Ap4A is not an efficient Zn(II) binding agent. A concerted potentiometric, calorimetric and NMR study.

PubMed

Wszelaka-Rylik, Małgorzata; Witkiewicz-Kucharczyk, Aleksandra; Wójcik, Jacek; Bal, Wojciech

2007-05-01

Diadenosine 5',5''-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo.

Kinetics and equilibria of cyanide binding to prostaglandin H synthase.

PubMed

MacDonald, I D; Dunford, H B

1989-09-01

Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.

Spectrophotometric study on binding of 2-thioxanthone acetic acid with ct-DNA.

PubMed

Ataci, Nese; Ozcelik, Elif; Arsu, Nergis

2018-06-02

Thioxanthone and its derivatives are the most remarkable molecules due to their vast variety of application such as radiation curing that is, until using them as a therapeutic drug. Therefore, in this study it was intended to use 2-Thioxanthone acetic acid with and without NaCl in Tris HCl buffer solution (pH:7.0) to represent the interaction with ct-DNA. The UV-vis absorption spectra of TXCH 2 COOH in the presence of ct-DNA showed hypochromism and the intrinstic binding constant (K b ) was determined as 6 × 10 3  L mol -1 . The fluoresence intensity of TXCH 2 COOH with ct-DNA clearly increased up to 101% which indicated that the fluorescence intensity was very sensitive to ct-DNA concentration. The binding constant (K) and the values of number of binding sites (n) and were calculated as 1.8 × 10 3  L mol -1 and 0.69, respectively. When the quenching constants (K sv ) of free TXCH 2 COOH and TXCH 2 COOH, which were bonded with ct-DNA were compared, slightly changed values of Ksv were seen. Moreover, displacement assay with Hoechst 33,258 and viscosity measurements in the presence and absence of NaCl salt also confirmed the binding mode which noted the electrostatic interaction following groove binding between TXCH 2 COOH and ct-DNA. Last but not least, the salt effect was examined on ct-DNA binding with TXCH 2 COOH. The results of the experiments indicated that the groove binding was strengthened by NaCl whereas in the high NaCl concentration, the binding ability of TXCH 2 COOH to ct-DNA was inversely affected. Copyright © 2018 Elsevier B.V. All rights reserved.

Proton and metal ion binding to natural organic polyelectrolytes-I. Studies with synthetic model compounds

USGS Publications Warehouse

Marinsky, J.A.; Reddy, M.M.

1984-01-01

A unified physico-chemical model, based on a modified Henderson-Hasselbalch equation, for the analysis of ion complexation reactions involving charged polymeric systems is presented and verified. In this model pH = pKa+p(??Ka) + log(??/1 - ??) where Ka is the intrinsic acid dissociation constant of the ionizable functional groups on the polymer, ??Ka is the deviation of the intrinsic constant due to electrostatic interaction between the hydrogen ion and the polyanion, and alpha (??) is the polyacid degree of ionization. Using this approach pKa values for repeating acidic units of polyacrylic (PAA) and polymethacrylic (PMA) acids were found to be 4.25 ?? 0.03 and 4.8 ?? 0.1, respectively. The polyion electrostatic deviation term derived from the potentiometric titration data (i.e. p(??Ka)) is used to calculate metal ion concentration at the complexation site on the surface of the polyanion. Intrinsic cobalt-polycarboxylate binding constants (7.5 for PAA and 5.6 for PMA), obtained using this procedure, are consistent with the range of published binding constants for cobalt-monomer carboxylate complexes. In two phase systems incorporation of a Donnan membrane potential term allows determination of the intrinsic pKa of a cross-linked PMA gel, pKa = 4.83, in excellent agreement with the value obtained for the linear polyelectrolyte and the monomer. Similarly, the intrinsic stability constant for cobalt ion binding to a PMA-gel (??CoPMA+ = 11) was found to be in agreement with the linear polyelectrolyte analogue and the published data for cobalt-carboxylate monodentate complexes. ?? 1984.

Binding site and affinity prediction of general anesthetics to protein targets using docking.

PubMed

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G

2012-05-01

The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the extracellular domain of GLIC. The predicted affinities correlated significantly with the known EC(50) values for the 6 frequently used anesthetics in GLIC for the site identified in the experimental crystal data (P = 0.006). However, predicted affinities in apoferritin, human serum albumin, and cytochrome C did not correlate with these 6 anesthetics' known experimental EC(50) values. A weak correlation between the predicted affinities and the octanol/water partition coefficients was observed for the sites in GLIC. We demonstrated that anesthetic binding sites and relative affinities can be predicted using docking calculations in an automatic docking server (AutoDock) for both water-soluble and membrane proteins. Correlation of predicted affinity and EC(50) for 6 frequently used general anesthetics was only observed in GLIC, a member of a protein family relevant to anesthetic mechanism.

Tc-99m galactosyl-neoglycoalbumin: in vitro characterization of receptor-mediated binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vera, D.R.; Krohn, K.A.; Stadalnik, R.C.

1984-07-01

Hepatic binding protein (HBP) is a membrane receptor that binds and transports plasma glycoproteins from hepatic blood to hepatocellular lysosomes. A characterization is made of the in vitro binding of Tc-99m galactosyl-neoglycoalbumin (Tc-NGA), a synthetic HBP ligand, to liver membrane. Structural modifications of NGA resulted in the alteration of the equilibrium constant, KA, and the forward-binding rate constant, kb. Binding was second-order; the relative amount of membrane-bound NGA depended on the initial concentrations of ligand and membrane. Membrane displacement studies, using carrier ligands in contrast to previously bound Tc-NGA or I-NGA, correlated with the binding characteristics of a native HBPmore » ligand, asialo-orosomucoid. Computer simulation was used to study the detectability of the changes in HBP concentration at different values of kb. The simulations indicated that radiopharmacokinetic sensitivity to alterations in (HBP) should be possible using a neoglycoalbumin preparation with a carbohydrate density within the range of 15 to 25 galactose units per albumin molecule.« less

Kinetic modeling of benzodiazepine receptor binding with PET and high specific activity [(11)C]Iomazenil in healthy human subjects.

PubMed

Bremner, J D; Horti, A; Staib, L H; Zea-Ponce, Y; Soufer, R; Charney, D S; Baldwin, R

2000-01-01

Quantitation of the PET benzodiazepine receptor antagonist, [(11)C]Iomazenil, using low specific activity radioligand was recently described. The purpose of this study was to quantitate benzodiazepine receptor binding in human subjects using PET and high specific activity [(11)C]Iomazenil. Six healthy human subjects underwent PET imaging following a bolus injection of high specific activity (>100 Ci/mmol) [(11)C]iomazenil. Arterial samples were collected at multiple time points after injection for measurement of unmetabolized total and nonprotein-bound parent compound in plasma. Time activity curves of radioligand concentration in brain and plasma were analyzed using two and three compartment model. Kinetic rate constants of transfer of radioligand between plasma, nonspecifically bound brain tissue, and specifically bound brain tissue compartments were fitted to the model. Values for fitted kinetic rate constants were used in the calculation of measures of benzodiazepine receptor binding, including binding potential (the ratio of receptor density to affinity), and product of BP and the fraction of free nonprotein-bound parent compound (V(3)'). Use of the three compartment model improved the goodness of fit in comparison to the two compartment model. Values for kinetic rate constants and measures of benzodiazepine receptor binding, including BP and V(3)', were similar to results obtained with the SPECT radioligand [(123)I]iomazenil, and a prior report with low specific activity [(11)C]Iomazenil. Kinetic modeling using the three compartment model with PET and high specific activity [(11)C]Iomazenil provides a reliable measure of benzodiazepine receptor binding. Synapse 35:68-77, 2000. Published 2000 Wiley-Liss, Inc.

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

PubMed

Rauf, Sakandar; Nawaz, Haq; Akhtar, Kalsoom; Ghauri, Muhammad A; Khalid, Ahmad M

2007-05-15

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: A comparative approach

NASA Astrophysics Data System (ADS)

Raman, N.; Sakthivel, A.; Pravin, N.

A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 102 to 105 indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.

A scale of metal ion binding strengths correlating with ionic charge, Pauling electronegativity, toxicity, and other physiological effects.

PubMed

Kinraide, Thomas B; Yermiyahu, Uri

2007-09-01

Equilibrium constants for binding to plant plasma membranes have been reported for several metal ions, based upon adsorption studies and zeta-potential measurements. LogK values for the ions are these: Al(3+), 4.30; La(3+), 3.34; Cu(2+), 2.60; Ca(2+) and Mg(2+), 1.48; Na(+) and K(+), 0 M(-1). These values correlate well with logK values for ion binding to many organic and inorganic ligands. LogK values for metal ion binding to 12 ligands were normalized and averaged to produce a scale for the binding of 49 ions. The scale correlates well with the values presented above (R(2)=0.998) and with ion binding to cell walls and other biomass. The scale is closely related to the charge (Z) and Pauling electronegativity (PE) of 48 ions (all but Hg(2+)); R(2)=0.969 for the equation (Scale values)=-1.68+Z(1.22+0.444PE). Minimum rhizotoxicity of metal ions appears to be determined by binding strengths: log a(PM,M)=1.60-2.41exp[0.238(Scale values)] determines the value of ion activities at the plasma membrane surface (a(PM,M)) that will ensure inhibition of root elongation. Additional toxicity appears to be related to softness, accounting for the great toxicity of Ag(+), for example. These binding-strength values correlate with additional physiological effects and are suitable for the computation of cell-surface electrical potentials.

Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall

PubMed Central

Thomas, Kieth J.; Rice, Charles V.

2014-01-01

Metals bind to the bacterial cell wall yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane (lipoteichoic acid) or covalently bound to the cell wall (wall teichoic acid). The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, contradicting previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger and previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, meaning that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 µmol/mg and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of there being a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent, suggesting a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations. PMID:25315444

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

PubMed Central

Land, B R; Harris, W V; Salpeter, E E; Salpeter, M M

1984-01-01

In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the falling phase of the MEPC. We find diffusion to be 4 X 10(-6) cm2 s-1, slightly slower than free diffusion, forward binding to be 3.3 X 10(7) M-1 s-1, and the distance from an average release site to the nearest exit from the cleft to be 1.6 micron. We also estimate the back reaction rates. From our values we can accurately describe the shape of MEPCs under different conditions of receptor and esterase concentration. Since we suggest that unbinding is slower than isomerization, we further predict that there should be several short "closing flickers" during the total open time for an AcCho-ligated receptor channel. PMID:6584895

Contacts between the factor TUF and RPG sequences.

PubMed

Vignais, M L; Huet, J; Buhler, J M; Sentenac, A

1990-08-25

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney.

PubMed

Gervais, A; Lane, L K; Anner, B M; Lindenmayer, G E; Schwartz, A

1977-01-01

Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+,K+-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.

Measuring Positive Cooperativity Using the Direct ESI-MS Assay. Cholera Toxin B Subunit Homopentamer Binding to GM1 Pentasaccharide

NASA Astrophysics Data System (ADS)

Lin, Hong; Kitova, Elena N.; Klassen, John S.

2014-01-01

Direct electrospray ionization mass spectrometry (ESI-MS) assay was used to investigate the stepwise binding of the GM1 pentasaccharide β- D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β- D-Gal p-(1→4)-β-D-Glc p (GM1os) to the cholera toxin B subunit homopentamer (CTB5) and to establish conclusively whether GM1os binding is cooperative. Apparent association constants were measured for the stepwise addition of one to five GM1os to CTB5 at pH 6.9 and 22 °C. The intrinsic association constant, which was established from the apparent association constant for the addition of a single GM1os to CTB5, was found to be (3.2 ± 0.2) × 106 M-1. This is in reasonable agreement with the reported value of (6.4 ± 0.3) × 106 M-1, which was measured at pH 7.4 and 25 °C using isothermal titration calorimetry (ITC). Analysis of the apparent association constants provides direct and unambiguous evidence that GM1os binding exhibits small positive cooperativity. Binding was found to be sensitive to the number of ligand-bound nearest neighbor subunits, with the affinities enhanced by a factor of 1.7 and 2.9 when binding occurs next to one or two ligand-bound subunits, respectively. These findings, which provide quantitative support for the binding model proposed by Homans and coworkers [14], highlight the unique strengths of the direct ESI-MS assay for measuring cooperative ligand binding.

Semi-empirical proton binding constants for natural organic matter

NASA Astrophysics Data System (ADS)

Matynia, Anthony; Lenoir, Thomas; Causse, Benjamin; Spadini, Lorenzo; Jacquet, Thierry; Manceau, Alain

2010-03-01

Average proton binding constants ( KH,i) for structure models of humic (HA) and fulvic (FA) acids were estimated semi-empirically by breaking down the macromolecules into reactive structural units (RSUs), and calculating KH,i values of the RSUs using linear free energy relationships (LFER) of Hammett. Predicted log KH,COOH and log KH,Ph-OH are 3.73 ± 0.13 and 9.83 ± 0.23 for HA, and 3.80 ± 0.20 and 9.87 ± 0.31 for FA. The predicted constants for phenolic-type sites (Ph-OH) are generally higher than those derived from potentiometric titrations, but the difference may not be significant in view of the considerable uncertainty of the acidity constants determined from acid-base measurements at high pH. The predicted constants for carboxylic-type sites agree well with titration data analyzed with Model VI (4.10 ± 0.16 for HA, 3.20 ± 0.13 for FA; Tipping, 1998), the Impermeable Sphere model (3.50-4.50 for HA; Avena et al., 1999), and the Stockholm Humic Model (4.10 ± 0.20 for HA, 3.50 ± 0.40 for FA; Gustafsson, 2001), but differ by about one log unit from those obtained by Milne et al. (2001) with the NICA-Donnan model (3.09 ± 0.51 for HA, 2.65 ± 0.43 for FA), and used to derive recommended generic values. To clarify this ambiguity, 10 high-quality titration data from Milne et al. (2001) were re-analyzed with the new predicted equilibrium constants. The data are described equally well with the previous and new sets of values ( R2 ⩾ 0.98), not necessarily because the NICA-Donnan model is overparametrized, but because titration lacks the sensitivity needed to quantify the full binding properties of humic substances. Correlations between NICA-Donnan parameters are discussed, but general progress is impeded by the unknown number of independent parameters that can be varied during regression of a model fit to titration data. The high consistency between predicted and experimental KH,COOH values, excluding those of Milne et al. (2001), gives faith in the proposed semi-empirical structural approach, and its usefulness to assess the plausibility of proton stability constants derived from simulations of titration data.

Evidence of bovine serum albumin-viologen herbicide binding interaction and associated structural modifications

NASA Astrophysics Data System (ADS)

Roy, Swarup; Saxena, Shailendra K.; Mishra, Suryakant; Yogi, Priyanka; Sagdeo, P. R.; Kumar, Rajesh

2017-07-01

The binding ability of viologen herbicide with bovine serum albumin (BSA) has been investigated to understand viologen associated hazards by investigating ethyl viologen's (EV) binding using various spectroscopies and in-silico molecular docking approaches. Apparent association constant (1.3 × 104 L/mol), calculated using UV-Vis spectra indicating a moderate complex formation between BSA and EV. A static mode of fluorescence quenching has been observed as evident from inverse temperature dependence of Stern-Volmer quenching constant which also confirms an EV-BSA complex formation. Emission and time resolved fluorescence studies reveal that the emission quenching of BSA with EV is initiated by static quenching mechanism. A moderately strong binding affinity between EV and BSA has been observed (binding constant value of 7.58 × 104 L/Mol) using fluorescence quenching titration, obtained at 298 K. Quantitative measurements of thermodynamic parameters like enthalpy and entropy changes clearly indicates hydrophobic force responsible for EV-BSA complex formation. The binding distance between EV and BSA was found to be 4.48 nm are involved in non-radiative energy transfer process. Furthermore, from the circular dichroism spectra it was observed that addition of EV is also found to change the secondary structure of BSA which leads to decrease in α-helix. Above mentioned results are found to be in consonance with molecular docking simulations and supports the EV-BSA binding.

Comprehensive insight into the binding of sunitinib, a multi-targeted anticancer drug to human serum albumin

NASA Astrophysics Data System (ADS)

Kabir, Md. Zahirul; Tee, Wei-Ven; Mohamad, Saharuddin B.; Alias, Zazali; Tayyab, Saad

2017-06-01

Binding studies between a multi-targeted anticancer drug, sunitinib (SU) and human serum albumin (HSA) were made using fluorescence, UV-vis absorption, circular dichroism (CD) and molecular docking analysis. Both fluorescence quenching data and UV-vis absorption results suggested formation of SU-HSA complex. Moderate binding affinity between SU and HSA was evident from the value of the binding constant (3.04 × 104 M-1), obtained at 298 K. Involvement of hydrophobic interactions and hydrogen bonds as the leading intermolecular forces in the formation of SU-HSA complex was predicted from the thermodynamic data of the binding reaction. These results were in good agreement with the molecular docking analysis. Microenvironmental perturbations around Tyr and Trp residues as well as secondary and tertiary structural changes in HSA upon SU binding were evident from the three-dimensional fluorescence and circular dichroism results. SU binding to HSA also improved the thermal stability of the protein. Competitive displacement results and molecular docking analysis revealed the binding locus of SU to HSA in subdomain IIA (Sudlow's site I). The influence of a few common ions on the binding constant of SU-HSA complex was also noticed.

Determination of the parameters of binding between lipopolysaccharide and chitosan and its N-acetylated derivative using a gravimetric piezoquartz biosensor.

PubMed

Naberezhnykh, G A; Gorbach, V I; Kalmykova, E N; Solov'eva, T F

2015-03-01

The interaction of endotoxin (lipopolysaccharide - LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization. Copyright © 2015 Elsevier B.V. All rights reserved.

Modulation of the platelet serotonin transporter by thermal balneotherapy: a study in healthy subjects.

PubMed

Baroni, S; Marazziti, D; Consoli, G; Picchetti, M; Catena-Dell'Osso, M; Galassi, A

2012-05-01

Although the beneficial effects of balneotherapy have been recognized since a long time, a few information is available on the biological mechanisms underlying them and the subjective feelings of increased well-being and mood. The links between the serotonin (5-HT) system and mood prompted us to investigate the 5-HT platelet transporter (SERT), which is considered a reliable, peripheral marker of the same structure present in presynaptic neurons, in 30 healthy volunteers before (t0) and 30 minutes after (t1) thermal balneotherapy with ozonized water, as compared with a similar group who underwent a bath in non-mineral water. MATERIALS AN METHODS: The SERT was evaluated by means of the specific binding of 3H-paroxetine (3H-Par) to platelet membranes. Equilibrium-saturation binding data, the maximal binding capacity (Bmax) and the dissociation constant (Kd), were obtained by means of the Scatchard analysis. The results showed that, while Bmax values did not change in both groups, the Kd values decreased significantly at t1 only in those subjects who bathed in ozonized water. The results of this study, while showing a decrease of the dissociation constant (Kd) which is the inverse of affinity constant, of 3H-Par binding to SERT in all subjects after balneotherapy and not in those bathing in normal water, suggest that SERT modifications may be related to a specific effect of ozonized water and, perhaps, also to the increased sense of well-being.

Thermal balneotherapy induces changes of the platelet serotonin transporter in healthy subjects.

PubMed

Marazziti, Donatella; Baroni, Stefano; Giannaccini, Gino; Catena Dell'Osso, Mario; Consoli, Giorgio; Picchetti, Michela; Carlini, Marina; Massimetti, Gabriele; Provenzano, Serafina; Galassi, Antonio

2007-10-01

Although the beneficial effects of balneotherapy have been recognized since a long time, a few information is available on the biological mechanisms underlying them and the subjective feelings of increased well-being and mood. The links between the serotonin (5-HT) system and mood prompted us to investigate the 5-HT platelet transporter (SERT), which is considered a reliable, peripheral marker of the same structure present in presynaptic neurons, in 20 healthy volunteers before (t0) and 30 min after (t1) thermal balneotherapy with ozonized water of Montecatini spa, as compared with a similar group who underwent a bath in non-mineral water. The SERT was evaluated by means of the specific binding of (3)H-paroxetine ((3)H-Par) to platelet membranes. Equilibrium-saturation binding data, the maximal binding capacity (Bmax) and the dissociation constant (Kd), were obtained by means of the Scatchard analysis. The results showed that, while Bmax values did not change in both groups, the Kd values decreased significantly at t1 only in those subjects who bathed in ozonized water. The results of this study, while showing a decrease of the dissociation constant (Kd) which is the inverse of affinity constant, of (3)H-Par binding to SERT in all subjects after balneotherapy and not in those bathing in normal water, suggest that SERT modifications may be related to a specific effect of ozonized water and, perhaps, also to the increased sense of well-being.

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

PubMed

Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

2013-09-17

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

Binding of /sup 3/H-acetylcholine to cholinergic receptors in bovine cerebral arteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimohama, S.; Tsukahara, T.; Taniguchi, T.

Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, /sup 3/H-acetylcholine (ACh), as the ligand. Specific binding of /sup 3/H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (K/sub D/) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of /sup 3/H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC/sub 50/ values of cholinergic drugs for /sup 3/H-ACh binding were as follows: atropine, 38.5 nM;more » ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC/sub 50/ values of nicotinic cholinergic agents such as nicotine, cytisine and ..cap alpha..-bungarotoxin exceeded 50 ..mu..M. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic reporters in these arteries. 18 references, 2 figures, 2 tables.« less

Intrinsic Thermodynamics and Structures of 2,4- and 3,4-Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases.

PubMed

Zubrienė, Asta; Smirnov, Alexey; Dudutienė, Virginija; Timm, David D; Matulienė, Jurgita; Michailovienė, Vilma; Zakšauskas, Audrius; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

2017-01-20

The goal of rational drug design is to understand structure-thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and selectivity for a target protein. In this study we explored the contribution of added functionalities of benzenesulfonamide inhibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affinities were found to be very different, spanning a range from -90 to +10 kJ mol -1 , and are compensated by a similar opposing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reactions. The sulfonamide group pK a values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho-substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dissociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all applied in this work. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

New mixed ligand palladium(II) complexes based on the antiepileptic drug sodium valproate and bioactive nitrogen-donor ligands: Synthesis, structural characterization, binding interactions with DNA and BSA, in vitro cytotoxicity studies and DFT calculations

NASA Astrophysics Data System (ADS)

Tabrizi, Leila; Chiniforoshan, Hossein; Tavakol, Hossein

2015-04-01

The complexes [Pd(valp)2(imidazole)2] (1), [Pd(valp)2(pyrazine)2] (2) (valp is sodium valproate) have been synthesized and characterized using IR, 1H NMR, 13C{1H} NMR and UV-Vis spectrometry. The interaction of complexes with CT-DNA has been investigated using spectroscopic tools and viscosity measurement. In each case, the association constant (Kb) was deduced from the absorption spectral study and the number of binding sites (n) and the binding constant (K) were calculated from relevant fluorescence quenching data. As a result, a non-covalent interaction between the metal complex and DNA was suggested, which could be assigned to an intercalative binding. In addition, the interaction of 1 and 2 was ventured with bovine serum albumin (BSA) with the help of absorption and fluorescence spectroscopy measurements. Through these techniques, the apparent association constant (Kapp) and the binding constant (K) could be calculated for each complex. Evaluation of cytotoxic activity of the complexes against four different cancer cell lines proved that the complexes exhibited cytotoxic specificity and significant cancer cell inhibitory rate. Moreover, density functional theory (DFT) calculations were employed to provide more evidence about the observed data. The majority of trans isomers were supported not only by energies, but also by the similarity of its calculated IR frequencies, UV adsorptions and NMR chemical shifts to the experimental values.

