KD values for B.1.1.7 and B.1.351 suggest that the antibody produced after vaccination can successfully protect the population from the alpha (B.1.1.7) and beta (B.1.351) SARS-CoV-2 mutations. The steric factors (Ps) obtained for all three types of spike proteins showed a 100-fold lower requirement for the formation of an immune complex when compared with nucleocapsid protein.

On Computing Equilibrium Binding Constants for Protein-Protein Association in Membranes.

Protein association in lipid membranes is fundamental to membrane protein function and of great biomedical relevance. All-atom and coarse-grained models have been extensively used to understand the protein-protein interactions in the membrane and to compute equilibrium association constants. However, slow translational and rotational diffusion of protein in membrane presents challenges to the effective sampling of conformations defining the ensembles of free and bound states contributing to the association equilibrium and the free energy of dimerization. We revisit the homodimerization equilibrium of the TM region of glycophorin A. Conformational sampling is performed using umbrella sampling along previously proposed one-dimensional collective variables and compared with sampling over a two-dimensional collective variable space using the MARTINI v2.2 force field. We demonstrate that the one-dimensional collective variables suffer from restricted sampling of the native homodimer conformations leading to a biased free energy landscape. Conversely, simulations along the two-dimensional collective variable effectively characterize the thermodynamically relevant native and non-native interactions contributing to the association equilibrium. These results demonstrate the challenges associated with accurately characterizing binding equilibria when multiple poses contribute to the bound state ensemble.

A strategy for determining the equilibrium constants for heteromeric ion channels in a complex model.

Ligand-gated ion channels are oligomers containing several binding sites for the ligands. However, the signal transmission from the ligand binding site to the pore has not yet been fully elucidated for any of these channels. In heteromeric channels, the situation is even more complex than in homomeric channels. Using published data for concatamers of heteromeric cyclic nucleotide-gated channels, we show that, on theoretical grounds, multiple functional parameters of the individual subunits can be determined with high precision. The main components of our strategy are (1) the generation of a defined subunit composition by concatenating multiple subunits, (2) the construction of 16 concatameric channels, which differ in systematically permutated binding sites, (3) the determination of respectively differing concentration-activation relationships, and (4) a complex global fit analysis with corresponding intimately coupled Markovian state models. The amount of constraints in this approach is exceedingly high. Furthermore, we propose a stochastic fit analysis with a scaled unitary start vector of identical elements to avoid any bias arising from a specific start vector. Our approach enabled us to determine 23 free parameters, including 4 equilibrium constants for the closed-open isomerizations, 4 disabling factors for the mutations of the different subunits, and 15 virtual equilibrium-association constants in the context of a 4-D hypercube. From the virtual equilibrium-association constants, we could determine 32 equilibrium-association constants of the subunits at different degrees of ligand binding. Our strategy can be generalized and is therefore adaptable to other ion channels.

Interaction of methylene blue with SDS in the premicellar solution of CPC in the aqueous and methanol-water system

Interactions between sodium dodecyl sulfate (SDS) and methylene blue (MB) have been studied in presence of cetylpyridinium chloride (CPC) in aqueous and methanol-water binary solvents via conductometric and tensiometric methods. The experiment was conducted to see the possibility of a synergistic effect of the SDS+CPC system and changes in the interactional behavior of SDS with MB in presence of CPC. The value of the molecular interaction parameter for micellization (βM) was found to be negative from the experimental results and the value of |βM| was found to be greater than ln(CMCSDS / CMCCPC), which didn’t describe synergism completely but indicated the strong striking interactions between the oppositely charged surfactants. However, the CMC of the SDS+CPC system was found to be very low as compared to the CMC of SDS alone. This result of CMC based on striking interactions between oppositely charged surfactants was used to calculate various parameters like Gibb’s free energy of micellization (∆Gmo), surface excess(Γmax), minimum surface area (Amin), surface pressure πCMC, standard free energy of adsorption (∆Gadso), pC20, minimum surface free energy (Gmins), packing parameter (P) and association equilibrium constant (K). The addition of methanol as a cosolvent enhances the CMC and α value of the SDS+CPC as well as the SDS+CPC-MB system. Correlation of ∆Gmo with different solvent parameters namely Gordon parameter (G), Reichardt's parameter (ET (30)), viscosity (ηo), and solvophobic parameter (SP) has also been evaluated.

