Determination Of Dissociation Constants Research Articles

Determination of chromatographic and spectrophotometric dissociation constants of some beta lactam antibiotics

In this work, dissociation constants values of seven beta lactam antibiotics in water and acetonitrile-water mixtures using spectrophotometric and reversed phase liquid chromatography methods were determined. The dissociation constant values of these compounds were calculated by NLREG and STAR programs. Aqueous pK(a) values of beta lactam antibiotics were calculated with extrapolation by means of the Yasuda-Shedlovsky and mole fraction equations. Finally, application of the different techniques was compared to the determination of aqueous pK(a) values of investigated compounds.

Protein interaction affinity determination by quantitative FRET technology

The dissociation constant, K(d) , is an important parameter for characterizing protein-protein interaction affinities. SUMOylation is one of the important protein post-translational modifications and it involves a multi-step enzymatic cascade reaction, resulting in peptide activation and substrate conjugation. Multiple covalent and non-covalent protein-protein interactions are involved in this cascade. Techniques involving Förster resonance energy transfer (FRET) have been widely used in biological studies in vitro and in vivo, and they are very powerful tools for elucidating protein interactions in many regulatory cascades. In our previous studies, we reported the attempt to develop a new method for the determination of the K(d) by FRET assay using the interaction of SUMO1 and its E2 ligase, Ubc9 as a test system. However, the generality and specifications of this new method have not been fully determined. Here we report a systematic approach for determining the dissociation constant (K(d) ) in the SUMOylation cascade and for further sensitivity and accuracy testing by the FRET technology. From a FRET donor to acceptor concentration ratio range of 4-40, the K(d) s of SUMO1 and Ubc9 consistently agree well with values from surface plasmon resonance and isothermal titration calorimetry. These results demonstrate the high sensitivity and accuracy of the FRET-based K(d) determination approach. This technology, therefore, can be used in general for protein-protein interaction dissociation constant determination.

Effect of pH on the Photophysical and Redox Properties of a Ruthenium(II) Mixed Chelate Derived from Imidazole-4,5-dicarboxylic Acid and 2,2′-Bipyridine: An Experimental and Theoretical Investigation

Combined experimental and DFT-TD-DFT computational studies were utilized to investigate the structural and electronic properties of mixed-ligand monometallic ruthenium(II) complexes of compositions [(bpy)(2)Ru(H(2)Imdc)](+) (1(+)), its N-H deprotonated form [(bpy)(2)Ru(HImdc)] (1), and COOH deprotonated form [(bpy)(2)Ru(Imdc)](-) (1(-)), where H(3)Imdc = imidazole-4,5-dicarboxylic acid and bpy = 2,2'-bipyridine. The optimized geometrical parameters for the complexes computed both in the gas phase and in solution are reported and compared with the previously reported X-ray data. The influence of pH on the absorption, emission, and redox properties of [(bpy)(2)Ru(H(2)Imdc)](+) (1(+)) has been thoroughly investigated. The absorption titration data were used to determine the ground state pK values, whereas the luminescence data were utilized for the determination of excited state acid dissociation constants. The proton-coupled redox activity of 1(+) has been studied over the pH range 2-12 in acetonitrile-water (3:2). From the E(1/2) versus pH profile, the equilibrium constants of the variously deprotonated complex species in Ru(II) and Ru(III) oxidation states have been determined. As compared to the protonated complex (1(+)), which undergoes reversible oxidation at 0.96 V (vs Ag/AgCl) in acetonitrile, the redox potential of the fully deprotonated complex (1(-)) is shifted to a much lower value, viz., 0.52 V. Density functional theory (DFT) and time-dependent DFT (TD-DFT) study provides insight into the nature of the ground and excited states with resulting detailed assignments of the orbitals involved in absorption and emission transitions. In particular, the red-shifts of the absorption and emission bands and the cathodic shift in the oxidation potential of 1(+) compared to 1 and 1(-) are also reproduced by our calculations.