Rate Constants and Mechanisms of Protein–Ligand Binding

PubMed Central

Pang, Xiaodong; Zhou, Huan-Xiang

2017-01-01

Whereas protein–ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms. PMID:28375732

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs.

PubMed

Alonso, A; Almendral, M J; Curto, Y; Criado, J J; Rodríguez, E; Manzano, J L

2006-08-15

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

Cyanide binding to ferrous and ferric microperoxidase-11.

PubMed

Ascenzi, Paolo; Sbardella, Diego; Santucci, Roberto; Coletta, Massimo

2016-07-01

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c (cytc). MP11 is characterized by a covalently linked solvent-exposed heme group, the heme-Fe atom being axially coordinated by a histidyl residue. Here, the reactions of ferrous and ferric MP11 (MP11-Fe(II) and MP11-Fe(III), respectively) with cyanide have been investigated from the kinetic and thermodynamic viewpoints, at pH 7.0 and 20.0 °C. Values of the second-order rate constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 4.5 M(-1) s(-1) and 8.9 × 10(3) M(-1) s(-1), respectively. Values of the first-order rate constant for cyanide dissociation from ligated MP11-Fe(II) and MP11-Fe(III) are 1.8 × 10(-1) s(-1) and 1.5 × 10(-3) s(-1), respectively. Values of the dissociation equilibrium constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 3.7 × 10(-2) and 1.7 × 10(-7) M, respectively, matching very well with those calculated from kinetic parameters so that no intermediate species seem to be involved in the ligand-binding process. The pH-dependence of cyanide binding to MP11-Fe(III) indicates that CN(-) is the only binding species. Present results have been analyzed in parallel with those of several heme-proteins, suggesting that (1) the ligand accessibility to the metal center and cyanide ionization may modulate the formation of heme-Fe-cyanide complexes, and (2) the general polarity of the heme pocket and/or hydrogen bonding of the heme-bound ligand may affect cyanide exit from the protein matrix. Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c. Penta-coordinated MP11 displays a very high reactivity towards cyanide, whereas the reactivity of hexa-coordinated horse heart cytochrome c is very low.

Gallium(III)/4-(2-pyridylazo)resorcinol system in water and SDS solution: kinetics and thermodynamics.

PubMed

Biver, T; Boggioni, A; Secco, F; Venturini, M

2008-01-01

The equilibria and kinetics of the complex formation and dissociation reaction between gallium(III) and PAR [4-(2-pyridylazo)resorcinol] have been investigated in water and in the presence of SDS micelles. The reactive form of Ga(III) is GaOH2+ in both cases. The addition of SDS results in an increase of both the binding affinity and velocity, the maximum accelerating effect being observed just above the cmc value of SDS that, under the conditions of the experiments, is 5.6 x 10-3 M. At pH = 3.2, the maximum value of the equilibrium constant ratio Kapp(SDS)/Kapp(H2O) is 27.4, whereas that of the binding rate constants kf(SDS)/kf(H2O) is 16. The results are interpreted in terms of increased concentrations of the reactants on the micelle surface and on competition of PAR and SDS for GaOH2+.

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

PubMed

Ojeda, Paola; Pérez, Alejandra; Ojeda, Lorena; Vargas-Uribe, Mauricio; Rivas, Coralia I; Salas, Monica; Vera, Juan Carlos; Reyes, Alejandro M

2012-09-01

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.

Possibility of using Rhodamine B dye in diagnosis of some men's diseases

NASA Astrophysics Data System (ADS)

Khodjayev, Gayrat; Ismailov, Zafar F.; Kurtaliev, Eldar N.; Nizomov, Negmat; Khaydarova, Feruza U.; Hamidov, Zariddin; Khakimova, Dilorom P.

2007-09-01

The functional differences of human blood serum albumin in norm and at different patologic process were studied by spectral-luminescent method by comparison of binding constant (K) and concentration of binding sites (N) values of rhodamine B dye with blood serum. It was shown that K and N of rhodamine B dye with blood serum of sick men is decreased as compared to that for healthy men.

Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

PubMed Central

Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

2009-01-01

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex.

PubMed

Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

2013-07-01

A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy=2,2-bipyridine, Hbcpip=2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5±1.4)×10(5) M(-1) in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived. Copyright © 2013 Elsevier B.V. All rights reserved.

The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding.

PubMed

Acharya, Bipul R; Bhattacharyya, Bhabatarak; Chakrabarti, Gopal

2008-07-29

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.

Sensing of hydrophobic cavity of serum albumin by an adenosine analogue: fluorescence correlation and ensemble spectroscopic studies.

PubMed

Nag, Moupriya; Bera, Kallol; Chakraborty, Sandipan; Basak, Soumen

2013-10-05

Adenosine is a naturally occurring purine nucleoside that plays important role in various biochemical processes. We have studied the binding of TNP-Ado (trinitrophenylated-adenosine), a fluorescent analogue of adenosine (which itself is a weak fluorophore), with a model transport protein, bovine serum albumin (BSA). The binding affinity was determined using Fluorescence correlation spectroscopy (FCS) and compared with its value obtained from macroscopic fluorescence spectroscopic studies. Fluorescence and circular dichroism (CD) spectroscopies were employed together with molecular docking study to locate the probable binding site of TNP-Ado on BSA and its effect on the conformation and stability of BSA. Fluorescence studies showed that TNP-Ado binds to BSA in 1:1 stoichiometry via an entropically favoured process. Induced CD spectra revealed that a chiro-optical switching of TNP-Ado occurs upon binding to BSA. Results on urea-induced denaturation of BSA and docking study suggested that the binding site for the ligand is in the hydrophobic subdomain IIA of BSA, consistent with the results of other measurements. This study establishes TNP-Ado as a sensor of hydrophobic regions in proteins like serum albumin, having the capability of detecting a minimum concentration of 140ng/ml protein. FCS measurement of binding interaction of rhodamine-labeled TNP-Ado (RTNP-Ado) with BSA yielded an association constant of KFCS=(1.03±0.06) × 10(4)M(-1). The association constants (Ka) obtained for binding of BSA with rhodamine-free (i.e. TNP-Ado) and rhodamine-labeled (RTNP-Ado) ligands, obtained using the ensemble spectroscopic technique, were (2.3±0.06) × 10(5)M(-1) and (3.4±0.03) × 10(4)M(-1), respectively. The difference between the values of Ka for the free and labeled ligands suggests that fluorescent labeling of small molecules perceptibly interferes with the binding process. On the other hand, the difference in Ka obtained by FCS and ensemble techniques is due to the fact that while the former measures the change in the diffusion constant (i.e. size) of RTNP-Ado upon binding to BSA, the latter focuses on the change of tryptophan emission properties of BSA due to the presence of bound RTNP-Ado. Copyright © 2013 Elsevier B.V. All rights reserved.

Binding of KATP channel modulators in rat cardiac membranes

PubMed Central

Löffler-Walz, Cornelia; Quast, Ulrich

1998-01-01

The binding of [3H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K+ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [3H]-P1075 binding with an EC50 value of 100 μM and a Hill coefficient of 1.4.In saturation experiments [3H]-P1075 binding was homogeneous with a KD value of 6±1 nM and a binding capacity (Bmax) of 33±3 fmol mg−1 protein.Upon addition of an excess of unlabelled P1075, the [3H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min−1. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM−1 min−1. From the kinetic experiments the KD value was calculated as 4.7±0.6 nM.Openers of the ATP-sensitive K+ channel belonging to different structural classes inhibited specific [3H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [3H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC50 value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC50 value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.It is concluded that binding studies with [3H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [3H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR2A) and vascular smooth muscle cells (SUR2B), differs from that expected for SUR2A and SUR2B. PMID:9579735

Rate constants for proteins binding to substrates with multiple binding sites using a generalized forward flux sampling expression

NASA Astrophysics Data System (ADS)

Vijaykumar, Adithya; ten Wolde, Pieter Rein; Bolhuis, Peter G.

2018-03-01

To predict the response of a biochemical system, knowledge of the intrinsic and effective rate constants of proteins is crucial. The experimentally accessible effective rate constant for association can be decomposed in a diffusion-limited rate at which proteins come into contact and an intrinsic association rate at which the proteins in contact truly bind. Reversely, when dissociating, bound proteins first separate into a contact pair with an intrinsic dissociation rate, before moving away by diffusion. While microscopic expressions exist that enable the calculation of the intrinsic and effective rate constants by conducting a single rare event simulation of the protein dissociation reaction, these expressions are only valid when the substrate has just one binding site. If the substrate has multiple binding sites, a bound enzyme can, besides dissociating into the bulk, also hop to another binding site. Calculating transition rate constants between multiple states with forward flux sampling requires a generalized rate expression. We present this expression here and use it to derive explicit expressions for all intrinsic and effective rate constants involving binding to multiple states, including rebinding. We illustrate our approach by computing the intrinsic and effective association, dissociation, and hopping rate constants for a system in which a patchy particle model enzyme binds to a substrate with two binding sites. We find that these rate constants increase as a function of the rotational diffusion constant of the particles. The hopping rate constant decreases as a function of the distance between the binding sites. Finally, we find that blocking one of the binding sites enhances both association and dissociation rate constants. Our approach and results are important for understanding and modeling association reactions in enzyme-substrate systems and other patchy particle systems and open the way for large multiscale simulations of such systems.

Simultaneous addition of two ligands: a potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry.

PubMed

Henzl, Michael T; Markus, Lindsey A; Davis, Meredith E; McMillan, Andrew T

2013-03-01

Capable of providing a detailed thermodynamic picture of noncovalent association reactions, isothermal titration calorimetry (ITC) has become a popular method for studying protein-ligand interactions. We routinely employ the technique to study divalent ion-binding by two-site EF-hand proteins from the parvalbumin- and polcalcin lineages. The combination of high Ca(2+) affinity and relatively low Mg(2+) affinity, and the attendant complication of parameter correlation, conspire to make the simultaneous extraction of binding constants and -enthalpies for both ions challenging. Although global analysis of multiple ITC experiments can overcome these hurdles, our current experimental protocol includes upwards of 10 titrations - requiring a substantial investment in labor, machine time, and material. This paper explores the potential for using a smaller suite of experiments that includes simultaneous titrations with Ca(2+) and Mg(2+) at different ratios of the two ions. The results obtained for four proteins, differing substantially in their divalent ion-binding properties, suggest that the approach has merit. The Ca(2+)- and Mg(2+)-binding constants afforded by the streamlined analysis are in reasonable agreement with those obtained from the standard analysis protocol. Likewise, the abbreviated analysis provides comparable values for the Ca(2+)-binding enthalpies. However, the streamlined analysis can yield divergent values for the Mg(2+)-binding enthalpies - particularly those for lower affinity sites. This shortcoming can be remedied, in large measure, by including data from a direct Ca(2+) titration in the presence of a high, fixed Mg(2+) concentration. Copyright © 2013. Published by Elsevier Inc.

Determination of Noncovalent Binding Using a Continuous Stirred Tank Reactor as a Flow Injection Device Coupled to Electrospray Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Santos, Inês C.; Waybright, Veronica B.; Fan, Hui; Ramirez, Sabra; Mesquita, Raquel B. R.; Rangel, António O. S. S.; Fryčák, Petr; Schug, Kevin A.

2015-07-01

Described is a new method based on the concept of controlled band dispersion, achieved by hyphenating flow injection analysis with ESI-MS for noncovalent binding determinations. A continuous stirred tank reactor (CSTR) was used as a FIA device for exponential dilution of an equimolar host-guest solution over time. The data obtained was treated for the noncovalent binding determination using an equimolar binding model. Dissociation constants between vancomycin and Ac-Lys(Ac)-Ala-Ala-OH peptide stereoisomers were determined using both the positive and negative ionization modes. The results obtained for Ac- L-Lys(Ac)- D-Ala- D-Ala (a model for a Gram-positive bacterial cell wall) binding were in reasonable agreement with literature values made by other mass spectrometry binding determination techniques. Also, the developed method allowed the determination of dissociation constants for vancomycin with Ac- L-Lys(Ac)- D-Ala- L-Ala, Ac- L-Lys(Ac)- L-Ala- D-Ala, and Ac- L-Lys(Ac)- L-Ala- L-Ala. Although some differences in measured binding affinities were noted using different ionization modes, the results of each determination were generally consistent. Differences are likely attributable to the influence of a pseudo-physiological ammonium acetate buffer solution on the formation of positively- and negatively-charged ionic complexes.

New cobalt(II) and nickel(II) complexes of benzyl carbazate Schiff bases: Syntheses, crystal structures, in vitro DNA and HSA binding studies.

PubMed

Nithya, Palanivelu; Helena, Sannasi; Simpson, Jim; Ilanchelian, Malaichamy; Muthusankar, Aathi; Govindarajan, Subbiah

2016-12-01

In the present study, new Schiff base complexes with the composition [M(NCS) 2 (L1) 2 ]·nH 2 O, where M=Co (n=0) (1) and Ni (n=2) (2); [M(NCS) 2 (L2) 2 ], M=Co (3) and Ni (4) as well as [M(NCS) 2 (L3) 2 ], M=Co (5) and Ni (6); (L1=benzyl 2-(propan-2-ylidene)hydrazinecarboxylate, L2=benzyl 2-(butan-2-ylidene)hydrazinecarboxylate and L3=benzyl 2-(pentan-3-ylidene)hydrazinecarboxylate) have been synthesized by a template method. The complexes were characterised by analytical methods, spectroscopic studies, thermal and X-ray diffraction techniques. The structures of all the complexes explore that the metal(II) cation has a trans-planar coordination environment, the monomeric units containing a six-coordinated metal center in octahedral geometry with N-bound isothiocyanate anions coordinated as terminal ligands. Furthermore, the binding of the two Schiff base ligands to the metal centers involves the azomethine nitrogen and the carbonyl oxygen in mutually trans configuration. The binding interactions of all the complexes with Calf thymus-deoxyribonucleic acid (CT-DNA) and human serum albumin (HSA) have been investigated using absorption and emission spectral techniques. The CT-DNA binding properties of these complexes reveal that they bind to CT-DNA through a partial intercalation mode and the binding constant values were calculated using the absorption and emission spectral data. The binding constant values (~10×10 6 moldm -3 ) indicate strong binding of metal complexes with CT-DNA. HSA binding interaction studies showed that the cobalt and nickel complexes can quench the intrinsic fluorescence of HSA through static quenching process. Also, molecular docking studies were supported out to apprehend the binding interactions of these complexes with DNA and HSA which offer new understandings into the experimental model observations. Copyright © 2016 Elsevier B.V. All rights reserved.

Ontogenesis of the angiotensin II (ANGII) receptor system in the duck brain.

PubMed

Müller, A R; Gerstberger, R

1994-03-18

The ontogenetic development of the central nervous angiotensin II (ANGII) receptor system in the duck was studied at embryonic days E20 and E27 and at postnatal days P3 and P14 by computerized semiquantitative autoradiography employing the receptor antagonist 125I[1Sar,8Ile]ANGII as radioligand. For circumventricular structures involved in the sensing of brain-intrinsic (AV3V region) or blood-borne (subfornical organ, SFO) ANGII, binding sites for 125I[1Sar,8Ile]ANGII were first detectable at E27, with a steady rise in binding density up to P14. The choroid plexus of the lateral (PCVL) and third (PCVIII) cerebral ventricles responsible for cerebrospinal fluid (CSF) production were endowed with maximal ANGII receptor densities at E20 with subsequent reduction to constant medium (PCVIII) or low (PCVL) values. Besides the choroid plexus, the magnocellular paraventricular nucleus (PVN) was the only structure presenting ANGII specific binding sites at E20, although at low density. As for the SFO and AV3V region, labelling of ANGII binding sites in the PVN increased continuously during development to high values at P14. Nuclear components of the limbic system (archistriatum, amygdala and habenular complex) did not reveal specific labelling by the radioligand at E20 with constant, moderate binding densities evaluated for E27, P3 and P14. In the duck brain, functionally related structures exhibited a homogeneous ontogenetic development of their ANGII receptor system.

Predicting Stability Constants for Uranyl Complexes Using Density Functional Theory

DOE PAGES

Vukovic, Sinisa; Hay, Benjamin P.; Bryantsev, Vyacheslav S.

2015-04-02

The ability to predict the equilibrium constants for the formation of 1:1 uranyl:ligand complexes (log K 1 values) provides the essential foundation for the rational design of ligands with enhanced uranyl affinity and selectivity. We also use density functional theory (B3LYP) and the IEFPCM continuum solvation model to compute aqueous stability constants for UO 2 2+ complexes with 18 donor ligands. Theoretical calculations permit reasonably good estimates of relative binding strengths, while the absolute log K 1 values are significantly overestimated. Accurate predictions of the absolute log K 1 values (root mean square deviation from experiment < 1.0 for logmore » K 1 values ranging from 0 to 16.8) can be obtained by fitting the experimental data for two groups of mono and divalent negative oxygen donor ligands. The utility of correlations is demonstrated for amidoxime and imide dioxime ligands, providing a useful means of screening for new ligands with strong chelate capability to uranyl.« less

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

NASA Astrophysics Data System (ADS)

Cubrilovic, Dragana; Biela, Adam; Sielaff, Frank; Steinmetzer, Torsten; Klebe, Gerhard; Zenobi, Renato

2012-10-01

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solution.

Quantitative production of compound I from a cytochrome P450 enzyme at low temperatures. Kinetics, activation parameters, and kinetic isotope effects for oxidation of benzyl alcohol.

PubMed

Wang, Qin; Sheng, Xin; Horner, John H; Newcomb, Martin

2009-08-05

Cytochrome P450 enzymes are commonly thought to oxidize substrates via an iron(IV)-oxo porphyrin radical cation transient termed Compound I, but kinetic studies of P450 Compounds I are essentially nonexistent. We report production of Compound I from cytochrome P450 119 (CYP119) in high conversion from the corresponding Compound II species at low temperatures in buffer mixtures containing 50% glycerol by photolysis with 365 nm light from a pulsed lamp. Compound I was studied as a reagent in oxidations of benzyl alcohol and its benzylic mono- and dideuterio isotopomers. Pseudo-first-order rate constants obtained at -50 degrees C with concentrations of substrates between 1.0 and 6.0 mM displayed saturation kinetics that gave binding constants for the substrate in the Compound I species (K(bind)) and first-order rate constants for the oxidation reactions (k(ox)). Representative results are K(bind) = 214 M(-1) and k(ox) = 0.48 s(-1) for oxidation of benzyl alcohol. For the dideuterated substrate C(6)H(5)CD(2)OH, kinetics were studied between -50 and -25 degrees C, and a van't Hoff plot for complexation and an Arrhenius plot for the oxidation reaction were constructed. The H/D kinetic isotope effects (KIEs) at -50 degrees C were resolved into a large primary KIE (P = 11.9) and a small, inverse secondary KIE (S = 0.96). Comparison of values extrapolated to 22 degrees C of both the rate constant for oxidation of C(6)H(5)CD(2)OH and the KIE for the nondeuterated and dideuterated substrates to values obtained previously in laser flash photolysis experiments suggested that tunneling could be a significant component of the total rate constant at -50 degrees C.

Comparative evaluation of in vitro efficacy of colesevelam hydrochloride tablets.

PubMed

Krishnaiah, Yellela S R; Yang, Yongsheng; Bykadi, Srikant; Sayeed, Vilayat A; Khan, Mansoor A

2014-09-01

Colesevelam hydrochloride is used as an adjunct to diet and exercise to reduce elevated low-density lipoprotein (LDL) cholesterol in patients with primary hyperlipidemia as well as to improve glycemic control in patients with type 2 diabetes. This is likely to result in submission of abbreviated new drug applications (ANDA). This study was conducted to compare the efficacy of two tablet products of colesevelam hydrochloride based on the in vitro binding of bile acid sodium salts of glycocholic acid (GC), glycochenodeoxycholic acid (GCDA) and taurodeoxycholic acid (TDCA). Kinetic binding study was carried out with constant initial bile salt concentrations as a function of time. Equilibrium binding studies were conducted under conditions of constant incubation time and varying initial concentrations of bile acid sodium salts. The unbound concentration of bile salts was determined in the samples of these studies. Langmuir equation was utilized to calculate the binding constants k1 and k2. The amount of the three bile salts bound to both the products reached equilibrium at 3 h. The similarity factor (f2) was 99.5 based on the binding profile of total bile salts to the test and reference colesevelam tablets as a function of time. The 90% confidence interval for the test to reference ratio of k2 values were 96.06-112.07 which is within the acceptance criteria of 80-120%. It is concluded from the results that the test and reference tablets of colesevelam hydrochloride showed a similar in vitro binding profile and capacity to bile salts.

Binding and Energetics of Electron Transfer between an Artificial Four-Helix Mn-Protein and Reaction Centers from Rhodobacter sphaeroides.

PubMed

Espiritu, Eduardo; Olson, Tien L; Williams, JoAnn C; Allen, James P

2017-12-12

The ability of an artificial four-helix bundle Mn-protein, P1, to bind and transfer an electron to photosynthetic reaction centers from the purple bacterium Rhodobacter sphaeroides was characterized using optical spectroscopy. Upon illumination of reaction centers, an electron is transferred from P, the bacteriochlorophyll dimer, to Q A , the primary electron acceptor. The P1 Mn-protein can bind to the reaction center and reduce the oxidized bacteriochlorophyll dimer, P + , with a dissociation constant of 1.2 μM at pH 9.4, comparable to the binding constant of c-type cytochromes. Amino acid substitutions of surface residues on the Mn-protein resulted in increases in the dissociation constant to 8.3 μM. The extent of reduction of P + by the P1 Mn-protein was dependent on the P/P + midpoint potential and the pH. Analysis of the free energy difference yielded a midpoint potential of approximately 635 mV at pH 9.4 for the Mn cofactor of the P1 Mn-protein, a value similar to those found for other Mn cofactors in proteins. The linear dependence of -56 mV/pH is consistent with one proton being released upon Mn oxidation, allowing the complex to maintain overall charge neutrality. These outcomes demonstrate the feasibility of designing four-helix bundles and other artificial metalloproteins to bind and transfer electrons to bacterial reaction centers and establish the usefulness of this system as a platform for designing sites to bind novel metal cofactors capable of performing complex oxidation-reduction reactions.

Fluorescence studies on binding of pyrene and its derivatives to humic acid

NASA Astrophysics Data System (ADS)

Nakashima, K.; Maki, M.; Ishikawa, F.; Yoshikawa, T.; Gong, Y.-K.; Miyajima, T.

2007-07-01

Binding of pyrene (PyH) and its derivatives to humic acid (HA) has been studied by fluorescence spectroscopy. The nature of the interaction between HA and pyrene derivatives are extensively investigated by employing three derivatives ranging from anionic to cationic compounds: 1-pyrenebutylic acid (PyA), 1-pyrenemethanol (PyM), and 1-pyrenebutyltrimethylammonium bromide (PyB). Binding constants between HA and PyX (X = H, A, M, B) are obtained by steady-state fluorescence quenching techniques, and it is found that PyB has a markedly large binding constant among the pyrene family. This is attributed to a strong electrostatic interaction between cationic PyB and anionic HA. The result suggests that an electrostatic interaction plays a dominant role in binding of pyrenes to humic acid. The importance of electrostatic interaction was also confirmed by a salt effect on the binding constant. Influence of collisional quenching on the binding constant, which causes overestimation of the binding constant, was examined by time-resolved fluorescence spectroscopy as well as temperature effect in steady-state fluorescence measurements. It is elucidated that collisional quenching does not much bring overestimation into the binding constants.