Nanopore-Based Single-Entity Electrochemistry for the Label-Free Monitoring of Single-Molecule Glycoprotein-Boronate Affinity Interaction and Its Sensing Application.

Nanopipettes provide a promising confined space that enables advances in single-molecule analysis, and their unique conical tubular structure is also suitable for single-cell analysis. In this work, functionalized-nanopore-based single-entity electrochemistry (SEE) analysis tools were developed for the label-free monitoring of single-molecule glycoprotein-boronate affinity interaction for the first time, and immunoglobulin G (IgG, one of the important biomarkers for many diseases such as COVID-19 and cancers) was employed as the model glycoprotein. The principle of this method is based on a single glycoprotein molecule passing through 4-mercaptophenylboronic acid (4-MPBA)-modified nanopipettes under a bias voltage and in the meantime interacting with the boronate group from modified 4-MPBA. This translocation and affinity interaction process can generate distinguishable current blockade signals. Based on the statistical analysis of these signals, the equilibrium association constant (κa) of single-molecule glycoprotein-boronate affinity interaction was obtained. The results show that the κa of IgG in the confined nanopore at the single-molecule level is much larger than that measured in the open system at the ensemble level, which is possibly due to the enhanced multivalent synergistic binding in the restricted space. Moreover, the functionalized-nanopore-based SEE analysis tools were further applied for the label-free detection of IgG, and the results indicate that our method has potential application value for the detection of glycoproteins in real samples, which also paves way for the single-cell analysis of glycoproteins.

Method of kinetic characterization of immunoreagents for development of express high-sensitive assays for detection of ochratoxin A and heart fatty acids binding protein

Development of rapid and sensitive immunoassays is a task of great importance in a variety of fields ranging from clinical practice and urgent diagnostics to food quality control and environmental monitoring. High attention of researches is paid to methods of screening, selection, and kinetic characterization of antibodies that enable fast, specific, and effective formation of immunocomplexes. Herein, we present a method for direct investigation of kinetics of immunoreagents during developments of express high sensitive lateral flow assays. As model biomolecules to be detected, the following substances were tested: ochratoxin A (OTA), which is one of the most dangerous mycotoxins naturally present in many vegetable raw materials; and heart fatty acids binding protein (hFABP), which is a cardiac marker used in differential diagnosis of acute myocardial infarction. The kinetic constants of association (kon) and dissociation (koff) with monoclonal antibodies are determined along with the corresponding equilibrium constants (KA and KD). The obtained values are as follows: for the anti-OTA antibodies – kon = 4.54*103 M−1s−1; koff = 3.32*10−4 s−1; KA = 1.37*107 M−1; KD = 7.31*10−8 M; and for the anti-hFABP antibodies – kon = 7.28*103 M−1s−1; koff = 1.97*10−4 s−1; KA = 3.70*107 M−1; KD = 2.70*10−8 M. The proposed method can be employed in combination with the immunochromatographic assays based on magnetic biolabels.•Investigation of immunoreagent kinetics for development of express high sensitive lateral flow assays•Kinetic characterization of monoclonal antibodies against OTA and hFABP for their rapid and sensitive detection•Both kinetic and equilibrium constants of association and dissociation are determined

Association of rhodamine cations with tetraphenylborate-anion in aqueous solution: structural properties of ions and stability of associates

The ability of rhodamine cations to form cation-anionic associates with tetraphenylborate-anion in an aqueous solution has been studied. The thermodynamic values of the equilibrium association constants Kasт were determined spectrophotometrically. The values of a number of properties of dye molecules (surface area, effective volume, hydrophobicity index log P, standard enthalpy of hydration ΔНohydr) were calculated using the QSAR model. The possibility of dependence of the value of Kasт on these factors has been reviewed. It was shown that changes in lg Kasт in the series of rhodamine associates are consistent with changes in the parameters log P and ΔНohydr.