Increased precision for analysis of protein–ligand dissociation constants determined from chemical shift titrations

NMR is ideally suited for the analysis of protein-protein and protein ligand interactions with dissociation constants ranging from ~2 μM to ~1 mM, and with kinetics in the fast exchange regime on the NMR timescale. For the determination of dissociation constants (K ( D )) of 1:1 protein-protein or protein-ligand interactions using NMR, the protein and ligand concentrations must necessarily be similar in magnitude to the K ( D ), and nonlinear least squares analysis of chemical shift changes as a function of ligand concentration is employed to determine estimates for the parameters K ( D ) and the maximum chemical shift change (Δδ(max)). During a typical NMR titration, the initial protein concentration, [P (0)], is held nearly constant. For this condition, to determine the most accurate parameters for K ( D ) and Δδ(max) from nonlinear least squares analyses requires initial protein concentrations that are ~0.5 × K ( D ), and a maximum concentration for the ligand, or titrant, of ~10 × [P (0)]. From a practical standpoint, these requirements are often difficult to achieve. Using Monte Carlo simulations, we demonstrate that co-variation of the ligand and protein concentrations during a titration leads to an increase in the precision of the fitted K ( D ) and Δδ(max) values when [P (0)] > K ( D ). Importantly, judicious choice of protein and ligand concentrations for a given NMR titration, combined with nonlinear least squares analyses using two independent variables (ligand and protein concentrations) and two parameters (K ( D ) and Δδ(max)) is a straightforward approach to increasing the accuracy of measured dissociation constants for 1:1 protein-ligand interactions.

Arginyl-tRNA Synthetase Facilitates Complex Formation Between Seryl-tRNA Synthetase and its Cognate Transfer RNA

Several studies have revealed the involvement of multi aminoacyl-tRNA synthetase complexes (MSC) in archaeal and eukaryotic translation. Here we analyzed interactions of atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS), transfer RNA (tRNASer) and arginyl-tRNA synthetase (ArgRS). Surface plasmon resonance (SPR) was used to determine dissociation constants for the MtSerRS:tRNASer complex and the results were consistent with cooperative binding of tRNASer. This finding was supported by the ability of MtSerRS to bind two tRNAs in gel mobility shift assay. Notably, the MtSerRS:tRNASer complex formation was stimulated by MtArgRS, previously determined interacting partner of MtSerRS. MtArgRS decreases Kd for MtSerRS:tRNASer two-fold, but does not affect cooperative properties or stoichiometry of the complex. Further investigation of complex formation between MtSerRS and tRNASer showed that this molecular interaction is salt-dependent. The most pronounced improvements in binding were determined at high ionic strength, using Tris as a buffering agent, while the addition of Mg2+ ions led to the same SPR response.

Molecular Interactions between Glycopeptide Vancomycin and Bacterial Cell Wall Peptide Analogues

The molecular interactions of the glycopeptide antibiotic vancomycin (Van) with bacterial cell wall analogues N,N'-diacetyl-L-Lys-D-Ala-D-Ala (Ac(2) KdAdA) and N,N'-diacetyl-L-Lys-D-Ala-D-Lac (Ac(2) KdAdL) were investigated in neat water, phosphate buffer and HEPES buffer by using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. The FCS determined dissociation constants (k(d)) show that the intrinsic binding affinity between Van and the drug-sensitive peptide ligand Ac(2)KdAdA remains invariant when the solvent is changed from neat water to either PBS or HEPES buffer; this demonstrates that there are no obvious solvent effects on the association between Van and Ac(2)KdAdA due to the strong intermolecular interaction between the two moieties. When compared to Ac(2)KdAdA, a significantly larger k(d) value was observed for the binding between the drug-resistant peptide ligand Ac(2)KdAdL and Van. Furthermore, the k(d) increased by about 8- to 11-times when the solvent was changed from neat water to 10 mM phosphate/HEPES buffer. The stability of the Ac(2)KdAdL-Van complex was dependent on the concentration of the buffer and k(d) increases as the concentration of either phosphate ions or HEPES increased until an equilibrium was attained. Both FCS and MD simulation studies clearly showed that the components constituting the buffer solution (e.g., phosphate ions and HEPES) are involved in molecular interactions with the binding pocket of Van and they profoundly affect the intrinsic stability of the complex formed between the low-affinity Ac(2)KdAdL and Van. These results could help us to better understand the detailed structure and activity of glycopeptide antibiotic derivatives toward bacterial cell wall peptide analogues, and can further facilitate the development of new drug candidates against drug-resistant bacterial strains.