Computational scheme for pH-dependent binding free energy calculation with explicit solvent.

PubMed

Lee, Juyong; Miller, Benjamin T; Brooks, Bernard R

2016-01-01

We present a computational scheme to compute the pH-dependence of binding free energy with explicit solvent. Despite the importance of pH, the effect of pH has been generally neglected in binding free energy calculations because of a lack of accurate methods to model it. To address this limitation, we use a constant-pH methodology to obtain a true ensemble of multiple protonation states of a titratable system at a given pH and analyze the ensemble using the Bennett acceptance ratio (BAR) method. The constant pH method is based on the combination of enveloping distribution sampling (EDS) with the Hamiltonian replica exchange method (HREM), which yields an accurate semi-grand canonical ensemble of a titratable system. By considering the free energy change of constraining multiple protonation states to a single state or releasing a single protonation state to multiple states, the pH dependent binding free energy profile can be obtained. We perform benchmark simulations of a host-guest system: cucurbit[7]uril (CB[7]) and benzimidazole (BZ). BZ experiences a large pKa shift upon complex formation. The pH-dependent binding free energy profiles of the benchmark system are obtained with three different long-range interaction calculation schemes: a cutoff, the particle mesh Ewald (PME), and the isotropic periodic sum (IPS) method. Our scheme captures the pH-dependent behavior of binding free energy successfully. Absolute binding free energy values obtained with the PME and IPS methods are consistent, while cutoff method results are off by 2 kcal mol(-1) . We also discuss the characteristics of three long-range interaction calculation methods for constant-pH simulations. © 2015 The Protein Society.

Vitamin K3 disrupts the microtubule networks by binding to tubulin: a novel mechanism of its antiproliferative activity.

PubMed

Acharya, Bipul R; Choudhury, Diptiman; Das, Amlan; Chakrabarti, Gopal

2009-07-28

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

Tertiary structural changes and iron release from human serum transferrin.

PubMed

Mecklenburg, S L; Donohoe, R J; Olah, G A

1997-08-01

Iron release from human serum transferrin was investigated by comparison of the extent of bound iron, measured by charge transfer absorption band intensity (465 nm), with changes observed by small-angle solution X-ray scattering (SAXS) for a series of equilibrated samples between pH 5.69 and 7.77. The phosphate buffers used in this study promote iron release at relatively high pH values, with an empirical pK of 6.9 for the convolved release from the two sites. The spectral data reveal that the N-lobe release is nearly complete by pH 7.0, while the C-lobe remains primarily metal-laden. Conversely, the radius of gyration, Rg, determined from the SAXS data remains constant between pH 7.77 and 7.05, and the evolution of Rg between its value observed for the diferric protein at pH 7.77 (31.2+/-0.2 A) and that of the apo protein at pH 5.69 (33.9+/-0.4 A) exhibits an empirical pK of 6.6. While Rg is effectively constant in the pH range associated with iron release from the N-lobe, the radius of gyration of cross-section, Rc, increases from 16.9+/-0.2 A to 17.6+/-0.2 A. Model simulations suggest that two different rotations of the NII domain relative to the NI domain about a hinge deep in the iron-binding cleft of the N-lobe, one parallel with and one perpendicular to the plane of the iron-binding site, can be significantly advanced relative to their holo protein positions while yielding constant Rg and increased Rc values consistent with the scattering data. Rotation of the CII domain parallel with the C-lobe iron-binding site plane can partially account for the increased Rg values measured at low pH; however, no reasonable combined repositioning of the NII and CII domains yields the experimentally observed increase in Rg.

Using resonance light scattering and UV/vis absorption spectroscopy to study the interaction between gliclazide and bovine serum albumin.

PubMed

Zhang, Qiu-Ju; Liu, Bao-Sheng; Li, Gai-Xia; Han, Rong

2016-08-01

At different temperatures (298, 310 and 318 K), the interaction between gliclazide and bovine serum albumin (BSA) was investigated using fluorescence quenching spectroscopy, resonance light scattering spectroscopy and UV/vis absorption spectroscopy. The first method studied changes in the fluorescence of BSA on addition of gliclazide, and the latter two methods studied the spectral change in gliclazide while BSA was being added. The results indicated that the quenching mechanism between BSA and gliclazide was static. The binding constant (Ka ), number of binding sites (n), thermodynamic parameters, binding forces and Hill's coefficient were calculated at three temperatures. Values for the binding constant obtained using resonance light scattering and UV/vis absorption spectroscopy were much greater than those obtained from fluorescence quenching spectroscopy, indicating that methods monitoring gliclazide were more accurate and reasonable. In addition, the results suggest that other residues are involved in the reaction and the mode 'point to surface' existed in the interaction between BSA and gliclazide. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Diffusion coefficients in systems with inclusion compounds. 1. alpha. -Cyclodextrin-L-phenylalanine-water at 25 degree C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paduano, L.; Sartorio, R.; Vitagliano, V.

Diffusion coefficients in the ternary system {alpha}-cyclodextrin (at one concentration)-L-phenylalanine (at four concentrations)-water have been measured by using the Gouy interferometric technique. The effect of the inclusion equilibrium on the cross-term diffusion coefficients was observed. The measured diffusion coefficients in the ternary systems were used to calculate values of the binding constants. These values are in good agreement with the value obtained from calorimetric studies.

Metal binding characterization and conformational studies using Raman microscopy of resin-bound poly(aspartic acid).

PubMed

Stair, Jacqueline L; Holcombe, James A

2007-03-01

The metal binding capacities, conditional stability constants, and secondary structure of immobilized polyaspartic acid (PLAsp) (n = 6, 20, and 30) on TentaGel resin were determined when binding Mg2+, Co2+, Cd2+, and Ni2+. Metal binding to the synthesized peptides was evaluated using breakthrough curves from a packed microcolumn and flame atomic absorption spectrophotometry (FAAS) detection. The metal capacities reached values of 590, 2160, and 3710 mumol of metal/g of resin for the 6-mer, 20-mer, and 30-mer, respectively, and this resulted in 2-3 residues per metal for all peptides and metals tested. Surprisingly, the concentrated environment of the resin along with the spatial distribution of attachment groups allowed for most residues to participate in metal binding regardless of the peptide length. Conditional stability constants calculated using single metal binding isotherms indicated that binding strength decreased as the chain length increased on the resin. Raman microscopy on single beads was used to determine PLAsp secondary structure, and all peptides were of a mixed conformation (i.e., beta-sheets, alpha-helices, random chain, etc.) during neutral conditioning and metal binding. Uniquely, the longer 20-mer and 30-mer peptides showed a distinct change from a mixed conformation to beta-sheets and alpha-helices during metal release with acid. This study confirms that metal release by longer immobilized peptides is often assisted by a conformational change, which easily spoils the binding cavity, while shorter peptides may release metal primarily by H+ displacement.

Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

PubMed

Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

2014-12-16

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

PubMed Central

2015-01-01

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain. PMID:25453233

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

PubMed

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

2016-05-15

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. Copyright © 2015 Elsevier B.V. All rights reserved.

Entrapment of Alpha1-Acid Glycoprotein in High-Performance Affinity Columns for Drug-Protein Binding Studies

PubMed Central

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S.

2015-01-01

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (± 0.5) × 105 M−1, which agreed with a previously reported value of 1.0 (± 0.1) × 105 M−1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. PMID:26627938

Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin.

PubMed

Choudhury, Diptiman; Ganguli, Arnab; Dastidar, Debabrata Ghosh; Acharya, Bipul R; Das, Amlan; Chakrabarti, Gopal

2013-06-01

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC50 value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC50 value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10(-7) M(-1) s(-1) and 4.0 × 10(-9) M(-1) s(-1) for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin-apigenin binding was 1:1 and binding the binding constant (Kd) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G2/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α-β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent, pinostrobin

PubMed Central

FEROZ, Shevin R.; SUMI, Rumana A.; MALEK, Sri N.A.; TAYYAB, Saad

2014-01-01

The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, Ka in the range of 1.49 – 6.12 × 104 M−1, with 1:1 binding stoichiometry. Based on the PS–albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics. PMID:25519455

Probing the General Time Scale Question of Boronic Acid Binding with Sugars in Aqueous Solution at Physiological pH

PubMed Central

Ni, Nanting; Laughlin, Sarah; Wang, Yingji; Feng, You; Zheng, Yujun

2012-01-01

The boronic acid group is widely used in chemosensor design due to its ability to reversibly bind diol-containing compounds. The thermodynamic properties of the boronic acid-diol binding process have been investigated extensively. However, there are few studies of the kinetic properties of such binding processes. In this report, stopped-flow method was used for the first time to study the kinetic properties of the binding between three model arylboronic acids, 4-, 5-, and 8-isoquinolinylboronic acids, and various sugars. With all the boronic acid-diol pair sexamined, reactions were complete within seconds. The kon values with various sugars follow the order of D-fructose >D-tagatose>D-mannose >D-glucose. This trend tracks the thermodynamic binding affinities for these sugars and demonstrates that the “on” rate is the key factor determining the binding constant. PMID:22464680

Nonspecific DNA Binding and Bending by HUαβ: Interfaces of the Three Binding Modes Characterized by Salt Dependent Thermodynamics

PubMed Central

Koh, Junseock; Shkel, Irina; Saecker, Ruth M.; Record, M. Thomas

2011-01-01

Previous ITC and FRET studies demonstrated that Escherichia coli HUαβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34 bp mode which interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it 140° and thereby increasing its flexibility, and two weaker, modestly cooperative small-site-size modes (10 bp, 6 bp) useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and deduce that DNA in the 34 bp mode is bent around but not wrapped on the body of HU, in contrast to specific binding of IHF. Analyses of binding isotherms (8, 15, 34 bp DNA) and initial binding heats (34, 38, 160 bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Ski) even though their binding site sizes differ greatly; most probable values of Ski on 34 bp or larger DNA are − 7.5 ± 0.5. From the similarity of Ski values, we conclude that binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent-DNA 34 bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6 bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Ski values are proposed. PMID:21513716

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight.

PubMed

Shamsi, Anas; Ahmed, Azaj; Bano, Bilqees

2018-05-01

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 10 4  M -1 implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.

Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

PubMed

Hoggett, J G; Kellett, G L

1976-06-15

A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.

Principal component analysis of chemical shift perturbation data of a multiple-ligand-binding system for elucidation of respective binding mechanism.

PubMed

Konuma, Tsuyoshi; Lee, Young-Ho; Goto, Yuji; Sakurai, Kazumasa

2013-01-01

Chemical shift perturbations (CSPs) in NMR spectra provide useful information about the interaction of a protein with its ligands. However, in a multiple-ligand-binding system, determining quantitative parameters such as a dissociation constant (K(d) ) is difficult. Here, we used a method we named CS-PCA, a principal component analysis (PCA) of chemical shift (CS) data, to analyze the interaction between bovine β-lactoglobulin (βLG) and 1-anilinonaphthalene-8-sulfonate (ANS), which is a multiple-ligand-binding system. The CSP on the binding of ANS involved contributions from two distinct binding sites. PCA of the titration data successfully separated the CSP pattern into contributions from each site. Docking simulations based on the separated CSP patterns provided the structures of βLG-ANS complexes for each binding site. In addition, we determined the K(d) values as 3.42 × 10⁻⁴ M² and 2.51 × 10⁻³ M for Sites 1 and 2, respectively. In contrast, it was difficult to obtain reliable K(d) values for respective sites from the isothermal titration calorimetry experiments. Two ANS molecules were found to bind at Site 1 simultaneously, suggesting that the binding occurs cooperatively with a partial unfolding of the βLG structure. On the other hand, the binding of ANS to Site 2 was a simple attachment without a significant conformational change. From the present results, CS-PCA was confirmed to provide not only the positions and the K(d) values of binding sites but also information about the binding mechanism. Thus, it is anticipated to be a general method to investigate protein-ligand interactions. Copyright © 2012 Wiley Periodicals, Inc.

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms*

PubMed Central

Radnai, László; Rapali, Péter; Hódi, Zsuzsa; Süveges, Dániel; Molnár, Tamás; Kiss, Bence; Bécsi, Bálint; Erdödi, Ferenc; Buday, László; Kardos, József; Kovács, Mihály; Nyitray, László

2010-01-01

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with Kd values of 9 and 40 μm, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy- and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: Kd values of 37 and 3.5 nm for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent Kd value (3 μm). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network. PMID:20889982

All human Na(+)-K(+)-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides.

PubMed

Wang, J; Velotta, J B; McDonough, A A; Farley, R A

2001-10-01

Three alpha-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (K(d)) for all samples calculated using the single-site model was 18 +/- 6 nM (mean +/- SD). No difference in K(d) was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only approximately 50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha-isoforms are similar to each other, although calculated K(d) values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

Humic Acid Complexation of Th, Hf and Zr in Ligand Competition Experiments: Metal Loading and Ph Effects

NASA Technical Reports Server (NTRS)

Stern, Jennifer C.; Foustoukos, Dionysis I.; Sonke, Jeroen E.; Salters, Vincent J. M.

2014-01-01

The mobility of metals in soils and subsurface aquifers is strongly affected by sorption and complexation with dissolved organic matter, oxyhydroxides, clay minerals, and inorganic ligands. Humic substances (HS) are organic macromolecules with functional groups that have a strong affinity for binding metals, such as actinides. Thorium, often studied as an analog for tetravalent actinides, has also been shown to strongly associate with dissolved and colloidal HS in natural waters. The effects of HS on the mobilization dynamics of actinides are of particular interest in risk assessment of nuclear waste repositories. Here, we present conditional equilibrium binding constants (Kc, MHA) of thorium, hafnium, and zirconium-humic acid complexes from ligand competition experiments using capillary electrophoresis coupled with ICP-MS (CE- ICP-MS). Equilibrium dialysis ligand exchange (EDLE) experiments using size exclusion via a 1000 Damembrane were also performed to validate the CE-ICP-MS analysis. Experiments were performed at pH 3.5-7 with solutions containing one tetravalent metal (Th, Hf, or Zr), Elliot soil humic acid (EHA) or Pahokee peat humic acid (PHA), and EDTA. CE-ICP-MS and EDLE experiments yielded nearly identical binding constants for the metal- humic acid complexes, indicating that both methods are appropriate for examining metal speciation at conditions lower than neutral pH. We find that tetravalent metals form strong complexes with humic acids, with Kc, MHA several orders of magnitude above REE-humic complexes. Experiments were conducted at a range of dissolved HA concentrations to examine the effect of [HA]/[Th] molar ratio on Kc, MHA. At low metal loading conditions (i.e. elevated [HA]/[Th] ratios) the ThHA binding constant reached values that were not affected by the relative abundance of humic acid and thorium. The importance of [HA]/[Th] molar ratios on constraining the equilibrium of MHA complexation is apparent when our estimated Kc, MHA values attained at very low metal loading conditions are compared to existing literature data. Overall, experimental data suggest that the tetravalent transition metal/-actinide-humic acid complexation is important over a wide range of pH values, including mildly acidic conditions, and thus, these complexes should be included in speciation models.

Ion sensing by charge transfer absorption variations of benzocrown-bipyridinium conjugates with an alkyl chain.

PubMed

Kuwabara, Tetsuo; Satake, Ryota; Guo, Haocheng

2015-01-01

Two benzocrown ether-bipyridinium conjugates, 1 and 2, each having a different length of alkyl chains with butyl and dodecyl groups, respectively, have been synthesized for the purpose of developing a new guest-responsive color-change chemosensor. Both 1 and 2 showed yellow colors with broad absorption bands around 400 nm in acetonitrile. These are associated with the intramolecular charge transfer (CT) absorption, in which the benzocrown ether and bipyridinium units act as the donor and acceptor, respectively. Upon addition of the guest; such as Na(+), they faded in color due to the blue shift in their intramolecular charge transfer absorption bands. These are associated with the formation of 1:1 host-guest inclusion complex. Analogues, 3 and 4, both being similar in structure to 1 and 2 with non-crown ether unit, also showed intramolecular CT absorptions around 400 nm, but did not change their absorption spectra upon addition of the guest because of the lack of guest-binding abilities. The guest-induced color change of 1 and 2 can be used for alkali and alkaline metal ion sensing. Both 1 and 2 could detect divalent cations such as Mg(2+) and Ca(2+) rather than univalent ones, Li(+), Na(+), K(+), Rb(+), and Cs(+). Although a marked difference between 1 and 2 was not observed in their guest sensing abilities, the remarkable recognition of 1 and 2 for Mg(2+) and Ca(2+) was found compared with that of 5, which has benzyl unit instead of alkyl chains of 1 and 2. The sensitivity values of 1 and 2 were roughly proportional to their binding constants, as shown by the binding constants with Li(+), Na(+), Mg(2+), and Ca(2+) with the values of 910, 260, 820, and 2300 M(-1) for 1 and 930, 290, 1270, and 2790 M(-1) for 2, while the binding constants of 5 were estimated to be 930, 440, 210, and 1200 M(-1) for Li(+), Na(+), Mg(2+), and Ca(2+), respectively. The limit concentration of detection of 2 for Ca(2+) was estimated to be 0.016 mM, which was the smallest value in this system.

Ligand-receptor binding affinities from saturation transfer difference (STD) NMR spectroscopy: the binding isotherm of STD initial growth rates.

PubMed

Angulo, Jesús; Enríquez-Navas, Pedro M; Nieto, Pedro M

2010-07-12

The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental K(D) measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein-ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

Interaction of bovine serum albumin with N-acyl amino acid based anionic surfactants: Effect of head-group hydrophobicity.

PubMed

Ghosh, Subhajit; Dey, Joykrishna

2015-11-15

The function of a protein depends upon its structure and surfactant molecules are known to alter protein structure. For this reason protein-surfactant interaction is important in biological, pharmaceutical, and cosmetic industries. In the present work, interactions of a series of anionic surfactants having the same hydrocarbon chain length, but different amino acid head group, such as l-alanine, l-valine, l-leucine, and l-phenylalanine with the transport protein, bovine serum albumin (BSA), were studied at low surfactant concentrations using fluorescence and circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The results of fluorescence measurements suggest that the surfactant molecules bind simultaneously to the drug binding site I and II of the protein subdomain IIA and IIIA, respectively. The fluorescence as well as CD spectra suggest that the conformation of BSA goes to a more structured state upon surfactant binding at low concentrations. The binding constants of the surfactants were determined by the use of fluorescence as well as ITC measurements and were compared with that of the corresponding glycine-derived surfactant. The binding constant values clearly indicate a significant head-group effect on the BSA-surfactant interaction and the interaction is mainly hydrophobic in nature. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescence Spectroscopic Studies on the Complexation of Antidiabetic Drugs with Glycosylated Serum Albumin

NASA Astrophysics Data System (ADS)

Seedher, N.; Kanojia, M.

2013-11-01

Glycosylation decreases the association constant values and hence the binding affinity of human serum albumin (HSA) for the antidiabetic drugs under study. The percentage of HAS-bound drug at physiological temperature was only about 21-38 % as compared to 46-74 % for non-glycosylated HSA. Thus the percentage of free drug available for an antihyperglycemic effect was about double (62-79 %) compared to the values for non-glycosylated HSA. Much higher free drug concentrations available for pharmacological effect can lead to the risk of hypoglycemia. Hydrophobic interactions were predominantly involved in the binding. In the binding of gliclazide, hydrogen bonding and electrostatic interactions were involved. Site specificity for glycosylated HSA was the same as that for non-glycosylated HSA; gliclazide and repaglinide bind only at site II whereas glimepiride and glipizide bind at both sites I and II. Glycosylation, however, caused conformational changes in albumin, and the binding region within site II was different for glycosylated and non-glycosylated albumin. Stern-Volmer analysis also indicated the conformational changes in albumin as a result of glycosylation and showed that the dynamic quenching mechanism was valid for fluorescence of both glycosylated and non-glycosylated HSA.

Spectroscopic and microcalorimetric studies on the molecular binding of food colorant acid red 27 with deoxyribonucleic acid.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2016-08-01

Interaction of the food colorant acid red 27 with double stranded DNA was investigated using spectroscopic and calorimetric methods. Absorbance and fluorescence studies suggested an intimate binding interaction between the dye and DNA. The quantum efficiency value testified an effective energy transfer from the DNA base pairs to the dye molecules. Minor groove displacement assay with Hoechst 33258 revealed that the binding occurs in the minor groove of DNA. Circular dichroism studies revealed that acid red 27 induces moderate conformational perturbations in DNA. Results of calorimetric studies suggested that the complexation process was driven largely by positive entropic contribution with a smaller favorable enthalpy contribution. The equilibrium constant of the binding was calculated to be (3.04 ± 0.09) × 10(4)  M(-1) at 298.15 K. Negative heat capacity value along with the enthalpy-entropy compensation phenomenon established the involvement of dominant hydrophobic forces in the binding process. Differential scanning calorimetry studies presented evidence for an increased thermal stability of DNA on binding of acid red 27. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.

2016-01-01

Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250 nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p: reinterpretation of recent single molecule experiments.

PubMed

Stigter, Dirk

2004-07-01

Brewer et al. (Biophys. J. 85 (2003) 2519-2524) have studied the compaction of dsDNA in a double flow cell by observing the extension of stained DNA tethered in buffer solutions with or without Abf2p. They use a Langmuir adsorption model in which one Abf2p molecule adsorbs on one site on the DNA, and the binding constant, K, is given as the ratio of the experimental rates of adsorption and desorption. This paper presents an improved interpretation. Instead of Langmuir adsorption we use the more appropriate McGhee-von Hippel (J. Mol. Biol. 86 (1974) 469-489) theory for the adsorption of large ligands to a one-dimensional lattice. We assume that each adsorbed molecule shortens the effective contour length of DNA by the foot print of Abf2p of 27 base pairs. When Abf2p adsorbs to DNA stretched in the flowing buffer solution, we account for a tension effect that decreases the adsorption rate and the binding constant by a factor of 2 to 4. The data suggest that the accessibility to Abf2p decreases significantly with increasing compaction of DNA, resulting in a lower adsorption rate and a lower binding constant. The kinetics reported by Brewer et al. (Biophys. J. 85 (2003) 2519-2524) lead to a binding constant K=3.6 x 10(6) M(-1) at the beginning, and to K=5 x 10(5) M(-1) near the end of a compaction run, more than an order of magnitude lower than the value K=2.57 x 10(7) M(-1) calculated by Brewer et al. (Biophys. J. 85 (2003) 2519-2524).

Influence of Analyte Concentration on Stability Constant Values Determined by Capillary Electrophoresis.

PubMed

Sursyakova, Viktoria V; Burmakina, Galina V; Rubaylo, Anatoly I

2016-08-01

The influence of analyte concentration when compared with the concentration of a charged ligand in background electrolyte (BGE) on the measured values of electrophoretic mobilities and stability constants (association, binding or formation constants) is studied using capillary electrophoresis (CE) and a dynamic mathematical simulator of CE. The study is performed using labile complexes (with fast kinetics) of iron (III) and 5-sulfosalicylate ions (ISC) as an example. It is shown that because the ligand concentration in the analyte zone is not equal to that in BGE, considerable changes in the migration times and electrophoretic mobilities are observed, resulting in systematic errors in the stability constant values. Of crucial significance is the slope of the dependence of the electrophoretic mobility decrease on the ligand equilibrium concentration. Without prior information on this dependence to accurately evaluate the stability constants for similar systems, the total ligand concentration must be at least >50-100 times higher than the total concentration of analyte. Experimental ISC peak fronting and the difference between the direction of the experimental pH dependence of the electrophoretic mobility decrease and the mathematical simulation allow assuming the presence of capillary wall interaction. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

PubMed

Hiner, Alexander N P; Sidrach, Lara; Chazarra, Soledad; Varón, Ramón; Tudela, José; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

2004-01-01

The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.