Evaluation of microcolumn stability in ultrafast affinity extraction for binding and rate studies

Ultrafast affinity extraction (UAE) has recently been developed and employed for measuring non-bound (or free) fractions and binding or rate constants for drugs and other targets with soluble binding agents such as serum proteins. This study examined the long-term stability of 10 mm × 2.1 mm i.d. affinity microcolumns when used in UAE at both low and high flow rates (e.g., 0.5 and 3.5 mL/min) over an extended series of injections. This stability was investigated by using immobilized human serum albumin (HSA) and samples containing the drug warfarin with or without soluble HSA as a model system. The free warfarin fractions measured at 0.5 mL/min in the presence of soluble HSA were stable up to 150 injections and changed by <10% at 3.5 mL/min. The association equilibrium constant for warfarin with HSA that was estimated by UAE at 3.5 mL/min had no significant change over 50 injections and a change of only ∼18–22% over 100–150 injections. The dissociation rate constant for warfarin from HSA was found by combining UAE results at 0.5 and 3.5 mL/min and employing a new two-point approach, with no significant changes in this value being seen even after 200 injections. The effects of extended microcolumn use on the retention time, peak width, and peak asymmetry for warfarin, and on the backpressure of the microcolumn, were also considered. These results indicated that UAE and HSA microcolumns could be used to provide consistent values for free solute fractions, binding constants, and rate constants over a large series of injections. These results should be useful in future work by providing guidelines for the assessment, further development, and use of UAE in characterizing interactions involving other drugs and binding agents in solution-based samples.

Elucidation of specific binding sites and extraction of toxic Gen X from HSA employing cyclodextrin

The presence of per and poly-fluoroalkyl substances (PFAS), commonly referred to as forever chemicals, in aquatic systems is a serious global health problem. While the remediation of PFAS from aqueous media has been extensively investigated, their interactions with and removal from biological systems have received far less attention. We report herein structural alterations to human serum albumin (HSA) upon addition of perfluoro(2-methyl-3-oxahexanoic) acid (Gen X) monitored by changes to the fluorescence and circular dichroism (CD) spectra of HSA. The equilibrium association constant for Gen X binding to HSA is 7( ± 1) × 103 M−1 determined from changes in HSA fluorescence emission data during titration. Site-specific HSA binding fluorophores, 8-anilinonaphthalene-1-sulfonic acid (1,8-ANS), warfarin and dansyl-L-proline were used to investigate the specific binding sites of Gen X on HSA. A competitive displacement study yields association constants for Gen X to HSA at the 1,8-ANS, warfarin, and dansyl-L-proline binding sites to be 6.25 ( ± 0.5) × 104 M−1, 1.1 × 106 M−1, and 2.5( ± 0.2) × 109 M−1 respectively. Addition of β-cyclodextrin (β-CD) and heptakis(6-deoxy-6-amino)-β-cyclodextrin heptahydrochloride to the HSA:Gen X complex leads to the effective extraction of Gen X from the complex with the return of HSA in its native form. Gen X also leads to displacement of site-specific binding fluorophores bound to HSA, while subsequent addition of β-CD extracts Gen X from HSA with the return of the characteristic fluorescence of the HSA bound site-specific agent. These results illustrate the strong and specific binding sites of Gen X on HSA and demonstrate the principles for the potential application of β-CD for the remediation of PFAS from biological systems.