‘Null Method’ Determination of Drug Biophase Concentration

PK/PD modeling is enhanced by improvements in the accuracy of its metrics. For PK/PD modeling of drugs and biologics that interact with enzymes or receptors, the equilibrium constant of the interaction can provide critical insight. Methodologies such as radioliogand binding and isolated tissue preparations can provide estimates of the equilibrium constants (as the dissociation constant, K value) for drugs and endogenous ligands that interact with specific enzymes and receptors. However, an impediment to further precision for PK/PD modeling is that it remains a problem to convert the concentration of drug in bulk solution (A) into an estimate of receptor occupation, since A is not necessarily the concentration (C) of drug in the biophase that yields fractional binding from the law of mass action, viz., C/(C + K). In most experimental studies A is much larger than K, so the use of administered instead of biophase concentration gives fractional occupancies very close to unity. We here provide a simple way to obtain an estimate of the factor that converts the total drug concentration into the biophase concentration in isolated tissue preparation. Our approach is an extension of the now classic 'null method' introduced and applied by Furchgott to determination of drug-receptor dissociation constants.

Crystal Structure of the Ubiquitin-associated (UBA) Domain of p62 and Its Interaction with Ubiquitin

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.

Site-directed mutagenesis of human cytosolic sulfotransferase (SULT) 2B1b to phospho-mimetic Ser348Asp results in an isoform with increased catalytic activity

Human SULT2B1b is distinct from other SULT isoforms due to the presence of unique amino (N)- and carboxy (C)-terminal peptides. Using site-directed mutagenesis, it was determined that phosphorylation of Ser348 was associated with nuclear localization. To investigate the effects of this phosphorylation of Ser348 on activity and cellular localization, an in silico molecular mimic was generated by mutating Ser348 to an Asp. The Asp residue mimics the shape and charge of a phospho-Ser and homology models of SULT2B1b-phospho-S348 and SULT2B1b-S348D suggest a similar significant structural rearrangement in the C-terminal peptide. To evaluate the functional consequences of this post-translational modification and predicted rearrangement, 6His-SULT2B1b-S348D was synthesized, expressed, purified and characterized. The 6His-SULT2B1b-S348D has a specific activity for DHEA sulfation ten-fold higher than recombinant 6His-SULT2B1b (209.6 and 21.8 pmol min −1 mg −1, respectively). Similar to native SULT2B1b, gel filtration chromatography showed SULT2B1b-S348D was enzymatically active as a homodimer. Stability assays comparing SULT2B1b and SUL2B1b-S348 demonstrated that SULT2B1b is 60% less thermostable than SULT2B1b-348D. The increased stability and sulfation activity allowed for better characterization of the sulfation kinetics for putative substrates as well as the determination of dissociation constants that were difficult to obtain with wild-type (WT) 6His-SULT2B1b. The K Ds for DHEA and PAPS binding to 6His-SULT2B1b-S348D were 650 ± 7 nM and 265 ± 4 nM, respectively, whereas K Ds for binding of substrates to the WT enzyme could not be determined. Characterization of the molecular mimic SULT2B1b-S348D provides a better understanding for the role of the unique structure of SULT2B1b and its effect on sulfation activity, and has allowed for improved kinetic characterization of the SULT2B1b enzyme.

Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument

Several methods for characterizing DNA-protein interactions are available, but none have demonstrated both high throughput and quantitative measurement of affinity. Here we describe 'high-throughput sequencing'-'fluorescent ligand interaction profiling' (HiTS-FLIP), a technique for measuring quantitative protein-DNA binding affinity at unprecedented depth. In this approach, the optics built into a high-throughput sequencer are used to visualize in vitro binding of a protein to sequenced DNA in a flow cell. Application of HiTS-FLIP to the protein Gcn4 (Gcn4p), the master regulator of the yeast amino acid starvation response, yielded ~440 million binding measurements, enabling determination of dissociation constants for all 12-mer sequences having submicromolar affinity. These data revealed a complex interdependency between motif positions, allowed improved discrimination of in vivo Gcn4p binding sites and regulatory targets relative to previous methods and showed that sets of genes with different promoter affinities to Gcn4p have distinct functions and expression kinetics. Broad application of this approach should increase understanding of the interactions that drive transcription.