Serum zinc, copper, retinol-binding protein, prealbumin, and ceruloplasmin concentrations in infants receiving intravenous zinc and copper supplementation.

PubMed

Lockitch, G; Godolphin, W; Pendray, M R; Riddell, D; Quigley, G

1983-02-01

One hundred twenty-seven newborn infants requiring parenteral nutrition were randomly assigned to receive differing amounts of zinc (40 to 400 micrograms/kg/day) and copper (20 or 40 micrograms/kg/day) supplementation within five birth weight groups (600 to 2,500 gm). The serum zinc concentration remained relatively constant in the group receiving the most zinc supplementation after two weeks of therapy, but declined sharply in the groups receiving less supplementation. No effect of increased copper intake was noted on ceruloplasmin values, but a difference in serum copper concentrations was noted at two weeks. No correlation was noted between serum zinc and copper values or among those for serum zinc, retinol-binding protein, and prealbumin. Reference ranges were defined for serum zinc, copper, retinol-binding protein, prealbumin, and ceruloplasmin in the preterm infant.

A Printed Equilibrium Dialysis Device with Integrated Membranes for Improved Binding Affinity Measurements.

PubMed

Pinger, Cody W; Heller, Andrew A; Spence, Dana M

2017-07-18

Equilibrium dialysis is a simple and effective technique used for investigating the binding of small molecules and ions to proteins. A three-dimensional (3D) printer was used to create a device capable of measuring binding constants between a protein and a small ion based on equilibrium dialysis. Specifically, the technology described here enables the user to customize an equilibrium dialysis device to fit their own experiments by choosing membranes of various material and molecular-weight cutoff values. The device has dimensions similar to that of a standard 96-well plate, thus being amenable to automated sample handlers and multichannel pipettes. The device consists of a printed base that hosts multiple windows containing a porous regenerated-cellulose membrane with a molecular-weight cutoff of ∼3500 Da. A key step in the fabrication process is a print-pause-print approach for integrating membranes directly into the windows subsequently inserted into the base. The integrated membranes display no leaking upon placement into the base. After characterizing the system's requirements for reaching equilibrium, the device was used to successfully measure an equilibrium dissociation constant for Zn 2+ and human serum albumin (K d = (5.62 ± 0.93) × 10 -7 M) under physiological conditions that is statistically equal to the constants reported in the literature.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, G.F.; Marks, B.H.

This study examines the beta adrenergic receptors of the rabbit detrusor smooth muscle, employing (/sup 125/I)iodocyanopindolol (ICYP) as a ligand for the binding of beta adrenergic receptors. Saturation binding experiments on the isolated membrane fraction yielded a KD for ICYP of 14.7 pM and a maximum binding of 147.6 fmol/mg of protein. Displacement of labeled ICYP by a series of beta adrenergic agents yielded the following KD values for the combined high and low affinity binding sites: I-propranolol, 0.76 nM; ICI 118,551, 1.7 nM; zinterol, 38.0 nM; metoprolol, 3.5 microM; and practolol, 61.4 microM. When these displacement experimental results weremore » compared to KD values from other reported binding studies with ICYP for beta adrenoreceptors, both the order of potency and the KD values indicated primarily beta-2 adrenergic receptor subtypes. Computer program Scatfit analysis of the displacement curves indicated a single slope and affinity constant for all five beta adrenergic agents. Hofstee plots for zinterol, ICI 118,551 and metoprolol, however, were not linear and indicated that minor populations of beta-1 adrenoreceptors were also present as both high and low affinity binding sites could be defined. It is concluded that the primary receptor population is beta-2 and that this tissue is heterogenous with a small population of beta-1 adrenoreceptors representing approximately 13 to 23% of the total beta adrenoreceptor population.« less

A calorimetric investigation of the interaction of the lac repressor with inducer.

PubMed

Donnér, J; Caruthers, M H; Gill, S J

1982-12-25

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.

Nickel-quinolones interaction. Part 4. Structure and biological evaluation of nickel(II)-enrofloxacin complexes compared to zinc(II) analogues.

PubMed

Skyrianou, Kalliopi C; Psycharis, Vassilis; Raptopoulou, Catherine P; Kessissoglou, Dimitris P; Psomas, George

2011-01-01

The nickel(II) complexes with the second-generation quinolone antibacterial agent enrofloxacin in the presence or absence of the nitrogen-donor heterocyclic ligands 1,10-phenanthroline, 2,2'-bipyridine or pyridine have been synthesized and characterized. Enrofloxacin acts as bidentate ligand coordinated to Ni(II) ion through the ketone oxygen and a carboxylato oxygen. The crystal structure of (1,10-phenanthroline)bis(enrofloxacinato)nickel(II) has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and bis(pyridine)bis(enrofloxacinato)nickel(II) exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the complexes have been evaluated in comparison to the corresponding Zn(II) enrofloxacinato complexes as well as Ni(II) complexes with the first-generation quinolone oxolinic acid. Copyright © 2010 Elsevier Inc. All rights reserved.

Steric Effects on the Binding of Phosphate and Polyphosphate Anions by Zinc(II) and Copper(II) Dinuclear Complexes of m-Xylyl-bis-cyclen.

PubMed

Esteves, Catarina V; Esteban-Gómez, David; Platas-Iglesias, Carlos; Tripier, Raphaël; Delgado, Rita

2018-05-11

The triethylbenzene-bis-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) compound (tbmce) was designed with an imposed structural rigidity at the m-xylyl spacer to be compared to a less restrained and known parent compound (bmce). The framework of both compounds differs only in the substituents of the m-xylyl spacer. The study was centered in the differences observed in the acid-base reactions of both compounds, their copper(II) and zinc(II) complexation behaviors, as well as in the uptake of phosphate and polyphosphate anions (HPPi 3- , ATP 4- , ADP 3- , AMP 2- , PhPO 4 2- , and HPO 4 2- ). On the one hand, the acid-base reactions showed lower values for the third and fourth protonation constants of tbmce than for bmce, suggesting that the ethyl groups of the spacer in tbmce force the two cyclen units to more conformational restricted positions. On the other hand, the stability constant values for copper(II) and zinc(II) complexes revealed that bmce is a better chelator than tbmce pointing out to additional conformational restraints imposed by the triethylbenzene spacer. The binding studies of phosphates by the dinuclear copper(II) and zinc(II) complexes showed much smaller effective association constants for the dicopper complexes. Single-crystal X-ray and computational (density functional theory) studies suggest that anion binding promotes the formation of tetranuclear entities in which anions are bridging the metal centers. Our studies also revealed the dinuclear zinc(II) complex of bmce as a promising receptor for phosphate anions, with the largest effective association constant of 5.94 log units being observed for the formation of [Zn 2 bmce(HPPi)] + . Accordingly, a colorimetric study via an indicator displacement assay to detect phosphates in aqueous solution found that the [Zn 2 bmce] 4+ complex acts as the best receptor for pyrophosphate displaying a detection limit of 2.5 nM by changes visible to naked eye.

p-( sup 125 I)iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerhardt, M.A.; Wade, S.M.; Neubig, R.R.

1990-08-01

The binding properties of p-(125I)iodoclonidine (( 125I)PIC) to human platelet membranes and the functional characteristics of PIC are reported. (125I)PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific (125I)PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. (125I)PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist (3H) bromoxidine (UK14,304), which representedmore » approximately 40% of the sites bound by the antagonist (3H)yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of (125I)PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for (125I)PIC binding was stereoselective. Competition for (3H)bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for (3H)yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. (125I)PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM.« less

Measurement of free glucocorticoids: quantifying corticosteroid-binding globulin binding affinity and its variation within and among mammalian species.

PubMed

Delehanty, Brendan; Hossain, Sabrina; Jen, Chao Ching; Crawshaw, Graham J; Boonstra, Rudy

2015-01-01

Plasma glucocorticoids (GCs) are commonly used as measures of stress in wildlife. A great deal of evidence indicates that only free GC (GC not bound by the specific binding protein, corticosteroid-binding globulin, CBG) leaves the circulation and exerts biological effects on GC-sensitive tissues. Free hormone concentrations are difficult to measure directly, so researchers estimate free GC using two measures: the binding affinity and the binding capacity in plasma. We provide an inexpensive saturation binding method for calculating the binding affinity (equilibrium dissociation constant, K d) of CBG that can be run without specialized laboratory equipment. Given that other plasma proteins, such as albumin, also bind GCs, the method compensates for this non-specific binding. Separation of bound GC from free GC was achieved with dextran-coated charcoal. The method provides repeatable estimates (12% coefficient of variation in the red squirrel, Tamiasciurus hudsonicus), and there is little evidence of inter-individual variation in K d (range 2.0-7.3 nM for 16 Richardson's ground squirrels, Urocitellus richardsonii). The K d values of 28 mammalian species we assessed were mostly clustered around a median of 4 nM, but five species had values between 13 and 61 nM. This pattern may be distinct from birds, for which published values are more tightly distributed (1.5-5.1 nM). The charcoal separation method provides a reliable and robust method for measuring the K d in a wide range of species. It uses basic laboratory equipment to provide rapid results at very low cost. Given the importance of CBG in regulating the biological activity of GCs, this method is a useful tool for physiological ecologists.

Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes.

PubMed

Anbazhagan, V; Sankhala, Rajeshwer S; Singh, Bhanu Pratap; Swamy, Musti J

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Isothermal Titration Calorimetric Studies on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109 with Phospholipid Membranes

PubMed Central

Anbazhagan, V.; Sankhala, Rajeshwer S.; Singh, Bhanu Pratap; Swamy, Musti J.

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process. PMID:22022488

Determination of human serum albumin using an intramolecular charge transfer fluorescence probe: 4'-dimethylamino-2,5-dihydroxychalcone.

PubMed

Xu, Zhicheng; Yang, Weibing; Dong, Chuan

2005-09-15

A new intramolecular charge transfer fluorescence probe, namely, 4'-dimethylamino-2,5-dihydroxychalcone (DMADHC), exhibited dramatic enhancement of fluorescence intensity with an accompanying blue shift of the emission maximum when the concentration of human serum albumin (HSA) was increased. Binding to HSA also caused a progressive shift in the absorption spectrum of DMADHC, and a clear isosbestic point appeared. The binding site number and binding constant were calculated. Thermodynamic parameters were given and possible binding site was speculated. The optimum conditions for the determination of HSA were also investigated. A new, fast, and simple spectrofluorimetric method for the determination of HSA was developed. In the detection of HSA in samples of human plasma, this method gave values close to that of the Erythrosin B method.

State-dependent compound inhibition of Nav1.2 sodium channels using the FLIPR Vm dye: on-target and off-target effects of diverse pharmacological agents.

PubMed

Benjamin, Elfrida R; Pruthi, Farhana; Olanrewaju, Shakira; Ilyin, Victor I; Crumley, Gregg; Kutlina, Elena; Valenzano, Kenneth J; Woodward, Richard M

2006-02-01

Voltage-gated sodium channels (NaChs) are relevant targets for pain, epilepsy, and a variety of neurological and cardiac disorders. Traditionally, it has been difficult to develop structure-activity relationships for NaCh inhibitors due to rapid channel kinetics and state-dependent compound interactions. Membrane potential (Vm) dyes in conjunction with a high-throughput fluorescence imaging plate reader (FLIPR) offer a satisfactory 1st-tier solution. Thus, the authors have developed a FLIPR Vm assay of rat Nav1.2 NaCh. Channels were opened by addition of veratridine, and Vm dye responses were measured. The IC50 values from various structural classes of compounds were compared to the resting state binding constant (Kr)and inactivated state binding constant (Ki)obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with Ki but not Kr. FLIPRIC50 values fell within 0.1-to 1.5-fold of EP Ki values, indicating that the assay generally reports use-dependent inhibition rather than resting state block. The Library of Pharmacologically Active Compounds (LOPAC, Sigma) was screened. Confirmed hits arose from diverse classes such as dopamine receptor antagonists, serotonin transport inhibitors, and kinase inhibitors. These data suggest that NaCh inhibition is inherent in a diverse set of biologically active molecules and may warrant counterscreening NaChs to avoid unwanted secondary pharmacology.

Direct Measurement of Equilibrium Constants for High-Affinity Hemoglobins

PubMed Central

Kundu, Suman; Premer, Scott A.; Hoy, Julie A.; Trent, James T.; Hargrove, Mark S.

2003-01-01

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (KD ≪ 1 μM) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and ∼100 μM−1 respectively, indicate that they are not capable of facilitating oxygen transport. PMID:12770899

Ternary borate-nucleoside complex stabilization by Ribonuclease A demonstrates phosphate mimicry

PubMed Central

Gabel, Scott A.; London, Robert E.

2010-01-01

Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate-ester recognition sites on proteins might exhibit significant affinity for non-enzymatically formed borate esters. 11B NMR studies and activity measurements on ribonuclease A in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A•3′-deoxycytidine-2′-borate ternary complex that mimics the complex formed between RNase A and a 2′-cytidine monophosphate (2′-CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2′-deoxycytidine mixture, demonstrating the critical importance of the 2′-hydroxyl group for complex formation. Titration of the ternary complex with 2′-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant of RNase A by 2′CMP. RNase A binding of a cyclic cytidine-2′,3′-borate ester, which is a structural homolog of the cytidine-2′,3′-cyclic phosphate substrate, could also be demonstrated. The apparent dissociation constant for the cytidine-2′,3′-borate•RNase A complex is 0.8 mM, which compares with a Michaelis constant of 11 mM for cCMP at pH 7, indicating considerably stronger binding. However, the value is 1000-fold larger than the reported dissociation constant of the RNase A complex with uridine-vanadate. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters support enzyme catalysis. PMID:17957392

Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters

PubMed Central

Hirayama, Bruce A; Díez-Sampedro, Ana; Wright, Ernest M

2001-01-01

Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7×12Å. Important hydrogen bond interactions occur at five positions bordering this surface. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site ∼8Å from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors. PMID:11588102

Quantitative analyses of bifunctional molecules.

PubMed

Braun, Patrick D; Wandless, Thomas J

2004-05-11

Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications.

Effect of long-chain Fatty acids on the binding of triflupromazine to human serum albumin: a spectrophotometric study.

PubMed

Kitamura, Keisuke; Takegami, Shigehiko; Tanaka, Rumi; Omran, Ahmed Ahmed; Kitade, Tatsuya

2014-01-01

Human serum albumin (HSA) in the blood binds long-chain fatty acids (LCFAs), and the number of bound LCFAs varies from 1 to 7 depending on the physical condition of the body. In this study, the influence of LCFA-HSA binding on drug-HSA binding was studied using triflupromazine (TFZ), a psychotropic phenothiazine drug, in a buffer (0.1 M NaCl, pH 7.40, 37°C) by a second-derivative spectrophotometric method which can suppress the residual background signal effects of HSA observed in the absorption spectra. The examined LCFAs were caprylic acid (CPA), lauric acid (LRA), oleic acid (OLA), and linoleic acid (LNA), respectively. Using the derivative intensity change of TFZ induced by the addition of HSA containing LCFA, the binding mode of TFZ was predicted to be a partition-like nonspecific binding. The binding constant (K M(-1)) showed an increase according to the LCFA content in HSA for LRA, OLA, and LNA up to an LCFA/HSA molar ratio of 3-4. However, at higher ratios the K value decreased, i.e. for OLA and LNA, at an LCFA/HSA ratio of 6-7, the K value decreased to 40% of the value for HSA alone. In contrast, CPA, having the shortest chain length (8 carbons) among the studied LCFAs, induced a 20% decrease in the K value regardless of its content in HSA. Since the pharmacological activity of a drug is closely related to the unbound drug concentration in the blood, the results of the present study are pharmaco-kinetically, pharmacologically, and clinically very important.

Deciphering the mechanism of interaction of edifenphos with calf thymus DNA

NASA Astrophysics Data System (ADS)

Ahmad, Ajaz; Ahmad, Masood

2018-01-01

Edifenphos is an important organophosphate pesticide with many antifungal and anti-insecticidal properties but it may cause potential hazards to human health. In this work, we have tried to explore the binding mode of action and mechanism of edifenphos to calf thymus DNA (CT-DNA). Several experiments such as ultraviolet-visible absorption spectra and emission spectroscopy showed complex formation between edifenphos and CT-DNA and low binding constant values supporting groove binding mode. These results were further confirmed by circular dichroism (CD), CT-DNA melting studies, viscosity measurements, density functional theory and molecular docking. CD study suggests that edifenphos does not alter native structure of CT-DNA. Isothermal calorimetry reveals that binding of edifenphos with CT-DNA is enthalpy driven process. Competitive binding assay and effect of ionic strength showed that edifenphos binds to CT-DNA via groove binding manner. Hence, edifenphos is a minor groove binder preferably interacting with A-T regions with docking score - 6.84 kJ/mol.

Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crankshaw, D.; Gaspar, V.; Pliska, V.

1990-01-01

The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The bindingmore » parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.« less

Interaction between Pin1 and its natural product inhibitor epigallocatechin-3-gallate by spectroscopy and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Xi, Lei; Wang, Yu; He, Qing; Zhang, Qingyan; Du, Linfang

2016-12-01

The binding of epigallocatechin-3-gallate (EGCG) to wild type Pin1 in solution was studied by spectroscopic methods and molecular dynamics simulations in this research to explore the binding mode and inhibition mechanism. The binding constants and number of binding sites per Pin1 for EGCG were calculated through the Stern-Volmer equation. The values of binding free energy and thermodynamic parameters were calculated and indicated that hydrogen bonds, electrostatic interaction and Van der Waals interaction played the major role in the binding process. The alterations of Pin1 secondary structure in the presence of EGCG were confirmed by far-UV circular dichroism spectra. The binding model at atomic-level revealed that EGCG was bound to the Glu12, Lys13, Arg14, Met15 and Arg17 in WW domain. Furthermore, EGCG could also interact with Arg69, Asp112, Cys113 and Ser114 in PPIase domain.

A Comparative Study on the Photophysics and Photochemistry of Xanthene Dyes in the Presence of Polyamidoamine (PAMAM) Dendrimers.

PubMed

Arbeloa, Ernesto Maximiliano; Previtali, Carlos Mario; Bertolotti, Sonia Graciela

2018-04-17

The photophysical and photochemical properties of the xanthene dyes Eosin Y, Erythrosin B, and Rose Bengal are evaluated in the presence of amino-terminated polyamidoamine (PAMAM) dendrimers of relatively high generation (G3-G5) in alkaline aqueous solution. UV/Vis absorption and fluorescence spectra of the dyes show bathochromic shifts, which correlate with the size of the dendrimer. Binding constants (K bind ) are calculated from absorption data. The resulting high K bind values indicate strong interactions between both molecules. Triplet-triplet absorption spectra of the dyes are recorded by laser flash photolysis, and a decrease in the triplet lifetimes is observed in the presence of dendrimers. At the same time, an increase in the absorption of the semireduced form of the dyes is observed. Rate constants for triplet quenching ( 3 k q ) and radical quantum yields (Φ R ) are obtained. The results are explained by a very efficient electron-transfer process from PAMAM to xanthene dyes for all of the dye/dendrimer couples that are evaluated. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

In silico studies and fluorescence binding assays of potential anti-prion compounds reveal an important binding site for prion inhibition from PrP(C) to PrP(Sc).

PubMed

Pagadala, Nataraj S; Perez-Pineiro, Rolando; Wishart, David S; Tuszynski, Jack A

2015-02-16

To understand the pharmacophore properties of 2-aminothiazoles and design novel inhibitors against the prion protein, a highly predictive 3D quantitative structure-activity relationship (QSAR) has been developed by performing comparative molecular field analysis (CoMFA) and comparative similarity analysis (CoMSIA). Both CoMFA and CoMSIA maps reveal the presence of the oxymethyl groups in meta and para positions on the phenyl ring of compound 17 (N-[4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-yl]quinolin-2-amine), is necessary for activity while electro-negative nitrogen of quinoline is highly favorable to enhance activity. The blind docking results for these compounds show that the compound with quinoline binds with higher affinity than isoquinoline and naphthalene groups. Out of 150 novel compounds retrieved using finger print analysis by pharmacophoric model predicted based on five test sets of compounds, five compounds with diverse scaffolds were selected for biological evaluation as possible PrP inhibitors. Molecular docking combined with fluorescence quenching studies show that these compounds bind to pocket-D of SHaPrP near Trp145. The new antiprion compounds 3 and 6, which bind with the interaction energies of -12.1 and -13.2 kcal/mol, respectively, show fluorescence quenching with binding constant (Kd) values of 15.5 and 44.14 μM, respectively. Further fluorescence binding assays with compound 5, which is similar to 2-aminothiazole as a positive control, also show that the molecule binds to the pocket-D with the binding constant (Kd) value of 84.7 μM. Finally, both molecular docking and a fluorescence binding assay of noscapine as a negative control reveals the same binding site on the surface of pocket-A near a rigid loop between β2 and α2 interacting with Arg164. This high level of correlation between molecular docking and fluorescence quenching studies confirm that these five compounds are likely to act as inhibitors for prion propagation while noscapine might act as a prion accelerator from PrP(C) to PrP(Sc). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Water miscible mono alcohols' effect on the proteolytic performance of Bacillus clausii serine alkaline protease.

PubMed

Duman, Yonca Avci; Kazan, Dilek; Denizci, Aziz Akin; Erarslan, Altan

2014-01-01

In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the Vm, kcat and kcat/Km values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the Km value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (KI) were in the range of 1.32-3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG(≠) and ΔG(≠)E-T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG(≠)ES value of the enzyme remained almost the same. The constant Km and ΔG(≠)ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The kcat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG(≠) and ΔG(≠)E-T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

PubMed

Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

2013-11-01

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Prediction of trivalent actinide amino(poly)carboxylate complex stability constants using linear free energy relationships with the lanthanide series

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uhnak, Nic E.

Prediction of Trivalent Actinide Amino(poly)carboxylate Complex Stability Constants Using Linear Free Energy Relationships with the Lanthanide Series Alternative title: LFER Based Prediction of An(III) APC Stability Constants There is a gap in the literature regarding the complexation of amino(poly)carboxylate (APC) ligands with trivalent actinides (An(III))). The chemistry of the An(III) is nearly identical to that of the trivalent lanthanides Lns, but the An(III) express a slight enhancement when binding APC ligands. Presented in this report is a simple method of predicting the stability constants of the An(III), Pu, Am, Cm, Bk and Cf by using linear free energy relationships (LFER)more » of the An and the lanthanide (Ln) series for 91 APCs. This method produced An stability constants within uncertainty to available literature values for most ligands.« less

Evaluation of DNA, BSA binding, and antimicrobial activity of new synthesized neodymium complex containing 29-dimethyl 110-phenanthroline.