Cucurbituril-mediated AIE: An unconventional indicator displacement assay for ketamine detection

Typical AIE luminogens with extensive π-electron conjugation are limited in use on account of their complex synthesis, high cost and environmental concerns. To this end, we report here a novel host-guest inclusion complex that is assembled from nonemissive ligands, and surprisingly exhibits notable AIE features in aqueous solution while maintaining good sensitivity for ketamine (KET) detection. Isothermal titration studies confirm that palmatine (PAL) binds to cucurbit[8]uril (Q8) in a 2:1 ratio with an equilibrium association constant of 1.75 × 1011 M−2, suggesting that the unconventional AIE effects can be attributed to the stacking of two PAL moieties inside a single Q8 cavity. Geometric constraints of the macrocycle result in dimeric coupling of PAL in the ground state with significant bathochromic shifts in absorption and emission, which can be readily excited to exhibit enhanced photoluminescence. The unusual upfield perturbation and broadening of Q8 signals in the 1H NMR spectrum upon binding with PAL convey decisive information that Q8 not only serves as a molecular container, but also participates in the electron delocalization during the AIE events. Upon addition of KET, fluorescence was switched off through guest exchange to generate KET@Q8 as well as free PAL. The significant AIE characteristics of 2*PAL@Q8 in combination with its specific molecular recognition towards KET gives rise to 5.0 ng/mL limit of detection with linear range from 16.8 to 500 ng/mL, and a pronounced selectivity over possible interfering substances in biological matrices. We demonstrate the first example of Q8-mediated AIE effects, where the photoluminescence of virtually nonemissive or feebly emissive ligands is remarkably intensified through clustering inside a macrocyclic host molecule, therefore offering great promise for further development into dedicated AIE-based sensors.

The Kiselev isotherm for adsorption at the liquid-solid interface: Solving the mystery of negative equilibrium constants

Equilibrium data for aqueous adsorption systems are often correlated using isotherm models originally developed for adsorption at the gas-solid interface. A prominent example is the Langmuir isotherm, which has been used in thousands of investigations to describe the adsorption of dissolved substances at the liquid-solid interface. Another example is the Kiselev isotherm, which was proposed to account for adsorption at the gas-solid interface when adsorbate-adsorbate interactions are significant. Researchers who have applied the Kiselev isotherm to adsorption data for liquid-solid systems were puzzled by the fact that data fittings in most cases returned negative values for the adsorbate-adsorbate association equilibrium constant. It is shown in the present study that the data fitting problem is a direct consequence of misusing the Kiselev isotherm. In all previous studies, the Kiselev isotherm was used to fit type I isotherm data. Because the concept of adsorbate-adsorbate interactions is incompatible with a type I isotherm, fitting the Kiselev isotherm to such data can lead to nonsensical parameter estimates. The Kiselev isotherm was devised to account for type V isotherms exhibiting a sigmoid or S-shaped data trend. Physically meaningful parameter estimates can be obtained if the Kiselev isotherm is applied to type V isotherm data.

Strontium Binding to α-Parvalbumin, a Canonical Calcium-Binding Protein of the “EF-Hand” Family

Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr2+ interference with Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA (‘CD’ and ‘EF’ sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M−1 and 4 × 109 M−1 for Ca2+, and 2 × 107 M−1 and 2 × 106 M−1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M−1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO− groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.

Moment Theory of Chromatography for the Analysis of Reaction Kinetics of Intermolecular Interactions.

Moment theory was applied to the kinetic study of intermolecular interactions. The association equilibrium constant (KA) and association (ka) and dissociation (kd) rate constants of chemical reactions were analytically determined on the basis of the moment theory from elution peak profiles measured by high-performance liquid chromatography (HPLC). The HPLC data were measured under the conditions that neither immobilization nor fluorescence labeling of solute and ligand molecules is required. These are the advantages of the moment analysis method for determining accurate values of KA, ka, and kd. Moment equations were developed on the basis of the Einstein equation for diffusion, the random walk model, and the general rate model of chromatography. The moment analysis method was applied to the inclusion complex formation system between dibenzo-18-crown-6 or dibenzo-15-crown-5 and alkali metal cations. It was demonstrated that the values of KA, ka, and kd can be determined on the assumption that the stoichiometry between crown ethers and cations is 1:1 or 2:1. The influence of the difference in the size between the inner cavity of crown ethers and cations on the association and dissociation of the inclusion complex was considered. The moment analysis method using HPLC is effective for analyzing intermolecular interactions from various perspectives because it is based on the separation technique and has different characteristics from other methods such as spectroscopy. The results of this study contribute to the dissemination of an opportunity for studying intermolecular interactions from equilibrium and kinetic points of view to many researchers because HPLC is widespread.