Determination of microscopic dissociation constants of 3-hydroxy-α-(methylamino)methyl-benzenemethanol by a spectral deconvolution method

Microscopic dissociation constants of 3-hydroxy-alpha-(methylamino)methyl-benzenemethanol have been calculated from the titration spectrophotomeric data (c = 3.8 x 10(-4) M. Ionic strength = 0.16; buffer system: H3BO3/KOH) by application of a spectral deconvolution method. The results found (pKa = 9.48; pKb = 9.71; pKc = 10.12 and pKd = 9.88) are in good concordance with those obtained from the conventional regression linear method (pKa = 9.45; pKb = 9.77; pKc = 10.14 and pKd = 9.81).

Determination of dissociation constants of aristolochic acid I and II by capillary electrophoresis with carboxymethyl chitosan-coated capillary

Aristolochic acid-I and aristolochic acid-II have been proved to be the main bioactive and toxic component in Aristolochia plants. As a result, the determination of their dissociation constants, which are important property parameters for weak acids, is highly desired for related pharmacological and toxicological studies. In this work, the dissociation constant values of aristolochic acid-I and aristolochic acid-II were determined by capillary electrophoresis using carboxymethyl chitosan-coated capillary, based on their electrophoretic mobilities by using nonlinear regression as well as linear regression, showing that the two models give comparable results. The data were also compared with those obtained by capillary electrophoresis with polybrene-coated capillary, and no conspicuous difference was observed. The correlation coefficients were all higher than 0.998 for both linear and nonlinear regression model. The pKa values were found to be 3.3±0.1 for aristolochic acid-I and 3.2±0.1 for aristolochic acid-II.

Solvatochromaticity and pH dependence of the electronic absorption spectra of some purines and pyrimidines and their metal complexes

The solvatochromic responses of uric acid (Ua), 6-amino-2-thiouracil (ATU) and a series of their complexes dissolved in ten solvents of different polarity have been measured. The solvent-dependent UV/Vis spectroscopic absorption maxima, λ max, are assigned to the corresponding electronic transitions and analyzed using SPSS program, regression analysis and Kamlet and Taft methods. The observed solvatochromism is discussed using various solute–solvent interaction mechanisms. The electronic absorption spectra of ATU were investigated in aqueous buffer solutions of varying pH and utilized for the determination of dissociation constants. The ranges of pH, where individual ionic species are predominant have been determined.

Specific allowance for antibody bivalence in the determination of dissociation constants by kinetic exclusion assay

Theory that takes rigorous account of antibody bivalence in the characterization of immunospecific reactions by kinetic exclusion assay is presented. In addition to reinforcing the basic correctness of quantitative expressions currently being used for the determination of dissociation constants ( K d) by this method, the current study highlights a requirement for conformity of the system with critical assumptions/approximations therein. Published results for the interaction between the extracellular domain of human insulin-like growth factor (hIGFR) and anti-hIGFR are used to illustrate aspects of the theoretical predictions for a system to which those assumptions/approximations may well apply; and those for a cadmium–ethylenediaminetetraacetic acid (Cd–EDTA) antibody interaction to emphasize the consequences of adopting the same analytical procedure in a situation where one of those assumptions does not apply. The major weakness of current protocols for the characterization of antigen–antibody interactions by kinetic exclusion assay is an absence of any check on the likely magnitude of the probability of antibody capture by the affinity beads—a parameter that needs to be 5% or lower for validity of the quantitative expression on which the analysis is based.

Binding of d-mannose-containing glycoproteins to d-mannose-specific lectins studied by surface plasmon resonance

Binding of selected glycoproteins containing α- d-mannose sites (invertase, glucoamylase, transferrin, glucose oxidase, and albumin α- d-mannopyranosylphenyl isothiocyanate) with two lectins selective for α- d-mannose branching glycans, concanavalin A (ConA), and Lens culinaris agglutinin (LCA) was studied on lectin biochips by microfluidic surface plasmon resonance (SPR). Lectin-containing biochips were prepared by covalent immobilization of lectins on a flat carboxymethylated gold surface. Both measurement as well as regeneration conditions were optimized. The determined dissociation constants of lectin–glycoprotein interactions were 10 −5 to 10 −7 mol/l. Dissociation constants K D of studied bindings were estimated by the steady state specific binding models based on both a binding to one site and the multiple binding sites model using Hill slope.