PubMed

Moradi, Zohreh; Khorasani-Motlagh, Mozhgan; Rezvani, Ali Reza; Noroozifar, Meissam

2018-02-01

In order to evaluate biological potential of a novel synthesized complex [Nd(dmp) 2 Cl 3 .OH 2 ] where dmp is 29-dimethyl 110-phenanthroline, the DNA-binding, cleavage, BSA binding, and antimicrobial activity properties of the complex are investigated by multispectroscopic techniques study in physiological buffer (pH 7.2).The intrinsic binding constant (K b ) for interaction of Nd(III) complex and FS-DNA is calculated by UV-Vis (K b  = 2.7 ± 0.07 × 10 5 ) and fluorescence spectroscopy (K b  = 1.13 ± 0.03 × 10 5 ). The Stern-Volmer constant (K SV ), thermodynamic parameters including free energy change (ΔG°), enthalpy change (∆H°), and entropy change (∆S°), are calculated by fluorescent data and Vant' Hoff equation. The experimental results show that the complex can bind to FS-DNA and the major binding mode is groove binding. Meanwhile, the interaction of Nd(III) complex with protein, bovine serum albumin (BSA), has also been studied by using absorption and emission spectroscopic tools. The experimental results show that the complex exhibits good binding propensity to BSA. The positive ΔH° and ∆S° values indicate that the hydrophobic interaction is main force in the binding of the Nd(III) complex to BSA, and the complex can quench the intrinsic fluorescence of BSA remarkably through a static quenching process. Also, DNA cleavage was investigated by agarose gel electrophoresis that according to the results cleavage of DNA increased with increasing of concentration of the complex. Antimicrobial screening test gives good results in the presence of Nd(III) complex system.

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

2011-11-01

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ( 1HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants Ka were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants KaI of PBZ-SA complex. Similarly, PBZ influences KaI of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of KaII values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of 1HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

Determination of the binding properties of p-cresyl glucuronide to human serum albumin.

PubMed

Yi, Dan; Monteiro, Elisa Bernardes; Chambert, Stéphane; Soula, Hédi A; Daleprane, Julio B; Soulage, Christophe O

2018-04-26

p-Cresyl glucuronide (p-CG) is a by-product of tyrosine metabolism that accumulates in patients with end-stage renal disease. p-CG binding to human serum albumin in physiological conditions (37°C, pH 7.40) was studied by ultrafiltration (MWCO 10 kDa) and data were analyzed assuming one binding site. The estimated value of the association constant was 2.77×10 3  M -1 and a maximal stoichiometry of 3.80 mol per mole. At a concentration relevant for end-stage renal patients, p-CG was 23% bound to albumin. Competition experiments, using fluorescent probes, demonstrated that p-CG did not bind to Sudlow's site I or site II. The p-CG did not interfere with the binding of p-cresyl-sulfate or indoxyl sulfate to serum albumin. Copyright © 2018. Published by Elsevier B.V.

Inner reorganization limiting electron transfer controlled hydrogen bonding: intra- vs. intermolecular effects.

PubMed

Martínez-González, Eduardo; Frontana, Carlos

2014-05-07

In this work, experimental evidence of the influence of the electron transfer kinetics during electron transfer controlled hydrogen bonding between anion radicals of metronidazole and ornidazole, derivatives of 5-nitro-imidazole, and 1,3-diethylurea as the hydrogen bond donor, is presented. Analysis of the variations of voltammetric EpIcvs. log KB[DH], where KB is the binding constant, allowed us to determine the values of the binding constant and also the electron transfer rate k, confirmed by experiments obtained at different scan rates. Electronic structure calculations at the BHandHLYP/6-311++G(2d,2p) level for metronidazole, including the solvent effect by the Cramer/Truhlar model, suggested that the minimum energy conformer is stabilized by intramolecular hydrogen bonding. In this structure, the inner reorganization energy, λi,j, contributes significantly (0.5 eV) to the total reorganization energy of electron transfer, thus leading to a diminishment of the experimental k.

Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach

NASA Astrophysics Data System (ADS)

Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

2016-04-01

In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 104. With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300K was calculated as - 5.234 kcal mol- 1 for CBZ-AAG interaction and - 6.237 kcal mol- 1 for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are - 9.553 kcal mol- 1 and - 14.618 cal mol- 1K- 1 respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol- 1 and 7.206 cal mol- 1K- 1 respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

Testing the Underlying Chemical Principles of the Biotic Ligand Model (BLM) to Marine Copper Systems: Measuring Copper Speciation Using Fluorescence Quenching.

PubMed

Tait, Tara N; McGeer, James C; Smith, D Scott

2018-01-01

Speciation of copper in marine systems strongly influences the ability of copper to cause toxicity. Natural organic matter (NOM) contains many binding sites which provides a protective effect on copper toxicity. The purpose of this study was to characterize copper binding with NOM using fluorescence quenching techniques. Fluorescence quenching of NOM with copper was performed on nine sea water samples. The resulting stability constants and binding capacities were consistent with literature values of marine NOM, showing strong binding with [Formula: see text] values from 7.64 to 10.2 and binding capacities ranging from 15 to 3110 nmol mg [Formula: see text] Free copper concentrations estimated at total dissolved copper concentrations corresponding to previously published rotifer effect concentrations, in the same nine samples, were statistically the same as the range of free copper calculated for the effect concentration in NOM-free artificial seawater. These data confirms the applicability of fluorescence spectroscopy techniques for NOM and copper speciation characterization in sea water and demonstrates that such measured speciation is consistent with the chemical principles underlying the biotic ligand model approach for bioavailability-based metals risk assessment.

The Binding Constant of Estradiol to Bovine Serum Albumin: An Upper-Level Experiment Utilizing Tritium-Labeled Estradiol and Liquid Scintillation Counting

ERIC Educational Resources Information Center

Peihong Liang; Adhyaru, Bhavin; Pearson, Wright L.; Williams, Kathryn R.

2006-01-01

The experiment used [to the third power]H-labeled estradiol to determine the binding constant of estradiol to bovine serum albumin. Estradiol must complex with serum proteins for the transport in the blood stream because of its low solubility in aqueous systems and estradiol-protein binding constant, where K[subscript B] is important to understand…

Kinetic and thermodynamic studies of bovine serum albumin interaction with ascorbyl palmitate and ascorbyl stearate food additives using surface plasmon resonance.

PubMed

Fathi, Farzaneh; Mohammadzadeh-Aghdash, Hossein; Sohrabi, Yousef; Dehghan, Parvin; Ezzati Nazhad Dolatabadi, Jafar

2018-04-25

Ascorbyl palmitate (AP) and ascorbyl stearate (AS) are examples of food additives, which have extensive use in food industry. In this study, we evaluated the interaction of bovine serum albumin (BSA) with AP and AS using surface plasmon resonance (SPR). In order to immobilize BSA, carboxymethyl dextran hydrogel (CMD) Au chip was used. After activation of carboxylic groups, BSA was immobilized onto the CMD chip through covalent amide binding formation. AP and AS binding to immobilized BSA at different concentrations was assessed. The dose-response sensorgrams of BSA upon increasing concentration of AP and AS have been shown. The low value of equilibrium dissociation constant or affinity unit (K D ) showed high affinity of both AP and AS to BSA. The K D value for binding of AP and AS to BSA were 4.09 × 10 -5 and 1.89 × 10 -5 , at 25 °C. Overall, the attained results showed that AP and AS molecules can bind to BSA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Milk β-casein as a vehicle for delivery of bis(indolyl)methane: Spectroscopy and molecular docking studies

NASA Astrophysics Data System (ADS)

Dezhampanah, Hamid; Esmaili, Masoomeh; Khorshidi, Alireza

2017-05-01

The interaction of bis(indolyl)methane with bovine milk β-casein was investigated using spectroscopy and molecular docking studies at different temperatures (25-37 °C). The circular dichroism and Fourier transform infrared spectroscopic data demonstrated that β-casein interacts with BIM molecule mainly via both the hydrophobic and hydrophilic interactions with a minor change in the secondary structure of β-casein. The fluorescence quenching measurements revealed that the presence of a single binding site on β-casein for BIM with the binding constant value of ∼104 M-1. The negative values of entropy and enthalpy changes confirm the predominate role of hydrogen binding and van der Waals interactions in the binding process. Fӧrster energy transfer measurement suggested that the distance between bound BIM and Trp residue is higher than the respective critical distance. Hence, the static quenching is more likely responsible for the fluorescence quenching rather than the mechanism of non-radiative. Docking study showed that BIM molecule forms three hydrogen bonds and several van der Waals contacts with β-casein.

Evaluation of BTEX and phenol removal from aqueous solution by multi-solute adsorption onto smectite organoclay.

PubMed

Carvalho, M N; da Motta, M; Benachour, M; Sales, D C S; Abreu, C A M

2012-11-15

The removal process of BTEX and phenol was evaluated. The smectite organoclay for single-solute system reached removal was evaluated by adsorption on smectite organoclay adsorbent by kinetic and equilibrium efficiencies between 55 and 90% while was reached between 30 and 90% for multi-solute system at 297 K and pH 9. The Langmuir-Freundlich model was used to fit the experimental data with correlation coefficient between 0.98 and 0.99 providing kinetic and equilibrium parameter values. Phenol and ethylbenzene presented high maximum adsorbed amount, 8.28 and 6.67 mg/g, respectively, compared to the other compounds for single-solute. Toluene and p-xylene presented high values of adsorption constant which indicates a high adsorption affinity of compounds to organoclay surface and high binding energy of adsorption. Phenol presented low kinetic adsorption constant value indicating slow rate of adsorption. Copyright © 2012 Elsevier B.V. All rights reserved.

Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bray, J.J.; Drachman, D.B.

1982-01-01

Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the rangemore » of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.« less

Binding affinities of cationic dyes in the presence of activated charcoal and anionic surfactant in the premicellar region

NASA Astrophysics Data System (ADS)

Ali, Farman; Ibrahim, Muhammad; Khan, Fawad; Bibi, Iram; Shah, Syed W. H.

2018-03-01

Binding preferences of cationic dyes malachite green and methylene blue in a mixed charcoal-sodium dodecyl sulfate system have been investigated using UV-visible absorption spectroscopy. The dye adsorption shows surfactant-dependent patterns, indicating diverse modes of interactions. At low surfactant concentration, a direct binding to charcoal is preferred. Comparatively greater quantities of surfactant lead to attachment of dye-surfactant complex to charcoal through hydrophobic interactions. A simple model was employed for determination of equilibrium constant K eq and concentration of dye-surfactant ion pair N DS for both dyes. The values of binding parameters revealed that malachite green was directly adsorbed onto charcoal, whereas methylene blue was bound through surfactant monomers. The model is valid for low surfactant concentrations in the premicellar region. These findings have significance for material and environmental sciences.

Processes and kinetics of Cd2+ sorption by a calcareous aquifer sand

USGS Publications Warehouse

Fuller, C.C.; Davis, J.A.

1987-01-01

The rate of Cd2+ sorption by a calcareous aquifer sand was characterized by two reaction steps, with the first step reaching completion in 24 hours. The second step proceeded at a slow and nearly constant rate for at least seven days. The first step includes a fast adsorption reaction which is followed by diffusive transport into either a disordered surface film of hydrated calcium carbonate or into pore spaces. After 24 hours the rate of Cd2+ sorption was constant and controlled by the rate of surface coprecipitation, as a solid solution of CdCO3 in CaCO3 formed in recrystallizing material. Desorption of Cd2+ from the sand was slow. Clean grains of primary minerals, e.g. quartz and aluminosilicates. sorbed much less Cd2+ than grains which had surface patches of secondary minerals, e.g. carbonates, iron and manganese oxides. Calcite grains sorbed the greatest amount of Cd2+ on a weight-normalized basis despite the greater abundance of quartz. A method is illustrated for determining empirical binding constants for trace metals at in situ pH values without introducing the experimental problem of supersaturation. The binding constants are useful for solute transport models which include a computation of aqueous speciation. ?? 1987.

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

PubMed Central

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-01-01

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

PubMed

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-07-16

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Spectroscopic analyses and studies on respective interaction of cyanuric acid and uric acid with bovine serum albumin and melamine

NASA Astrophysics Data System (ADS)

Chen, Dandan; Wu, Qiong; Wang, Jun; Wang, Qi; Qiao, Heng

2015-01-01

In this work, the fluorescence quenching was used to study the interaction of cyanuric acid (CYA) and uric acid (UA) with bovine serum albumin (BSA) at two different temperatures (283 K and 310 K). The bimolecular quenching constant (Kq), apparent quenching constant (Ksv), effective binding constant (KA) and corresponding dissociation constant (KD), binding site number (n) and binding distance (r) were calculated by adopting Stern-Volmer, Lineweaver-Burk, Double logarithm and overlap integral equations. The results show that CYA and UA are both able to obviously bind to BSA, but the binding strength order is BSA + CYA < BSA + UA. And then, the interactions of CYA and UA with melamine (MEL) under the same conditions were also studied by using similar methods. The results indicates that both CYA and UA can bind together closely with melamine (MEL). It is wished that these research results would facilitate the understanding the formation of kidney stones and gout in the body after ingesting excess MEL.

Yeast hexokinase. A fluorescence temperature-jump study of the kinetics of the binding of glucose to the monomer forms of hexokinases P-I and P-II.

PubMed

Hoggett, J G; Kellett, G L

1976-09-15

The binding of glucose to the monomeric forms of hexokinases P-I and P-II in Tris and phosphate buffers at pH 8.0 in the presence of 1 mol l-1 KCl has been studied using the fluorescence temperature-jump technique. For both isozymes only one relaxation time was observed; values of tau-1 increased linearly with increasing concentration of free reacting partners. The apparent second-order rate constant for association was about 2 X 10(6) 1 mol-1 s-1 for both isozymes; the differences in the stabilities of the complexes with P-I and P-II are entirely attributable to the fact that glucose dissociates more slowly from its complex with P-I than P-II (approximately 300 s-1 and 1100 s-1 respectively). Although the kinetic data are compatible with a single-step mechanism for glucose binding the association rate constant was much lower than that expected for a diffusion-limited rate of encounter. Other mechanisms for describing an induced-fit are discussed. It is shown that the data are incompatible with a slow 'prior-isomerization' pathway of substrate binding, but are consistent with a 'substrate-guided' pathway involving isomerization of the enzyme-substrate complex.

First-Principles Study on the Gilbert Damping Constants of Transition Metal Alloys, Fe--Ni and Fe--Pt Systems

NASA Astrophysics Data System (ADS)

Sakuma, Akimasa

2012-08-01

We adapt the tight-binding linear muffin-tin orbital (TB-LMTO) method to the torque-correlation model for the Gilbert damping constant α and perform the first-principles calculation for disordered transition metal alloys, Fe--Ni and Fe--Pt systems, within the framework of the CPA. Quantitatively, the calculated α values are about one-half of the experimental values, whereas the variations in the Fermi level dependence of α are much larger than these discrepancies. As expected, we confirm in the (Fe--Ni)1-XPtX and FePt systems that Pt atoms certainly enhance α owing to their large spin--orbit coupling. For the disordered alloys, we find that α decreases with increasing chemical degree of order in a wide range.

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme.

PubMed

Chou, W Y; Chang, H P; Huang, C H; Kuo, C C; Tong, L; Chang, G G

2000-02-01

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

Comparison of solute-binding properties of plastic materials used as pharmaceutical product containers.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy

2010-01-01

Material/water equilibrium binding constants (E(b)) were determined for 11 organic solutes and 2 plastic materials commonly used in pharmaceutical product containers (plasticized polyvinyl chloride and polyolefin). In general, solute binding by the plasticized polyvinyl chloride material was greater, by nearly an order of magnitude, than the binding by the polyolefin (on an equal weight basis). The utilization of the binding constants to facilitate container compatibility assessments (e.g., drug loss by container binding) for drug-containing products is discussed.

A novel methodological approach for the analysis of host-ligand interactions.

PubMed

Strat, Daniela; Missailidis, Sotiris; Drake, Alex F

2007-02-02

Traditional analysis of drug-binding data relies upon the Scatchard formalism. These methods rely upon the fitting of a linear equation providing intercept and gradient data that relate to physical properties, such as the binding constant, cooperativity coefficients and number of binding sites. However, the existence of different binding modes with different binding constants makes the implementation of these models difficult. This article describes a novel approach to the binding model of host-ligand interactions by using a derived analytical function describing the observed signal. The benefit of this method is that physically significant parameters, that is, binding constants and number of binding sites, are automatically derived by the use of a minimisation routine. This methodology was utilised to analyse the interactions between a novel antitumour agent and DNA. An optical spectroscopy study confirms that the pentacyclic acridine derivative (DH208) binds to nucleic acids. Two binding modes can be identified: a stronger one that involves intercalation and a weaker one that involves oriented outer-sphere binding. In both cases the plane of the bound acridine ring is parallel to the nucleic acid bases, orthogonal to the phosphate backbone. Ultraviolet (UV) and circular dichroism (CD) data were fitted using the proposed model. The binding constants and the number of binding sites derived from the model remained consistent across the different techniques used. The different wavelengths at which the measurements were made maintained the coherence of the results.

Biophysical study on the interaction of ceftriaxone sodium with bovine serum albumin using spectroscopic methods.

PubMed

Pan, Jiongwei; Ye, Zaiting; Cai, Xiaoping; Wang, Liangxing; Cao, Zhuo

2012-12-01

The interaction of ceftriaxone sodium (CS), a cephalosporin antibiotic, with the major transport protein, bovine serum albumin (BSA), was investigated using different spectroscopic techniques such as fluorescence, circular dichroism (CD), and UV-vis spectroscopy. Values of binding parameters for BSA-CS interaction in terms of binding constant and number of binding sides were found to be 9.00 × 10(3), 3.24 × 10(3), and 2.30 × 10(3) M(-1) at 281, 301, and 321 K, respectively. Thermodynamic analysis of the binding data obtained at different temperatures showed that the binding process was spontaneous and was primarily mediated by van der Waals force or hydrogen bonding. CS binding to BSA caused secondary structural alterations in the protein as revealed by CD results. The distance between CS and Trp of BSA was determined as 3.23 nm according to the Förster resonance energy transfer theory. © 2012 Wiley Periodicals, Inc.

Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques

NASA Astrophysics Data System (ADS)

Katrahalli, Umesha; Jaldappagari, Seetharamappa; Kalanur, Shankara S.

2010-01-01

The interaction between human serum albumin (HSA) and fluoxetine hydrochloride (FLX) have been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism and FTIR under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of FLX to HSA. The values of binding constant, K of FLX-HSA were evaluated at 289, 300 and 310 K and were found to be 1.90 × 10 3, 1.68 × 10 3 and 1.45 × 10 3 M -1, respectively. The number of binding sites, n was noticed to be almost equal to unity thereby indicating the presence of a single class of binding site for FLX on HSA. Based on the thermodynamic parameters, Δ H0 and Δ S0 nature of binding forces operating between HSA and FLX were proposed. Spectral results revealed the conformational changes in protein upon interaction. Displacement studies indicated the site I as the main binding site for FLX on HSA. The effect of common ions on the binding of FLX to HSA was also investigated.

Lectins as endocytic ligands: an assessment of lectin binding and uptake to rabbit conjunctival epithelial cells.

PubMed

Qaddoumi, Mohamed; Lee, Vincent H L

2004-07-01

To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.

The Use of Hammett Constants to Understand the Non-Covalent Binding of Aromatics

PubMed Central

Lewis, Michael; Bagwill, Christina; Hardebeck, Laura K. E.; Wireduaah, Selina

2012-01-01

Non-covalent interactions of aromatics are important in a wide range of chemical and biological applications. The past two decades have seen numerous reports of arene-arene binding being understood in terms Hammett substituent constants, and similar analyses have recently been extended to cation-arene and anion-arene binding. It is not immediately clear why electrostatic Hammett parameters should work so well in predicting the binding for all three interactions, given that different intermolecular forces dominate each interaction. This review explores such anomalies, and summarizes how Hammett substituent constants have been employed to understand the non-covalent binding in arene-arene, cation-arene and anion-arene interactions. PMID:24688634

Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method.

PubMed

Ma, Weina; Yang, Liu; Lv, Yanni; Fu, Jia; Zhang, Yanmin; He, Langchong

2017-06-23

The equilibrium dissociation constant (K D ) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the K D value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative K D values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The K D values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the K D values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity. Copyright © 2017 Elsevier B.V. All rights reserved.

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirose, J.; Ando, S.; Kidani, Y.

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The K/sub i/ values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 x 10/sup -5/ M, very close to the K/sub i/ valuesmore » above. With arsanilazotyrosine-248 carboxypeptidase A ((Azo-CPD)Zn)), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the K/sub i/ values were (3.0-3.5) x 10/sup -5/ M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 x 10/sup -5/ M and is similar to the K/sub i/ values for ((Azo-CPD)Zn). The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates. This behavior is believed to arise by the excess zinc ions fixing the enzyme in a conformation to which the substrates cannot bind.« less

Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

PubMed Central

Alder, J Tracy; Hacksell, Uli; Strange, Philip G

2003-01-01

Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT1A serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). Ligand binding studies were used to determine dissociation constants for agonist binding to the 5-HT1A receptor: Ki values for agonists were determined in competition versus the binding of the agonist [3H]-8-OH DPAT. Competition data were all fitted best by a one-binding site model.Ki values for agonists were also determined in competition versus the binding of the antagonist [3H]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (Kh) and lower affinity (Kl) sites were determined. The ability of the agonists to activate the 5-HT1A receptor was determined using stimulation of [35S]-GTPγS binding. Maximal effects of agonists (Emax) and their potencies (EC50) were determined from concentration/response curves for stimulation of [35S]-GTPγS binding. Kl/Kh determined from ligand binding assays correlated with the relative efficacy (relative Emax) of agonists determined in [35S]-GTPγS binding assays. There was also a correlation between Kl/Kh and Kl/EC50 for agonists determined from ligand binding and [35S]-GTPγS binding assays. Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of Kl/Kh for predicting the relative efficacy of agonists. PMID:12684269

The structure and energetics of Cr(CO)6 and Cr(CO)5

NASA Technical Reports Server (NTRS)

Barnes, Leslie A.; Liu, Bowen; Lindh, Roland

1992-01-01

The geometric structure of Cr(CO)6 is optimized at the modified coupled pair functional (MCPF), single and double excitation coupled-cluster (CCSD) and CCSD(T) levels of theory (including a perturbational estimate for connected triple excitations), and the force constants for the totally symmetric representation are determined. The geometry of Cr(CO)5 is partially optimized at the MCPF, CCSD, and CCSD(T) levels of theory. Comparison with experimental data shows that the CCSD(T) method gives the best results for the structures and force constants, and that remaining errors are probably due to deficiencies in the one-particle basis sets used for CO. The total binding energies of Cr(CO)6 and Cr(CO)5 are also determined at the MCPF, CCSD, and CCSD(T) levels of theory. The CCSD(T) method gives a much larger total binding energy than either the MCPF or CCSD methods. An analysis of the basis set superposition error (BSSE) at the MCPF level of treatment points out limitations in the one-particle basis used. Calculations using larger basis sets reduce the BSSE, but the total binding energy of Cr(CO)6 is still significantly smaller than the experimental value, although the first CO bond dissociation energy of Cr(CO)6 is well described. An investigation of 3s3p correlation reveals only a small effect. In the largest basis set, the total CO binding energy of Cr(CO)6 is estimated to be 140 kcal/mol at the CCSD(T) level of theory, or about 86 percent of the experimental value. The remaining discrepancy between the experimental and theoretical value is probably due to limitations in the one-particle basis, rather than limitations in the correlation treatment. In particular an additional d function and an f function on each C and O are needed to obtain quantitative results. This is underscored by the fact that even using a very large primitive set (1042 primitive functions contracted to 300 basis functions), the superposition error for the total binding energy of Cr(CO)6 is 22 kcal/mol at the MCPF level of treatment.

Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes.

PubMed Central

Cutsforth, G A; Koppaka, V; Krishnaswamy, S; Wu, J R; Mann, K G; Lentz, B R

1996-01-01

The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid-specific sites on the factor Va molecule. Images FIGURE 5 PMID:8744332

Energetics and kinetics of cooperative cofilin-actin filament interactions.