Anticancer activity and cell death mechanism of Pt(II) complexes: Their in vitro bio-transformation to Pt(II)-DNA adduct formation and BSA binding study by spectroscopic method

Pt(II) complex cis-[Pt(PEA)(OH2)2] X2, C-2 (where, PEA = 2-Pyridylethylamine and X = ClO4− or NO3–) was synthesized by hydrolysis of cis-[Pt(PEA)Cl2] C-1. Glutathione (GSH) and DL-penicilamine (DL-pen) substituted complexes cis-[Pt(PEA)(GSH)],C-3 and cis-[Pt(PEA)DL-pen)]X C-4 were synthesized and characterized by spectroscopic methods. Kinetic studies were traced on complex C-2 with the thiols, GSH and DL-pen. Pt(II)-Sulfur adduct formation mechanisms of the substituted products C-3 and C-4 were established from the kinetic investigation. At pH 4.0, C-2 - thiols interactions follow two consecutive steps: the first step is dependent, and the second is independent of [thiol]. The association equilibrium constant (KE), substitution rate constants for both steps (k1 & k2), and activation parameters (ΔH‡ and ΔS‡) have been assessed to propose the mechanism. Agarose gel electrophoresis mobilization pattern of DNA with complexes was performed to visualize the interaction nature. CT-DNA and BSA binding activities of the complexes have been executed by electronic, fluorescence spectroscopy, and viscometric titration methods. Evaluation of thermodynamic parameters (ΔH0, ΔS0, and ΔG0) from BSA binding constants was executed to propose the driving forces of interaction between these species. A molecular docking study was performed to evaluate the binding mode of complexes with BDNA strands. Anticancer activity of the complexes C-1 to C-4 was explored on both A549 and HEp-2 cell lines, compared with approved anticancer drugs cisplatin, carboplatin, and oxaliplatin. All these complexes were tested by NBT assay on normal cell line skeletal muscle cells (L6 myotubes) to observe the adverse effects compared to recognized anticancer medications. The ultimate aim is to explore the role of anticancer agents on cell death mechanism, which has been performed by flow-cytometer on HEp-2 cell lines.

High-Performance affinity chromatographic studies of repaglinide and nateglinide interactions with normal and glyoxal- or methylglyoxal-modified human albumin serum

During diabetes human serum albumin (HSA), an important drug transport protein, can be modified by agents such as glyoxal (Go) and methylglyoxal (MGo) to form advanced glycation end-products. High-performance affinity microcolumns and zonal elution competition studies were used to compare interactions by the anti-diabetic drugs repaglinide and nateglinide with normal and Go- or MGo-modified HSA at Sudlow sites I and II of this protein. Both drugs had their strongest binding at Sudlow site II for the normal and modified forms of HSA. The association equilibrium constants at this site for repaglinide and nateglinide with normal HSA were 6.1 (± 0.2) × 104 M−1 and 7.1 (± 0.8) × 105 M−1, respectively, at pH 7.4 and 37⁰C; these values increased by up to 3.6-fold for repaglinide and decreased by up to 45–55 % for nateglinide when HSA was modified by Go or MGo at levels seen in prediabetes or diabetes. Both drugs were also found to bind at Sudlow site I, with association equilibrium constants at this site on normal HSA of 4.2 (± 0.3) × 104 M−1 for repaglinide and 5.0 (± 0.1) × 104 M−1 for nateglinide. The binding strength for repaglinide at Sudlow site I increased by 1.3- to 1.7-fold with the Go-modified HSA and decreased slightly (i.e., up to 19 %) for the MGo-modified HSA, while nateglinide showed only a small or insignificant change in binding with the same modified HSA samples. These results indicated that binding by repaglinide and nateglinide with HSA can be altered significantly by modification of this protein with Go or MGo, making these modifications of potential interest in the treatment of patients with these drugs during diabetes.

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.