Graphical determination of dissociation constant, pKa of non-polar amino acids

The dissociation constant (pKa) of non – polar amino acids including (alanine, glycine, valine phenylalanine and tryptophan) were determined by potentiometric titration technique. The pKa values obtained by extrapolation for alanine, glycine, and valine were 10.29, 9.87 and 9.91 respectively. The implications of the results are discussed and recommendations were made. Keywords: Dissociation constant, amino acids, non-polar, potentiometry.

Interaction of Escherichia coli RNA Polymerase σ70 Subunit with Promoter Elements in the Context of Free σ70, RNA Polymerase Holoenzyme, and the β′-σ70 Complex

Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ(70) from Escherichia coli, the β'-σ complex, and the σ(70) RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ(70), free or in the context of the holoenzyme, recognizes the -10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the -10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the -10 element. We further show that region 1 of σ(70) is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β' RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended -10 promoter element by σ(70). The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.

Development of FRET Assay into Quantitative and High-throughput Screening Technology Platforms for Protein–Protein Interactions

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein–protein interactions. Here we report the development of dissociation constant (K d) determination for protein–protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction K d determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The K d determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both K d determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications.

Physicochemical properties of hydroxylated polychlorinated biphenyls aid in predicting their interactions with rat sulfotransferase 1A1 (rSULT1A1)

Hydroxylated metabolites of polychlorinated biphenyls (OHPCBs) interact with rat sulfotransferase 1A1 (rSULT1A1) as substrates and inhibitors. Previous studies have shown that there are complex and incompletely understood structure–activity relationships governing the interaction of rSULT1A1 with these molecules. Furthermore, modification of the enzyme with glutathione disulfide (GSSG) results in the conversion of some OHPCBs from inhibitors to substrates. We have now examined estimated values for the acid-dissociation constant ( K a) and the octanol–water distribution coefficient ( D), as well as experimentally determined dissociation constants for enzyme complexes, to assist in the prediction of interactions of OHPCBs with rSULT1A1. Under reducing conditions, initial velocities for rSULT1A1-catalyzed sulfation exhibited a positive correlation with p K a and a negative correlation with log D of the OHPCBs. IC 50 values of inhibitory OHPCBs decreased with decreasing p K a values for both the glutathione (GSH)-pretreated and GSSG-pretreated forms of rSULT1A1. Comparison of GSH- and GSSG-pretreated forms of rSULT1A1 with respect to binding of OHPCB in the presence and absence of adenosine 3′,5′-diphosphate (PAP) revealed that the dissociation constants with the two redox states of the enzyme were similar for each OHPCB. Thus, p K a and log D values are useful in predicting the binding of OHPCBs to the two redox forms of rSULT1A1 as well as the rates of sulfation of those OHPCBs that are substrates. However, the differences in substrate specificity for OHPCBs that are seen with changes in redox status of the enzyme are not directly related to specific structural effects of individual OHPCBs within inhibitory enzyme–PAP–OHPCB complexes.

Using a Genetically Encoded Fluorescent Amino Acid as a Site-Specific Probe to Detect Binding of Low-Molecular-Weight Compounds

Development of enzyme inhibitors requires an activity assay for the identification of hits and lead compounds. To determine dissociation constants in a straightforward manner, we explored the use of a genetically encoded fluorescent amino acid for site-specific tagging of the target protein. The unnatural amino acid 7-(hydroxy-coumarin-4-yl) ethylglycine (Hco) was site-specifically incorporated in the target protein by cell-free protein synthesis using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair. Using the West Nile virus nonstructural protein 2B-nonstructural protein 3 protease as the target protein, the fluorescence of Hco-tagged samples proved to be exquisitely sensitive to the presence of inhibitors and small ligand molecules if they bind in the vicinity of the Hco residue. No significant change in fluorescence was observed when the ligand-binding site was far from the Hco residue. Hco-tagged proteins thus combine outstanding sensitivity with accurate information on the site of binding, making Hco labeling an attractive tool in drug discovery.