PubMed

Cao, Wenxiang; Goodarzi, Jim P; De La Cruz, Enrique M

2006-08-11

We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to actin filaments, accounting for contributions of ion-linked equilibria, and determined the kinetic basis of cooperative cofilin binding. Ions weaken non-contiguous (isolated, non-cooperative) cofilin binding to an actin filament without affecting cooperative filament interactions. Non-contiguous cofilin binding is coupled to the dissociation of approximately 1.7 thermodynamically bound counterions. Counterion dissociation contributes approximately 40% of the total cofilin binding free energy (in the presence of 50 mM KCl). The non-contiguous and cooperative binding free energies are driven entirely by large, positive entropy changes, consistent with a cofilin-mediated increase in actin filament structural dynamics. The rate constant for cofilin binding to an isolated site on an actin filament is slow and likely to be limited by filament breathing. Cooperative cofilin binding arises from an approximately tenfold more rapid association rate constant and an approximately twofold slower dissociation rate constant. The more rapid association rate constant is presumably a consequence of cofilin-dependent changes in the average orientation of subdomain 2, subunit angular disorder and filament twist, which increase the accessibility of a neighboring cofilin-binding site on an actin filament. Cooperative association is more rapid than binding to an isolated site, but still slow for a second-order reaction, suggesting that cooperative binding is limited also by binding site accessibility. We suggest that the dissociation of actin-associated ions weakens intersubunit interactions in the actin filament lattice that enhance cofilin-binding site accessibility, favor cooperative binding and promote filament severing.

Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5'-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

PubMed Central

Donoso, J; Muñoz, F; García Del Vado, A; Echevarría, G; García Blanco, F

1986-01-01

Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate. PMID:3099764

Probing the mechanism of interaction of metoprolol succinate with human serum albumin by spectroscopic and molecular docking analysis.

PubMed

Pawar, Suma K; Jaldappagari, Seetharamappa

2017-09-01

In the present work, the mechanism of the interaction between a β1 receptor blocker, metoprolol succinate (MS) and human serum albumin (HSA) under physiological conditions was investigated by spectroscopic techniques, namely fluorescence, Fourier transform infra-red spectroscopy (FT-IR), fluorescence lifetime decay and circular dichroism (CD) as well as molecular docking and cyclic voltammetric methods. The fluorescence and lifetime decay results indicated that MS quenched the intrinsic intensity of HSA through a static quenching mechanism. The Stern-Volmer quenching constants and binding constants for the MS-HSA system at 293, 298 and 303 K were obtained from the Stern-Volmer plot. Thermodynamic parameters for the interaction of MS with HSA were evaluated; negative values of entropy change (ΔG°) indicated the spontaneity of the MS and HSA interaction. Thermodynamic parameters such as negative ΔH° and positive ΔS° values revealed that hydrogen bonding and hydrophobic forces played a major role in MS-HSA interaction and stabilized the complex. The binding site for MS in HSA was identified by competitive site probe experiments and molecular docking studies. These results indicated that MS was bound to HSA at Sudlow's site I. The efficiency of energy transfer and the distance between the donor (HSA) and acceptor (MS) was calculated based on the theory of Fosters' resonance energy transfer (FRET). Three-dimensional fluorescence spectra and CD results revealed that the binding of MS to HSA resulted in an obvious change in the conformation of HSA. Cyclic voltammograms of the MS-HSA system also confirmed the interaction between MS and HSA. Furthermore, the effects of metal ions on the binding of MS to HSA were also studied. Copyright © 2017 John Wiley & Sons, Ltd.

Synthesis of β-arabinofuranoside glycolipids, studies of their binding to surfactant protein-A and effect on sliding motilities of M. smegmatis.

PubMed

Naresh, Kottari; Avaji, Prakash Gouda; Maiti, Krishnagopal; Bharati, Binod K; Syal, Kirtimaan; Chatterji, Dipankar; Jayaraman, Narayanaswamy

2012-04-01

Surfactant protein A (SP-A), which is a lung innate immune system component, is known to bind glycolipids present at the cell surface of a mycobacterial pathogen. Lipoarabinomannan (LAM), a component of mycobacterial thick, waxy cell wall, is one of the glycolipid ligands for SP-A. In order to assess binding of synthetic glycolipids with SP-A and the glycosidic linkage preferences for the interaction, β-arabinofuranoside trisaccharide glycolipids constituted with β-(1→2), β-(1→3) and β-(1→2), β-(1→5) linkages relevant to LAM were synthesized through chemical glycosylations. The efficacies of synthetic glycolipids to interact with SP-A were assessed by using the surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. The equilibrium binding constants of the interaction of two constitutionally varying β-arabinofuranoside glycolipids with SP-A were found to be in the millimolar range. A comparison of the results with few α-anomeric arabinofuranoside glycolipids showed that glycolipids with β-anomeric linkages were having relatively lower equilibrium binding constants than those with α-anomeric linkages in binding to the protein, whereas oligosaccharides alone, without lipidic chains, exhibited higher equilibrium binding constants. Further, the synthetic compounds inhibited the growth of mycobacteria and affected sliding motilities of the bacteria, although to an extent relatively lesser than that of synthetic compounds constituted with α-anomeric linkages.

Interaction of chlorogenic acids and quinides from coffee with human serum albumin.

PubMed

Sinisi, Valentina; Forzato, Cristina; Cefarin, Nicola; Navarini, Luciano; Berti, Federico

2015-02-01

Chlorogenic acids and their derivatives are abundant in coffee and their composition changes between coffee species. Human serum albumin (HSA) interacts with this family of compounds with high affinity. We have studied by fluorescence spectroscopy the specific binding of HSA with eight compounds that belong to the coffee polyphenols family, four acids (caffeic acid, ferulic acid, 5-O-caffeoyl quinic acid, and 3,4-dimethoxycinnamic acid) and four lactones (3,4-O-dicaffeoyl-1,5-γ-quinide, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, and 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide), finding dissociation constants of the albumin-chlorogenic acids and albumin-quinides complexes in the micromolar range, between 2 and 30μM. Such values are comparable with those of the most powerful binders of albumin, and more favourable than the values obtained for the majority of drugs. Interestingly in the case of 3,4-O-dicaffeoyl-1,5-γ-quinide, we have observed the entrance of two ligand molecules in the same binding site, leading up to a first dissociation constant even in the hundred nanomolar range, which is to our knowledge the highest affinity ever observed for HSA and its ligands. The displacement of warfarin, a reference drug binding to HSA, by the quinide has also been demonstrated. Copyright © 2014 Elsevier Ltd. All rights reserved.

The mechanism of tubulin-colchicine recognition--a kinetic study of the binding of a bicyclic colchicine analogue with a minor modification of the A ring.

PubMed

Dumortier, C; Potenziano, J L; Bane, S; Engelborghs, Y

1997-10-01

2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-1-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K1 and k2 values for MDC at 25 degrees C were 540 +/- 65 M(-1) and 70 +/- 6 s(-1) respectively. From the temperature dependence, a reaction enthalpy change (deltaH(o)1) of the initial binding of 49 +/- 11 kJ/mol(-1) and an activation energy for the second step of 28 +/- 9 kJ/mol(-1) were calculated. Displacement experiments of bound MDC by MTC allowed the determination of a rate constant of reverse isomerisation of 0.60 +/- 0.07 s(-1) at 25 degrees C and the activation energy of 81 +/- 6 kJ/mol(-1). The overall binding constant was (6.3 +/- 0.2) x 10(4) M(-1) at 25 degrees C. Combination of these results with the kinetic parameters for association gives a full characterisation of the enthalpy pathway for the binding of MDC. The pathway of MDC is shown to differ considerably from that of MTC binding. Since its structural difference is located in ring A, this result indicates the use of ring A in the first step. The kinetics of the binding of MDC in the presence of some A-ring colchicine analogues (podophyllotoxin, 3',4',5'-trimethoxyacetophenone and N-acetylmescaline) and a C-ring analogue (tropolone methyl ether) suggest that the A and C rings are involved in the binding of MDC.

Kinetics of Ca2+ binding to parvalbumin in bovine chromaffin cells: implications for [Ca2+] transients of neuronal dendrites

PubMed Central

Lee, Suk-Ho; Schwaller, Beat; Neher, Erwin

2000-01-01

The effect of parvalbumin (PV) on [Ca2+] transients was investigated by perfusing adrenal chromaffin cells with fura-2 and fluorescein isothiocyanate (FITC)-labelled PV. As PV diffused into cells, the decay of [Ca2+] transients was transformed from monophasic into biphasic. The proportion of the initial fast decay phase increased in parallel with the fluorescence intensity of FITC, indicating that PV is responsible for the initial fast decay phase.The relationship between the fast decay phase and the [Ca2+] level was investigated using depolarizing trains of stimuli. Within a train the relative amplitude of the fast decay phase was inversely dependent on the [Ca2+] level preceding a given stimulus.Based on these observations, we estimated the Ca2+ binding ratio of PV (κP), the apparent dissociation constant of PV for Ca2+ (Kdc,app), and the unbinding rate constant of Ca2+ from PV (kc-) in the cytosol of chromaffin cells. Assuming free [Mg2+] to be 0.14 mm, we obtained values of 51.4 ± 2.0 nm (n = 3) and 0.95 ± 0.026 s−1 (n = 3), for Kdc,app and kc-, respectively.With the parameters obtained in the perfusion study, we simulated [Ca2+] transients, using two different Ca2+ extrusion rates (γ) – 20 and 300 s−1– which represent typical values for chromaffin cells and neuronal dendrites, respectively. The simulation indicated that Ca2+ is pumped out before it is equilibrated with PV, when γ is comparable to the equilibration rates between PV and Ca2+, resulting in the fast decay phase of a biexponential [Ca2+] transient.From these results we conclude that Ca2+ buffers with slow kinetics, such as PV, may cause biexponential decays in [Ca2+] transients, thereby complicating the analysis of endogenous Ca2+ binding ratios (κS) based on time constants. Nevertheless, estimates of κS based on Ca2+ increments provide reasonable estimates for Ca2+ binding ratios before equilibration with PV. PMID:10835044

Cd and proton adsorption onto bacterial consortia grown from industrial wastes and contaminated geologic settings.

PubMed

Borrok, David M; Fein, Jeremy B; Kulpa, Charles F

2004-11-01

To model the effects of bacterial metal adsorption in contaminated environments, results from metal adsorption experiments involving individual pure stains of bacteria must be extrapolated to systems in which potentially dozens of bacterial species are present. This extrapolation may be made easier because bacterial consortia from natural environments appear to exhibit similar metal binding properties. However, bacteria that thrive in highly perturbed contaminated environments may exhibit significantly different adsorptive behavior. Here we measure proton and Cd adsorption onto a range of bacterial consortia grown from heavily contaminated industrial wastes, groundwater, and soils. We model the results using a discrete site surface complexation approach to determine binding constants and site densities for each consortium. The results demonstrate that bacterial consortia from different contaminated environments exhibit a range of total site densities (approximately a 3-fold difference) and Cd-binding constants (approximately a 10-fold difference). These ranges for Cd binding constants may be small enough to suggest that bacteria-metal adsorption in contaminated environments can be described using relatively few "averaged" bacteria-metal binding constants (in conjunction with the necessary binding constants for competing surfaces and ligands). However, if additional precision is necessary, modeling parameters must be developed separately for each contaminated environment of interest.

Genistein Binding to Copper(II)-Solvent Dependence and Effects on Radical Scavenging.

PubMed

Yang, Jing; Xu, Yi; Liu, Hao-Yu; Han, Rui-Min; Zhang, Jian-Ping; Skibsted, Leif H

2017-10-18

Genistein, but not daidzein, binds to copper(II) with a 1:2 stoichiometry in ethanol and with a 1:1 stoichiometry in methanol, indicating chelation by the 5-phenol and the 4-keto group of the isoflavonoid as demonstrated by the Jobs method and UV-visible absorption spectroscopy. In ethanol, the stability constants had the value 1.12 × 10 11 L²∙mol -2 for the 1:2 complex and in methanol 6.0 × 10⁵ L∙mol -1 for the 1:1 complex at 25 °C. Binding was not detected in water, as confirmed by an upper limit for the 1:1 stability constant of K = 5 mol -1 L as calculated from the difference in solvation free energy of copper(II) between methanol and the more polar water. Solvent molecules compete with genistein as demonstrated in methanol where binding stoichiometry changes from 1:2 to 1:1 compared to ethanol and methanol/chloroform (7/3, v / v ). Genistein binding to copper(II) increases the scavenging rate of the stable, neutral 2,2-diphenyl-1-picrylhydrazyl radical by more than a factor of four, while only small effects were seen for the short-lived but more oxidizing β -carotene radical cation using laser flash photolysis. The increased efficiency of coordinated genistein is concluded to depend on kinetic rather than on thermodynamic factors, as confirmed by the small change in reduction potential of -0.016 V detected by cyclic voltammetry upon binding of genistein to copper(II) in methanol/chloroform solutions.

Investigating the interaction of anticancer drug temsirolimus with human transferrin: Molecular docking and spectroscopic approach.

PubMed

Shamsi, Anas; Ahmed, Azaj; Khan, Mohd Shahnawaz; Husain, Fohad Mabood; Amani, Samreen; Bano, Bilqees

2018-05-16

In our present study, binding between an important anti renal cancer drug temsirolimus and human transferrin (hTF) was investigated employing spectroscopic and molecular docking approach. In the presence of temsirolimus, hyper chromaticity is observed in hTF in UV spectroscopy suggestive of complex formation between hTF and temsirolimus. Fluorescence spectroscopy revealed the occurrence of quenching in hTF in the presence of temsirolimus implying complex formation taking place between hTF and temsirolimus. Further, the mode of interaction between hTF and temsirolimus was revealed to be static by fluorescence quenching analysis at 3 different temperatures. Binding constant values obtained employing fluorescence spectroscopy depicts strong interaction between hTF and temsirolimus; temsirolimus binds to hTF at 298 K with a binding constant of .32 × 10 4  M -1 implying the strength of this interaction. The negative Gibbs free energy obtained through quenching experiments is evident of the fact that the binding is spontaneous. CD spectra of hTF also showed a downward shift in the presence of temsirolimus as compared with free hTF implying complex formation between hTF and temsirolimus. Molecular docking was performed with a view to find out which residues are key players in this interaction. The importance of our study stems from the fact it will provide an insight into binding pattern of commonly administered renal cancer drug with an important protein that plays a pivotal role in many physiological processes. Copyright © 2018 John Wiley & Sons, Ltd.

Structure and gelation properties of casein micelles doped with curcumin under acidic conditions.

PubMed

Khanji, Aya N; Michaux, Florentin; Jasniewski, Jordane; Petit, Jeremy; Lahimer, Emna; Cherif, Mohamed; Salameh, Dominique; Rizk, Toufic; Banon, Sylvie

2015-12-01

In this study, the ability of micellar casein (MC) to interact with curcumin during acidification and to produce acid gel was investigated. Steady-state fluorescence spectroscopy of curcumin variation and fluorescence quenching of caseins upon binding with curcumin molecules were evidenced. Increasing the temperature from 20 to 35 °C enhanced MC-curcumin interactions as reflected by the increase in the binding constant from 0.6 ± 0.3 × 10(4) to 6.6 ± 0.6 × 10(4) M(-1). From changes in entropy, enthalpy and Gibbs free energy, hydrophobic interactions were proposed as major binding forces. Static fluorescence MC quenching was demonstrated for the MC-curcumin complex during acidification. From pH 7.4 to pH 5.0, the binding site numbers varied in the range from 1.25 ± 0.05 to 1.49 ± 0.05 and the binding constant kb varied from 3.9 ± 0.4 × 10(4) to 7.5 ± 0.7 × 10(4) M(-1). Small angle X-ray scattering profiles demonstrated that the MC internal structure was unchanged upon curcumin binding. The ζ-potential value of curcumin-doped MC indicated that curcumin did not modify the global charge of MC particles. Acid gelation studied by oscillation rheology and static multiple light scattering at 20 and 35 °C led to a similar behavior for native and curcumin-doped MC suspensions. For the first time, it was demonstrated that the colloidal and functional properties of MC were unchanged when doped with curcumin during acidification.

Kinetic rate constant prediction supports the conformational selection mechanism of protein binding.

PubMed

Moal, Iain H; Bates, Paul A

2012-01-01

The prediction of protein-protein kinetic rate constants provides a fundamental test of our understanding of molecular recognition, and will play an important role in the modeling of complex biological systems. In this paper, a feature selection and regression algorithm is applied to mine a large set of molecular descriptors and construct simple models for association and dissociation rate constants using empirical data. Using separate test data for validation, the predicted rate constants can be combined to calculate binding affinity with accuracy matching that of state of the art empirical free energy functions. The models show that the rate of association is linearly related to the proportion of unbound proteins in the bound conformational ensemble relative to the unbound conformational ensemble, indicating that the binding partners must adopt a geometry near to that of the bound prior to binding. Mirroring the conformational selection and population shift mechanism of protein binding, the models provide a strong separate line of evidence for the preponderance of this mechanism in protein-protein binding, complementing structural and theoretical studies.

STUDIES OF VERAPAMIL BINDING TO HUMAN SERUM ALBUMIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

PubMed Central

Mallik, Rangan; Yoo, Michelle J.; Chen, Sike; Hage, David S.

2008-01-01

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g., the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 104 M−1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37°C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e., the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 104 M−1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with L-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body. PMID:18980867

Quantitative Assessment of the Interplay Between DNA Elasticity and Cooperative Binding of Ligands

NASA Astrophysics Data System (ADS)

Siman, L.; Carrasco, I. S. S.; da Silva, J. K. L.; de Oliveira, M. C.; Rocha, M. S.; Mesquita, O. N.

2012-12-01

Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of the total ligand concentration, which we apply to our data of the DNA-cationic β-cyclodextrin and to the DNA-HU protein data available in the literature, determining the values of the local persistence lengths, the dissociation constant, and the degree of cooperativity for each set of data. In both cases the persistence length behaves nonmonotonically as a function of ligand concentration and based on the results obtained we discuss some physical aspects of the interplay between DNA elasticity and cooperative binding of ligands.

Self-assembling choline mimicks with enhanced binding affinities to C-LytA protein

PubMed Central

Shi, Yang; Zhou, Hao; Zhang, Xiaoli; Wang, Jingyu; Long, Jiafu; Yang, Zhimou; Ding, Dan

2014-01-01

Streptococcus pneumoniae (pneumococcus) causes multiple illnesses in humans. Exploration of effective inhibitors with multivalent attachment sites for choline-binding modules is of great importance to reduce the pneumococcal virulence. In this work, we successfully developed two self-assembling choline mimicks, Ada-GFFYKKK' and Nap-GFFYKKK', which have the abilities to self-assemble into nanoparticles and nanofibers, respectively, yielding multivalent architectures. Additionally, the best characterized choline-binding module, C-terminal moiety of the pneumococcal cell-wall amidase LytA (C-LytA) was also produced with high purity. The self-assembling Ada-GFFYKKK' and Nap-GFFYKKK' show strong interactions with C-LytA, which possess much higher association constant values to the choline-binding modules as compared to the individual peptide Fmoc-K'. This study thus provides a self-assembly approach to yield inhibitors that are very promising for reducing the pneumococcal virulence. PMID:25315737

Influence of water chemistry and natural organic matter on active and passive uptake of inorganic mercury by gills of rainbow trout (Oncorhynchus mykiss).

PubMed

Klinck, Joel; Dunbar, Michael; Brown, Stephanie; Nichols, Joel; Winter, Anna; Hughes, Christopher; Playle, Richard C

2005-03-25

To distinguish physiologically regulated uptake from passive uptake of inorganic Hg in fish, rainbow trout (Oncorhynchus mykiss) were exposed to inorganic Hg (0.5, 1, or 2 microM total Hg) in ion-poor water with various treatments. Addition of ions to the water (mM concentrations of Ca, K, Cl) did not consistently alter Hg accumulation by trout gills, although there was a trend to higher Hg accumulation at higher ion concentrations. The apical Ca channel blockers Verapamil and lanthanum also did not consistently affect Hg accumulation by trout gills. Pre-treatment of trout with the Na channel blocker Phenamil decreased Hg uptake by about half. These results suggest a combination of physiologically regulated and passive uptake of Hg by trout gills. Strong complexing agents of Hg (EDTA, NTA, ethylenediamine, cysteine) decreased Hg-binding by trout gills in a dose-dependent manner. From these data, a conditional equilibrium binding constant for Hg to the gills was estimated as logK(Hg-gill) = 18.0, representing very strong binding of Hg to the gills. This value is a first step in creating a biotic ligand model (BLM) for inorganic Hg and fish. Natural organic matter (2-10 mg C/L) also decreased Hg-binding by trout gills, although mM concentrations of Na, K, and Cl interfered with this effect. At low concentrations of these ions, natural organic matter samples isolated from various sources bound Hg to similar degrees, as judged by Hg accumulation by trout gills. A conditional binding constant to natural organic matter (NOM) was estimated as logK(Hg-NOM) = 18.0 with about 0.5 micromol binding sites per mg C, representing strong binding of Hg to NOM.

Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. Dynamic binding capacity using lysozyme as a model.

PubMed

Boto, R E F; Anyanwu, U; Sousa, F; Almeida, P; Queiroz, J A

2009-09-01

A constant development of dye-affinity chromatography to replace more traditional techniques is verified, with the aim of increasing specificity in the purification of biomolecules. The establishment of a new dye-affinity chromatographic support imposes their complete characterization, namely with relation to the binding capacity for proteins, in order to evaluate its applicability on global purification processes. Following previous studies, the adsorption of lysozyme onto a thiacarbocyanine dye immobilized on beaded cellulose was investigated. The effect of different parameters, such as temperature, ionic strength, pH, protein concentration and flow rate, on the dynamic binding capacity of the support to retain lysozyme was also studied. Increasing the temperature and the lysozyme concentration had a positive effect on the dynamic binding capacity (DBC), whereas increasing the ionic strength and the flow rate resulted in the opposite. It was also discovered that the pH used had an important impact on the lysozyme binding onto the immobilized dye. The maximum DBC value obtained for lysozyme was 8.6 mg/mL, which was achieved at 30 degrees C and pH 9 with a protein concentration of 0.5 mg/mL and a flow rate of 0.05 mL/min. The dissociation constant (K(d)) obtained was 2.61 +/- 0.36 x 10(-5 )m, proving the affinity interaction between the thiacarbocyanine dye ligand and the lysozyme. Copyright (c) 2009 John Wiley & Sons, Ltd.

Determination of the side-reaction coefficient of the trihydroxamate siderophore desferrioxamine B in metal-free seawater

NASA Astrophysics Data System (ADS)

Schijf, J.; Burns, S. M.

2016-02-01

Desferrioxamines are a class of trihydroxamate siderophores, members of which occur in surface seawater at low-picomolar concentrations. The total synthesis of desferrioxamine B (DFOB), achieved in the late 1980s and prompted by its use in the treatment of human iron-overload disorders, has ensured a steady commercial supply enabling extensive laboratory studies of its properties. While highly specific for Fe3+, DFOB binds many di-, tri-, and tetravalent metals with substantial affinity and has consequently been employed as a model for strong organic ligands that ostensibly dominate the speciation of several bio-essential metals in the ocean, yet remain largely unidentified. Such comparisons are only meaningful if we know the side-reaction coefficient of DFOB in seawater, which accounts for its binding with the divalent cations Mg2+ and Ca2+. Although quite weak, this has a potentially important effect on the availability of the free ligand, due to the great abundance of these sea salt constituents. We have performed potentiometric titrations to measure the sequential binding of Mg and Ca to the three hydroxamate groups of DFOB, quantified by stability constants β1, β2, and β3. Values of β1 are reported for the first time, however no evidence was found for binding with the terminal amine of DFOB and the corresponding stability constant β4 was thus omitted from the regression model constructed to fit the titration curves. We also examined Mg and Ca binding to methanesulfonate (MSA), a common DFOB counter-ion, by measuring the stability of their complexes with acetohydroxamate in the presence and absence of MSA. Whereas stabilities of metal-MSA complexes have not been published, their similarity to sulfate complexes suggests that MSA may compete with DFOB for Mg and Ca in the titrations. Our calculated side-reaction coefficient is consistent with a previous estimate, but should properly be expressed in terms of protonated forms of DFOB, resulting in a much lower value.

Screening of biologically important Zn2 + by a chemosensor with fluorescent turn on-off mechanism

NASA Astrophysics Data System (ADS)

Khan, Tanveer A.; Sheoran, Monika; Nikhil Raj M., Venkata; Jain, Surbhi; Gupta, Diksha; Naik, Sunil G.

2018-01-01

Reported herein the synthesis, characterization and biologically important zinc ion binding propensity of a weakly fluorescent chemosensor, 4-methyl-2,6-bis((E)-(2-(4-phenylthiazol-2-yl)hydrazono)methyl)phenol (1). 1H NMR spectroscopic titration experiment reveals the binding knack of 1 to the essential Zn2 +. The photo-physical studies of 1 exhibit an enhancement in the fluorescence by several folds upon binding with the zinc ions attributed to PET-off process, with a binding constant value of 5.22 × 103 M- 1. 1 exhibits an excellent detection range for Zn2 + with lower detection limit value of 2.31 × 10- 8 M. The selectivity of 1 was studied with various mono and divalent metal cations and it was observed that most cations either quenches the fluorescence or remains unchanged except for Cd2 +, which shows a slight enhancement in fluorescence intensity of 1. The ratiometric displacement of Cd2 + ions by Zn2 + ions shows an excellent selectivity towards in-situ detection of Zn2 + ions. Photo-physical studies also support the reversible binding of 1 to Zn2 + ions having on and off mechanism in presence of EDTA. Such recognition of the biologically important zinc ions finds potential application in live cell imaging.

Interaction of Antitumor Agent Mitoxantrone with Double Helical Synthetic Polyribonucleotides Poly(G)ṡPoly(C) and Poly(I)ṡPoly(C)

NASA Astrophysics Data System (ADS)

Babayan, Yuri S.; Hakobyan, Sergey N.; Ghazaryan, Rusanna S.; Shahinyan, Mariam A.

The interaction of antitumor drug mitoxantrone (MTX) with double-stranded synthetic RNA homopolymers has been studied by means of spectroscopic (UV-Visible absorption, circular dichroism) techniques. The results show a base specificity in this interaction: the association constant with poly(G)ṡpoly(C) is higher than with poly(I)ṡpoly(C). Values of changes of the system enthalpy and entropy due to complex-formation were determined through the temperature dependence of the binding constant. Calculations show that due to the intercalation interaction of MTX, the values of changes of the system entropy and enthalpy differ from those obtained at ehtidium bromide interaction with synthetic polyribonucleotides, which shows that the intercalation interaction of MTX with double-stranded RNA significantly differs from that of ethidium bromide with RNA.

Structure and energetics of Cr(CO)6 and Cr(CO)5

NASA Technical Reports Server (NTRS)

Barnes, Leslie A.; Liu, Bowen; Lindh, Roland

1993-01-01

The geometric structures and energetics of Cr(CO)6 and Cr(CO)5 are determined at the modified coupled-pair functional, single and double excitation coupled-cluster (CCSD), and CCSD(T) levels of theory. For Cr(CO)6, the structure and force constants for the totally symmetric representation are in good agreement with experimental data once basis set constants are taken into account. In the largest basis set at the CCSD(T) level of theory, the total binding energy of CR(CO)6 is estimated at around 140 kcal/mol, or about 86 percent of the experimental value. In contrast, the first bond energy of Cr(CO)6 is very well described at the CCSD(T) level of theory, with the best estimated value of 38 kcal/mol being within the experimental uncertainty.

Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 Are Not a Requirement.

PubMed

Lindström, Ida; Dogan, Jakob

2017-08-15

A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered under native-like conditions. Intrinsically disordered proteins (IDPs) are common in protein-protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate-limiting transition state for binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2) by site-directed mutagenesis and kinetic experiments (Φ-value analysis) and found that the native protein-protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after the rate-limiting barrier has been crossed. The association rate constant in the absence of electrostatic enhancement was determined to be rather high. This is consistent with the Φ-value analysis, which showed that there are few or no obligatory native contacts. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has usually been observed for proteins that fold according to the so-called nucleation-condensation mechanism. Thus, native hydrophobic binding interactions at the rate-limiting transition state for association between TAD-STAT2 and TAZ1 are not a requirement, which is generally in agreement with previous findings on other IDP systems and might be a common mechanism for IDPs.

Investigation of flavonoids bearing different substituents on ring C and their cu2+ complex binding with bovine serum albumin: structure-affinity relationship aspects.

PubMed

Shi, Shuyun; Zhang, Yuping; Chen, Xiaoqin; Peng, Mijun

2011-10-12

The effects of 1:1 flavonoid-Cu(2+) complexes of four flavonoids with different C-ring substituents, quercetin (QU), luteolin (LU), taxifolin (TA), and (+)-catechin (CA), on bovine serum albumin (BSA) were investigated and compared with corresponding free flavonoids by spectroscopic analysis in an attempt to characterize the chemical association taking place. The results indicated that all of the quenching mechanisms were based on static quenching combined with nonradiative energy transfer. Cu(2+) chelation changed the binding constants for BSA depending on the structures of flavonoids and the detected concentrations. The reduced hydroxyl groups, increased steric hindrance, and hydrophilicity of Cu(2+) chelation may be the main reasons for the reduced binding constants, whereas the formation of stable flavonoid-Cu(2+) complexes and synergistic action could increase the binding constants. The changed trends of critical energy transfer distance (R(0)) for Cu(2+) chelation were contrary to those of binding constants.

Proton and metal ion binding to natural organic polyelectrolytes-II. Preliminary investigation with a peat and a humic acid

USGS Publications Warehouse

Marinsky, J.A.; Reddy, M.M.

1984-01-01

We summarize here experimental studies of proton and metal ion binding to a peat and a humic acid. Data analysis is based on a unified physico-chemical model for reaction of simple ions with polyelectrolytes employing a modified Henderson-Hasselbalch equation. Peat exhibited an apparent intrinsic acid dissociation constant of 10-4.05, and an apparent intrinsic metal ion binding constant of: 400 for cadmium ion; 600 for zinc ion; 4000 for copper ion; 20000 for lead ion. A humic acid was found to have an apparent intrinsic proton binding constant of 10-2.6. Copper ion binding to this humic acid sample occurred at two types of sites. The first site exhibited reaction characteristics which were independent of solution pH and required the interaction of two ligands on the humic acid matrix to simultaneously complex with each copper ion. The second complex species is assumed to be a simple monodentate copper ion-carboxylate species with a stability constant of 18. ?? 1984.

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate.

PubMed

Chétrite, G; Cassoly, R

1985-10-05

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.

Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

PubMed

Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

2008-12-25

Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

Alkoxy bridged binuclear rhenium (I) complexes as a potential sensor for β-amyloid aggregation.

PubMed

Sathish, Veerasamy; Babu, Eththilu; Ramdass, Arumugam; Lu, Zong-Zhan; Velayudham, Murugesan; Thanasekaran, Pounraj; Lu, Kuang-Lieh; Rajagopal, Seenivasan

2014-12-01

Alkoxy bridged binuclear rhenium(I) complexes are used as a probe for the selective and sensitive detection of aggregation of β-amyloid fibrils that are consorted with Alzheimer's disease (AD). The strong binding of the complexes is affirmed by the fluorescence enhancement and calculated binding constant value in the order of 10(5)M(-1) is obtained from the Scatchard plots. The binding of β-amyloid can be attributed to π-π stacking interaction of naphthalene moiety present in rhenium(I) complexes, and it is supported by docking studies. The selectivity is quite high towards other proteins and the formation of fibrils can be observed in the range of 30-40 nm through the AFM and TEM techniques. Copyright © 2014 Elsevier B.V. All rights reserved.

Binding energy of the donor impurities in GaAs-Ga 1- x Al x As quantum well wires with Morse potential in the presence of electric and magnetic fields

NASA Astrophysics Data System (ADS)

Aciksoz, Esra; Bayrak, Orhan; Soylu, Asim

2016-10-01

The behavior of a donor in the GaAs-Ga1-x Al x As quantum well wire represented by the Morse potential is examined within the framework of the effective-mass approximation. The donor binding energies are numerically calculated for with and without the electric and magnetic fields in order to show their influence on the binding energies. Moreover, how the donor binding energies change for the constant potential parameters (D e, r e, and a) as well as with the different values of the electric and magnetic field strengths is determined. It is found that the donor binding energy is highly dependent on the external electric and magnetic fields as well as parameters of the Morse potential. Project supported by the Turkish Science Research Council (TÜBİTAK) and the Financial Supports from Akdeniz and Nigde Universities.

Fluorenone based fluorescent probe for selective "turn-on" detection of pyrophosphate and alanine

NASA Astrophysics Data System (ADS)

Daniel Thangadurai, T.; Nithya, I.; Manjubaashini, N.; Bhuvanesh, N.; Bharathi, G.; Nandhakumar, R.; Nataraj, D.

2018-06-01

To sense biologically important entities with different size and dimensions, a fluorenone based fluorescent receptor was designed and synthesized. Probe 1 displayed a distinct fluorescence enhancement emission at 565 nm for pyrophosphate and 530 nm for alanine in polar solvent. The fluorescence titration experiments confirm 1:1 stoichiometric ratio with high-binding constant and very low limit of detection (LoD) values. Receptor 1 showed a highly selective and sensitive recognition to HP2O73 - and to alanine over other competitive anions and amino acids. In addition, the fluorescence lifetime measurement and reversible binding study results support the practical importance of 1.

Differential processing of the two subunits of human choriogonadotropin (hCG) by granulosa cells. I. Preparation and characterization of selectively labeled hCG

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landefeld, T.D.; Byrne, M.D.; Campbell, K.L.

1981-12-01

The alpha- and beta-subunits of hCG were radioiodinated and recombined with unlabeled complementary subunits. The resultant recombined hormones, selectively labeled in either the alpha- or beta-subunit, were separated from unrecombined subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, extracted with Triton X-100, and characterized by binding analysis. The estimates of maximum binding (active fraction) of the two resultant selectively labeled, recombined hCG preparations, determined with excess receptor were 0.41 and 0.59. These values are similar to those obtained when hCG is labeled as an intact molecule. The specific activities of the recombined preparations were estimated by four different methods, and themore » resulting values were used in combination with the active fraction estimates to determine the concentrations of active free and bound hormone. Binding analyses were run using varying concentrations of both labeled and unlabeled hormone. Estimates of the equilibrium dissociation binding constant (Kd) and receptor capacity were calculated in three different ways. The mean estimates of capacity (52.6 and 52.7 fmol/mg tissue) and Kd (66.6 and 65.7 pM) for the two preparations were indistinguishable. Additionally, these values were similar to values reported previously for hCG radioiodinated as an intact molecule. The availability of well characterized, selectively labeled hCG preparations provides new tools for studying the mechanism of action and the target cell processing of the subunits of this hormone.« less

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

PubMed Central

Liu, Yungang; Lehmler, Hans-Joachim; Robertson, Larry W.; Duffel, Michael W.

2010-01-01

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 (Ka) 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 pKa and a negative correlation with log D of the OHPCBs. IC50 values of inhibitory OHPCBs decreased with decreasing pKa 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, pKa 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. PMID:21130751

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycoclusters to lectins and tetanus toxin C-fragment

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Minoura, Norihiko

2011-03-01

We develop a fluorescent ruthenium metalloglycocluster for use as a powerful molecular probe in evaluating the binding between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analyses. Changes in the FE and FP of these metalloglycoclusters are measured following the addition of lectin [peanut agglutinin (PNA), Ricinus communis agglutinin 120, Concanavalin A (ConA), or wheat germ agglutinin] or tetanus toxin c-fragment (TCF). After the addition of PNA, the FE spectrum of [Ru(bpy-2Gal)3] shows a new emission peak and the FP value of [Ru(bpy-2Gal)3] increases. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] shows a new emission peak and the FP value increases on addition of ConA. Because other combinations of metalloglycoclusters and lectins show little change, specific binding of galactose to PNA and that of glucose to ConA are confirmed by the FE and FP measurements. Resulting dissociation constants (Kd) prove that the metalloglycoclusters with highly clustered carbohydrates show higher affinity for the respective lectins than those with less clustered carbohydrates. Furthermore, specific binding of [Ru(bpy-2Gal)3] to TCF was confirmed by the FP measurement.

The relationship between metal toxicity and biotic ligand binding affinities in aquatic and soil organisms: a review.

PubMed

Ardestani, Masoud M; van Straalen, Nico M; van Gestel, Cornelis A M

2014-12-01

The biotic ligand model (BLM) is a theoretical, potentially mechanistic approach to assess metal bioavailability in soil and aquatic systems. In a BLM, toxicity is linked to the fraction of biotic ligand occupied, which in turn, depends on the various components of the solution, including activity of the metal. Bioavailability is a key factor in determining toxicity and uptake of metals in organisms. In this study, the present status of BLM development for soil and aquatic organisms is summarized. For all species and all metals, toxicity was correlated with the conditional biotic ligand binding constants. For almost all organisms, values for Ag, Cu, and Cd were higher than those for Zn and Ni. The constants derived for aquatic systems seem to be equally valid for soil organisms, but in the case of soils, bioavailability from the soil solution is greatly influenced by the presence of the soil solid phase. Copyright © 2014 Elsevier Ltd. All rights reserved.

Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions.

PubMed

Sawas, Abdul H; Pentyala, Srinivas N; Rebecchi, Mario J

2004-10-05

This study directly examines the enthalpic contributions to binding in aqueous solution of closely related anesthetic haloethers (desflurane, isoflurane, enflurane, and sevoflurane), a haloalkane (halothane), and an intravenous anesthetic (propofol) to bovine and human serum albumin (BSA and HSA) using isothermal titration calorimetry. Binding to serum albumin is exothermic, yielding enthalpies (DeltaH(obs)) of -3 to -6 kcal/mol for BSA with a rank order of apparent equilibrium association constants (K(a) values): desflurane > isoflurane approximately enflurane > halothane >or= sevoflurane, with the differences being largely ascribed to entropic contributions. Competition experiments indicate that volatile anesthetics, at low concentrations, share the same sites in albumin previously identified in crystallographic and photo-cross-linking studies. The magnitude of the observed DeltaH increased linearly with increased reaction temperature, reflecting negative changes in heat capacities (DeltaC(p)). These -DeltaC(p) values significantly exceed those calculated for burial of each anesthetic in a hydrophobic pocket. The enhanced stabilities of the albumin/anesthetic complexes and -DeltaC(p) are consistent with favorable solvent rearrangements that promote binding. This idea is supported by substitution of D(2)O for H(2)O that significantly reduces the favorable binding enthalpy observed for desflurane and isoflurane, with an opposing increase of DeltaS(obs). From these results, we infer that solvent restructuring, resulting from release of water weakly bound to anesthetic and anesthetic-binding sites, is a dominant and favorable contributor to the enthalpy and entropy of binding to proteins.

Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

PubMed

Vippagunta, S R; Dorn, A; Matile, H; Bhattacharjee, A K; Karle, J M; Ellis, W Y; Ridley, R G; Vennerstrom, J L

1999-11-04

Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC(50) values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that antimalarial 4-aminoquinolines act by this mechanism.

Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance.

PubMed

Hou, Yuting; An, Jianhong; Deng, Chunyan; Chen, Shu; Xiang, Juan

2016-07-01

The interactions between the redox couple of cytochrome c (Cyt c) and cytochrome c oxidase (COX) were investigated at a mimic redox-modulated interface by using an electrochemical surface plasmon resonance (EC-SPR) system. Although early studies of the binding between COX and Cyt c have been conducted using several techniques in homogeneous solutions, a problem still inherent is that ferro-cytochrome c (Cyt c red), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt c ox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt c red is quantitatively electro-generated from Cyt c ox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt c red/Cyt c ox can be evaluated accurately. In this study, the values of association/dissociation rate constants (k a, k d) for both COX/Cyt c red and COX/Cyt c ox were obtained. The dissociation constants, K D, were finally calculated as 3.33 × 10(-8) mol · L(-1) for COX/Cyt c red and 4.25 × 10(-5) mol · L(-1) for COX/Cyt c ox, respectively. In-situ EC-SPR is promising for better mimicking the in vivo condition that COX is embedded in the inner mitochondrial membrane and Cyt c acts as an electron shuttle in the mobile phase. It is an effective method for the investigation of redox-dependent biomolecular interactions. Graphical Abstract Schematic representation of the experimental designs using EC-SPR system. (a) the Au-Cys-COX SPR chip with SAM layers. (b) redox-modulated Cyt c and its binding onto pre-immobilized COX.

Study on interaction between curcumin and pepsin by spectroscopic and docking methods.

PubMed

Ying, Ming; Huang, Fengwen; Ye, Haidong; Xu, Hong; Shen, Liangliang; Huan, Tianwen; Huang, Shitong; Xie, Jiangfeng; Tian, Shengli; Hu, Zhangli; He, Zhendan; Lu, Jun; Zhou, Kai

2015-08-01

The interaction between curcumin and pepsin was investigated by fluorescence, synchronous fluorescence, UV-vis absorption, circular dichroism (CD), and molecular docking. Under physiological pH value in stomach, the fluorescence of pepsin can be quenched effectively by curcumin via a combined quenching process. Binding constant (Ka) and binding site number (n) of curcumin to pepsin were obtained. According to the theory of Förster's non-radiation energy transfer, the distance r between pepsin and curcumin was found to be 2.45 nm within the curcumin-pepsin complex, which implies that the energy transfer occurs between curcumin and pepsin, leading to the quenching of pepsin fluorescence. Fluorescence experiments also suggest that curcumin is located more closely to tryptophan residues than tyrosine residues. CD spectra together with UV-vis absorbance studies show that binding of curcumin to pepsin results in the extension of peptide strands of pepsin with loss of some β-sheet structures. Thermodynamic parameters calculated from the binding constants at different temperatures reveal that hydrophobic force plays a major role in stabilizing the curcumin-pepsin complex. In addition, docking results support the above experimental findings and suggest the possible hydrogen bonds of curcumin with Thr-77, Thr-218, and Glu-287 of pepsin, which help further stabilize the curcumin-pepsin complex. Copyright © 2015 Elsevier B.V. All rights reserved.

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization ofmore » the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.« less

Concentration-Dependent Multiple Binding Sites on Saliva-Treated Hydroxyapatite for Streptococcus sanguis

PubMed Central

Gibbons, R. J.; Moreno, E. C.; Etherden, I.

1983-01-01

The influence of bacterial cell concentration on estimates of the number of binding sites and the affinity for the adsorption of a strain of Streptococcus sanguis to saliva-treated hydroxyapatite was determined, and the possible presence of multiple binding sites for this organism was tested. The range of concentrations of available bacteria varied from 4.7 × 106 to 5,960 × 106 cells per ml. The numbers of adsorbed bacteria increased over the entire range tested, but a suggestion of a break in an otherwise smooth adsorption isotherm was evident. Values for the number of binding sites and the affinity varied considerably depending upon the range of available bacterial concentrations used to estimate them; high correlation coefficients were obtained in all cases. The use of low bacterial cell concentrations yielded lower values for the number of sites and much higher values for the affinity constant than did the use of high bacterial cell concentrations. When data covering the entire range of bacterial concentrations were employed, values for the number of sites and the affinity were similar to those obtained by using only high bacterial cell concentrations. The simplest explanation for these results is that there are multiple binding sites for S. sanguis on saliva-treated hydroxyapatite surfaces. When present in low concentration, the streptococci evidently attach to more specific high-affinity sites which become saturated when higher bacterial concentrations are employed. The possibility of multiple binding sites was substantiated by comparing estimates of the adsorption parameters from a computer-simulated isotherm with those derived from the experimentally generated isotherm. A mathematical model describing bacterial adsorption to binary binding sites was further evidence for the existence of at least two classes of binding sites for S. sanguis. Far fewer streptococci adsorbed to experimental pellicles prepared from saliva depleted of bacterial aggregating activity when low numbers of streptococci were used, but the magnitude of this difference was considerably less when high streptococcal concentrations were employed. This suggests an association between salivary components which possess bacterial-aggregating activity and bacterial adsorption to high-affinity specific binding sites on saliva-treated hydroxyapatite surfaces. PMID:6822416

New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

PubMed

Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

2017-12-01

Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

Binding constant of cell adhesion receptors and substrate-immobilized ligands depends on the distribution of ligands

NASA Astrophysics Data System (ADS)

Li, Long; Hu, Jinglei; Xu, Guangkui; Song, Fan

2018-01-01

Cell-cell adhesion and the adhesion of cells to tissues and extracellular matrix, which are pivotal for immune response, tissue development, and cell locomotion, depend sensitively on the binding constant of receptor and ligand molecules anchored on the apposing surfaces. An important question remains of whether the immobilization of ligands affects the affinity of binding with cell adhesion receptors. We have investigated the adhesion of multicomponent membranes to a flat substrate coated with immobile ligands using Monte Carlo simulations of a statistical mesoscopic model with biologically relevant parameters. We find that the binding of the adhesion receptors to ligands immobilized on the substrate is strongly affected by the ligand distribution. In the case of ligand clusters, the receptor-ligand binding constant can be significantly enhanced due to the less translational entropy loss of lipid-raft domains in the model cell membranes upon the formation of additional complexes. For ligands randomly or uniformly immobilized on the substrate, the binding constant is rather decreased since the receptors localized in lipid-raft domains have to pay an energetic penalty in order to bind ligands. Our findings help to understand why cell-substrate adhesion experiments for measuring the impact of lipid rafts on the receptor-ligand interactions led to contradictory results.

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lach, E.; Trifilieff, A.; Landry, Y.

1991-01-01

The binding of the radiolabeled bombesin analogue ({sup 125}I-Tyr{sup 4})bombesin to guinea-pig lung membranes was investigated. Binding of ({sup 125}I-Tyr{sup 4})bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B{sub max} = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K{sub D} = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as ({sup 125}I-Tyr{sup 4})bombesin, neuromedin B and neuromedin C inhibited the binding of ({sup 125}I-Tyr{sup 4})bombesin inmore » an order of potencies as follows: ({sup 125}I-Tyr{sup 4})bombesin {gt} bombesin {ge} neuromedin C {much gt} neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.« less

Transient kinetic studies of pH-dependent hydrolyses by exo-type carboxypeptidase P on a 27-MHz quartz crystal microbalance.

PubMed

Furusawa, Hiroyuki; Takano, Hiroki; Okahata, Yoshio

2008-02-15

pH-Dependent kinetic parameters (k(on), k(off), and k(cat)) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme-substrate (ES) complex (k(on)), the decay of the ES complex (k(off)), and the formation of the product (k(cat)) could be analyzed by transient kinetics as mass changes on the QCM plate. The Kd (k(off)/k(on)) value was different from the Michaelis constant Km calculated from (k(off) + k(cat))/k(on) due to k(cat) > k(off). The rate-determining step was the binding step (k(on), and the catalytic rate k(cat) was faster than other k(on) and k(off) values. In the range of pH 2.5-5.0, values of k(on) gradually increased with decreasing pH showing a maximum at pH 3.7, values of k(off) were independent of pH, and k(cat) increased gradually with decreasing pH. As a result, the apparent rate constant (k(cat)/Km) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of k(on) is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of k(cat) at the lower pH correlated with the decrease of alpha-helix contents of the myoglobin substrate on the QCM.

Target and Tissue Selectivity Prediction by Integrated Mechanistic Pharmacokinetic-Target Binding and Quantitative Structure Activity Modeling.

PubMed

Vlot, Anna H C; de Witte, Wilhelmus E A; Danhof, Meindert; van der Graaf, Piet H; van Westen, Gerard J P; de Lange, Elizabeth C M

2017-12-04

Selectivity is an important attribute of effective and safe drugs, and prediction of in vivo target and tissue selectivity would likely improve drug development success rates. However, a lack of understanding of the underlying (pharmacological) mechanisms and availability of directly applicable predictive methods complicates the prediction of selectivity. We explore the value of combining physiologically based pharmacokinetic (PBPK) modeling with quantitative structure-activity relationship (QSAR) modeling to predict the influence of the target dissociation constant (K D ) and the target dissociation rate constant on target and tissue selectivity. The K D values of CB1 ligands in the ChEMBL database are predicted by QSAR random forest (RF) modeling for the CB1 receptor and known off-targets (TRPV1, mGlu5, 5-HT1a). Of these CB1 ligands, rimonabant, CP-55940, and Δ 8 -tetrahydrocanabinol, one of the active ingredients of cannabis, were selected for simulations of target occupancy for CB1, TRPV1, mGlu5, and 5-HT1a in three brain regions, to illustrate the principles of the combined PBPK-QSAR modeling. Our combined PBPK and target binding modeling demonstrated that the optimal values of the K D and k off for target and tissue selectivity were dependent on target concentration and tissue distribution kinetics. Interestingly, if the target concentration is high and the perfusion of the target site is low, the optimal K D value is often not the lowest K D value, suggesting that optimization towards high drug-target affinity can decrease the benefit-risk ratio. The presented integrative structure-pharmacokinetic-pharmacodynamic modeling provides an improved understanding of tissue and target selectivity.

Ionised concentrations in calcium and magnesium buffers: Standards and precise measurement are mandatory.

PubMed

McGuigan, John A S; Kay, James W; Elder, Hugh Y

2016-09-01

In Ca(2+) and Mg(2+) buffer solutions the ionised concentrations ([X(2+)]) are either calculated or measured. Calculated values vary by up to a factor of seven due to the following four problems: 1) There is no agreement amongst the tabulated constants in the literature. These constants have usually to be corrected for ionic strength and temperature. 2) The ionic strength correction entails the calculation of the single ion activity coefficient, which involves non-thermodynamic assumptions; the data for temperature correction is not always available. 3) Measured pH is in terms of activity i.e. pHa. pHa measurements are complicated by the change in the liquid junction potentials at the reference electrode making an accurate conversion from H(+) activity to H(+) concentration uncertain. 4) Ligands such as EGTA bind water and are not 100% pure. Ligand purity has to be measured, even when the [X(2+)] are calculated. The calculated [X(2+)] in buffers are so inconsistent that calculation is not an option. Until standards are available, the [X(2+)] in the buffers must be measured. The Ligand Optimisation Method is an accurate and independently verified method of doing this (McGuigan & Stumpff, Anal. Biochem. 436, 29, 2013). Lack of standards means it is not possible to compare the published [Ca(2+)] in the nmolar range, and the apparent constant (K(/)) values for Ca(2+) and Mg(2+) binding to intracellular ligands amongst different laboratories. Standardisation of Ca(2+)/Mg(2+) buffers is now essential. The parameters to achieve this are proposed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Spectroscopic studies on the interaction of chromium (VI) and chromium (III) with keyhole limpet hemocyanin.

PubMed

Fan, Yulan; Zeng, Guidi; Liu, Jingyi; Chen, Huifang; Xue, Jun; Wu, Yongquan; Li, Xun

2017-03-01

The interactions of keyhole limpet hemocyanin (KLH) with chromium nitrate, potassium dichromate, and chromate were investigated using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy under simulated physiological conditions. The experimental results showed that the different forms of chromium could quench the intrinsic fluorescence of KLH following a static quenching mechanism rather than by dynamic collision, which indicated that a Cr-KLH complex was formed. The Stern-Volmer quenching constants for the interaction indicated that the binding reaction of KLH with Cr(VI) was stronger the binding of KLH with Cr(III). The thermodynamic values for binding of Cr(VI) to KLH are ΔH > 0 and ΔS > 0. By contrast, the values for the interaction of Cr(III) with KLH are ΔH < 0 and ΔS < 0. The results of synchronous fluorescence, UV-vis absorption and CD spectroscopy showed that the α-helical secondary structure and conformation of KLH were altered by different forms of chromium. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A comparative study on the binding of single and double chain surfactant-cobalt(III) complexes with bovine serum albumin

NASA Astrophysics Data System (ADS)

Vignesh, G.; Sugumar, K.; Arunachalam, S.; Vignesh, S.; Arthur James, R.

2013-09-01

The comparative binding effect of single and double aliphatic chain containing surfactant-cobalt(III) complexes cis-[Co(bpy)2(DA)2](ClO4)3ṡ2H2O (1), cis-[Co(bpy)2(DA)Cl](ClO4)2ṡ2H2O (2), cis-[Co(phen)2(CA)2](ClO4)3ṡ2H2O (3), and cis-[Co(phen)2(CA)Cl](ClO4)2ṡ2H2O (4) with bovine serum albumin (BSA) under physiological condition was analyzed by steady state, time resolved fluorescence, synchronous, three-dimensional fluorescence, UV-Visible absorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of BSA through a static mechanism. The binding constants (Kb) and the number of binding sites were calculated and binding constant values are found in the range of 104-105 M-1. The results indicate that compared to single chain complex, double chain surfactant-cobalt(III) complex interacts strongly with BSA. Also the sign of thermodynamic parameters (ΔG°, ΔH°, and ΔS°) indicate that all the complexes interact with BSA through hydrophobic force. The binding distance (r) between complexes and BSA was calculated using Förster non-radiation energy transfer theory and found to be less than 7 nm. The results of synchronous, three dimensional fluorescence and circular dichroism spectroscopic methods indicate that the double chain surfactant-cobalt(III) complexes changed the conformation of the protein considerably than the respective single chain surfactant-cobalt(III) complexes. Antimicrobial studies of the complexes showed good activities against pathogenic microorganisms.

Quantitation of benzodiazepine receptor binding with PET [11C]iomazenil and SPECT [123I]iomazenil: preliminary results of a direct comparison in healthy human subjects.

PubMed

Bremner, J D; Baldwin, R; Horti, A; Staib, L H; Ng, C K; Tan, P Z; Zea-Ponce, Y; Zoghbi, S; Seibyl, J P; Soufer, R; Charney, D S; Innis, R B

1999-08-31

Although positron emission tomography (PET) and single photon emission computed tomography (SPECT) are increasingly used for quantitation of neuroreceptor binding, almost no studies to date have involved a direct comparison of the two. One study found a high level of agreement between the two techniques, although there was a systematic 30% increase in measures of benzodiazepine receptor binding in SPECT compared with PET. The purpose of the current study was to directly compare quantitation of benzodiazepine receptor binding in the same human subjects using PET and SPECT with high specific activity [11C]iomazenil and [123I]iomazenil, respectively. All subjects were administered a single bolus of high specific activity iomazenil labeled with 11C or 123I followed by dynamic PET or SPECT imaging of the brain. Arterial blood samples were obtained for measurement of metabolite-corrected radioligand in plasma. Compartmental modeling was used to fit values for kinetic rate constants of transfer of radioligand between plasma and brain compartments. These values were used for calculation of binding potential (BP = Bmax/Kd) and product of BP and the fraction of free non-protein-bound parent compound (V3'). Mean values for V3' in PET and SPECT were as follows: temporal cortex 23+/-5 and 22+/-3 ml/g, frontal cortex23+/-6 and 22+/-3 ml/g, occipital cortex 28+/-3 and 31+/-5 ml/g, and striatum 4+/-4 and 7+/-4 ml/g. These preliminary findings indicate that PET and SPECT provide comparable results in quantitation of neuroreceptor binding in the human brain.

Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

PubMed

Núñez-Montenegro, Ara; Carballo, Rosa; Vázquez-López, Ezequiel M

2014-11-01

The binding affinities towards estrogen receptors (ERs) α and β of a set of thiosemicarbazone ligands (HL(n)) and their rhenium(I) carbonyl complexes [ReX(HL(n))(CO)3] (X=Cl, Br) were determined by a competitive standard radiometric assay with [(3)H]-estradiol. The ability of the coordinated thiosemicarbazone ligands to undergo deprotonation and the lability of the ReX bond were used as a synthetic strategy to obtain [Re(hpy)(L(n))(CO)3] (hpy=3- or 4-hydroxypyridine). The inclusion of the additional hpy ligand endows the new thiosemicarbazonate complexes with an improved affinity towards the estrogen receptors and, consequently, the values of the inhibition constant (Ki) could be determined for some of them. In general, the values of Ki for both ER subtypes suggest an appreciable selectivity towards ERα. Copyright © 2014 Elsevier Inc. All rights reserved.

Mechanistic Studies of Human Spermine Oxidase: Kinetic Mechanism and pH Effects†

PubMed Central

Adachi, Maria S.; Juarez, Paul R.; Fitzpatrick, Paul F.

2009-01-01

In mammalian cells, the flavoprotein spermine oxidase (SMO) catalyzes the oxidation of spermine to spermidine and 3-aminopropanal. Mechanistic studies have been carried out with the recombinant human enzyme. The initial velocity pattern when the ratio between the concentrations of spermine and oxygen is kept constant establishes the steady-state kinetic pattern as ping-pong. Reduction of SMO by spermine in the absence of oxygen is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with a limiting value (k3) of 49 s−1 and an apparent Kd value of 48 µM at pH 8.3. The rate constant for the slow step is independent of the spermine concentration, with a value of 5.5 s−1, comparable to the kcat value of 6.6 s−1. The kinetics of the oxidative half-reaction depend on the aging time after spermine and enzyme are mixed in a double mixing experiment. At an aging time of 6 s the reaction is monophasic with a second order rate constant of 4.2 mM−1 s−1. At an aging time of 0.3 s the reaction is biphasic with two second order constants equal to 4.0 and 40 mM−1 s−1. Neither is equal to the kcat/KO2 value of 13 mM−1s−1. These results establish the existence of more than one pathway for the reaction of the reduced flavin intermediate with oxygen. The kcat/KM value for spermine exhibits a bell-shaped pH-profile, with an average pKa value of 8.3. This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for k3 shows a pKa value of 7.4 for a group that must be unprotonated. The pKi-pH profiles for the competitive inhibitors N,N’-dibenzylbutane-1,4-diamine and spermidine show that the fully protonated forms of the inhibitors and the unprotonated form of an amino acid residue with a pKa of about 7.4 in the active site are preferred for binding. PMID:20000632

Calorimetric and spectroscopic studies of the interaction between zidovudine and human serum albumin

NASA Astrophysics Data System (ADS)

Pîrnău, Adrian; Mic, Mihaela; Neamţu, Silvia; Floare, Călin G.; Bogdan, Mircea

2018-02-01

A quantitative analysis of the interaction between zidovudine (AZT) and human serum albumin (HSA) was achieved using Isothermal titration calorimetry (ITC) in combination with fluorescence and 1H NMR spectroscopy. ITC directly measure the heat during a biomolecular binding event and gave us thermodynamic parameters and the characteristic association constant. By fluorescence quenching, the binding parameters of AZT-HSA interaction was determined and location to binding site I of HSA was confirmed. Via T1 NMR selective relaxation time measurements the drug-protein binding extent was evaluated as dissociation constants Kd and the involvement of azido moiety of zidovudine in molecular complex formation was put in evidence. All three methods indicated a very weak binding interaction. The association constant determined by ITC (3.58 × 102 M- 1) is supported by fluorescence quenching data (2.74 × 102 M- 1). The thermodynamic signature indicates that at least hydrophobic and electrostatic type interactions played a main role in the binding process.

Binding of resveratrol with sodium caseinate in aqueous solutions.

PubMed

Acharya, Durga P; Sanguansri, Luz; Augustin, Mary Ann

2013-11-15

The interaction between resveratrol (Res) and sodium caseinate (Na-Cas) has been studied by measuring fluorescence quenching of the protein by resveratrol. Quenching constants were determined using Stern-Volmer equation, which suggests that both dynamic and static quenching occur between Na-Cas and Res. Binding constants for the complexation between Na-Cas and Res were determined at different temperatures. The large binding constants (3.7-5.1×10(5)M(-1)) suggest that Res has strong affinity for Na-Cas. This affinity decreases as the temperature is raised from 25 to 37°C. The binding involves both hydrogen bonding and hydrophobic interaction, as suggested by negative enthalpy change and positive entropy change for the binding reaction. The present study indicates that Na-Cas, a common food protein, may be used as a carrier of Res, a bioactive polyphenol which is insoluble in both water and oils. Copyright © 2013 Elsevier Ltd. All rights reserved.

Heterodimer Autorepression Loop: A Robust and Flexible Pulse-Generating Genetic Module

NASA Astrophysics Data System (ADS)

Lannoo, B.; Carlon, E.; Lefranc, M.

2016-07-01

We investigate the dynamics of the heterodimer autorepression loop (HAL), a small genetic module in which a protein A acts as an autorepressor and binds to a second protein B to form an A B dimer. For suitable values of the rate constants, the HAL produces pulses of A alternating with pulses of B . By means of analytical and numerical calculations, we show that the duration of A pulses is extremely robust against variation of the rate constants while the duration of the B pulses can be flexibly adjusted. The HAL is thus a minimal genetic module generating robust pulses with a tunable duration, an interesting property for cellular signaling.

Synthesis, characterization and biological application of four novel metal-Schiff base complexes derived from allylamine and their interactions with human serum albumin: Experimental, molecular docking and ONIOM computational study.

PubMed

Kazemi, Zahra; Rudbari, Hadi Amiri; Sahihi, Mehdi; Mirkhani, Valiollah; Moghadam, Majid; Tangestaninejad, Shahram; Mohammadpoor-Baltork, Iraj; Gharaghani, Sajjad

2016-09-01

Novel metal-based drug candidate including VOL2, NiL2, CuL2 and PdL2 have been synthesized from 2-hydroxy-1-allyliminomethyl-naphthalen ligand and have been characterized by means of elemental analysis (CHN), FT-IR and UV-vis spectroscopies. In addition, (1)H and (13)C NMR techniques were employed for characterization of the PdL2 complex. Single-crystal X-ray diffraction technique was utilized to characterise the structure of the complexes. The Cu(II), Ni(II) and Pd(II) complexes show a square planar trans-coordination geometry, while in the VOL2, the vanadium center has a distorted tetragonal pyramidal N2O3 coordination sphere. The HSA-binding was also determined, using fluorescence quenching, UV-vis spectroscopy, and circular dichroism (CD) titration method. The obtained results revealed that the HSA affinity for binding the synthesized compounds follows as PdL2>CuL2>VOL2>NiL2, indicating the effect of metal ion on binding constant. The distance between these compounds and HSA was obtained based on the Förster's theory of non-radiative energy transfer. Furthermore, computational methods including molecular docking and our Own N-layered Integrated molecular Orbital and molecular Mechanics (ONIOM) were carried out to investigate the HSA-binding of the compounds. Molecular docking calculation indicated the existence of hydrogen bond between amino acid residues of HSA and all synthesized compounds. The formation of the hydrogen bond in the HSA-compound systems leads to their stabilization. The ONIOM method was utilized in order to investigate HSA binding of compounds more precisely in which molecular mechanics method (UFF) and semi empirical method (PM6) were selected for the low layer and the high layer, respectively. The results show that the structural parameters of the compounds changed along with binding to HSA, indicating the strong interaction between the compounds and HSA. The value of binding constant depends on the extent of the resultant changes. This should be mentioned that both theoretical methods calculated the Kb values in the same sequence and are in a good agreement with the experimental data. Copyright © 2016 Elsevier B.V. All rights reserved.

Copper removal by algal biomass: biosorbents characterization and equilibrium modelling.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Pinheiro, José P S; Domingos, Rute F; Boaventura, Rui A R

2009-04-30

The general principles of Cu(II) binding to algal waste from agar extraction, composite material and algae Gelidium, and different modelling approaches, are discussed. FTIR analyses provided a detailed description of the possible binding groups present in the biosorbents, as carboxylic groups (D-glucuronic and pyruvic acids), hydroxyl groups (cellulose, agar and floridean starch) and sulfonate groups (sulphated galactans). Potentiometric acid-base titrations showed a heterogeneous distribution of two major binding groups, carboxyl and hydroxyl, following the quasi-Gaussian affinity constant distribution suggested by Sips, which permitted to estimate the maximum amount of acid functional groups (0.36, 0.25 and 0.1 mmol g(-1)) and proton binding parameters (pK(H)=5.0, 5.3 and 4.4; m(H)=0.43, 0.37, 0.33), respectively for algae Gelidium, algal waste and composite material. A non-ideal, semi-empirical, thermodynamically consistent (NICCA) isotherm fitted better the experimental ion binding data for different pH values and copper concentrations, considering only the acid functional groups, than the discrete model. Values of pK(M) (3.2; 3.6 and 3.3), n(M) (0.98, 0.91, 1.0) and p (0.67, 0.53 and 0.43) were obtained, respectively for algae Gelidium, algal waste and composite material. NICCA model reflects the complex macromolecular systems that take part in biosorption considering the heterogeneity of the biosorbent, the competition between protons and metals ions to the binding sites and the stoichiometry for different ions.

DNA binding by a new metallointercalator that contains a proflavine group bearing a hanging chelating unit.

PubMed

Bazzicalupi, Carla; Bencini, Andrea; Bianchi, Antonio; Biver, Tarita; Boggioni, Alessia; Bonacchi, Sara; Danesi, Andrea; Giorgi, Claudia; Gratteri, Paola; Ingraín, Antonio Marchal; Secco, Fernando; Sissi, Claudia; Valtancoli, Barbara; Venturini, Marcella

2008-01-01

The new bifunctional molecule 3,6-diamine-9-[6,6-bis(2-aminoethyl)-1,6-diaminohexyl]acridine (D), which is characterised by both an aromatic moiety and a separate metal-complexing polyamine centre, has been synthesised. The characteristics of D and its ZnII complex ([ZnD]) (protonation and metal-complexing constants, optical properties and self-aggregation phenomena) have been analysed by means of NMR spectroscopy, potentiometric, spectrophotometric and spectrofluorimetric techniques. The equilibria and kinetics of the binding process of D and [ZnD] to calf thymus DNA have been investigated at I=0.11 M (NaCl) and 298.1 K by using spectroscopic methods and the stopped-flow technique. Static measurements show biphasic behaviour for both D-DNA and [ZnD]-DNA systems; this reveals the occurrence of two different binding processes depending on the polymer-to-dye molar ratio (P/D). The binding mode that occurs at low P/D values is interpreted in terms of external binding with a notable contribution from the polyamine residue. The binding mode at high P/D values corresponds to intercalation of the proflavine residue. Stopped-flow, circular dichroism and supercoiled-DNA unwinding experiments corroborate the proposed mechanism. Molecular-modelling studies support the intercalative process and evidence the influence of NH+...O interactions between the protonated acridine nitrogen atom and the oxygen atoms of the polyanion; these interactions play a key role in determining the conformation of DNA adducts.

Copper(II) binding by dissolved organic matter: Importance of the copper-to-dissolved organic matter ratio and implications for the Biotic Ligand Model

USGS Publications Warehouse

Craven, Alison M.; Aiken, George R.; Ryan, Joseph N.

2012-01-01

The ratio of copper to dissolved organic matter (DOM) is known to affect the strength of copper binding by DOM, but previous methods to determine the Cu2+–DOM binding strength have generally not measured binding constants over the same Cu:DOM ratios. In this study, we used a competitive ligand exchange–solid-phase extraction (CLE-SPE) method to determine conditional stability constants for Cu2+–DOM binding at pH 6.6 and 0.01 M ionic strength over a range of Cu:DOM ratios that bridge the detection windows of copper-ion-selective electrode and voltammetry measurements. As the Cu:DOM ratio increased from 0.0005 to 0.1 mg of Cu/mg of DOM, the measured conditional binding constant (cKCuDOM) decreased from 1011.5 to 105.6 M–1. A comparison of the binding constants measured by CLE-SPE with those measured by copper-ion-selective electrode and voltammetry demonstrates that the Cu:DOM ratio is an important factor controlling Cu2+–DOM binding strength even for DOM isolates of different types and different sources and for whole water samples. The results were modeled with Visual MINTEQ and compared to results from the biotic ligand model (BLM). The BLM was found to over-estimate Cu2+ at low total copper concentrations and under-estimate Cu2+ at high total copper concentrations.

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction.

PubMed

Eble, Johannes A

2018-02-15

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ascenzi, Paolo; National Institute for Infectious Diseases I.R.C.C.S. 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Roma; Imperi, Francesco

Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k{sub off}) is reported. In the absence of drugs, the value of k{sub off} is (1.3 {+-} 0.2) x 10{sup -4} s{sup -1}. Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k{sub off} value increases to (8.6 {+-} 0.9) x 10{sup -4} s{sup -1}. From the dependence of k{sub off} on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NOmore » (i.e., K = (1.2 {+-} 0.2) x 10{sup -3} M and (6.2 {+-} 0.7) x 10{sup -5} M, respectively) were determined. The increase of k{sub off} values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow's site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.« less

Long-term bioconcentration kinetics of hydrophobic chemicals in Selenastrum capricornutum and Microcystis aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koelmans, A.A.; Woude, H. van der; Hattink, J.

1999-06-01

The bioconcentration of two chlorobenzenes (CBs) and of seven polychlorobiphenyls (PCBs) to Selenastrum capricornutum and Microcystis aeruginosa was studied with accumulation experiments followed by gas purge elimination experiments. Henry's law constants at 10 C were needed to interpret the gas purge results and were measured in control experiments. For the M. aerogunisa culture, steady-state uptake was reached within days, whereas uptake by S. capricornutum took several weeks. The relationships between the log bioconcentration factors (BCF) and log octanol-water partition coefficients (K[sub OW]) were nonlinear, with relatively low values for the more hydrophobic PCBs. Rate constants for the elimination of CBsmore » and PCBs from the algal cells were shown to be larger than 1 per day when calculated with a one-compartment model. With such large rate constants, it is unlikely that the curvature observed for these species is caused by slow kinetics or that algal growth affects BCF by dilution of CB or PCB concentrations. The log BCF-log K[sub OW] relationships could be described by a simple three-phase model that accounted for the binding of CBs and PCBs to dissolved organic carbon (DOC). Modeling bioconcentration of hydrophobic chemicals in phytoplankton should account for the binding to DOC.« less

Dye-induced aggregation of single stranded RNA: a mechanistic approach.

PubMed

Biver, Tarita; Ciatto, Carlo; Secco, Fernando; Venturini, Marcella

2006-08-15

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 degrees C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PD(I) and an internal complex PD(II) form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (C(P)/C(D)). For C(P)/C(D)<2.5, cooperative formation of stacks external to polymer strands prevails (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g=1), equilibrium constant for the nucleation step (K( *)=(1.4+/-0.6)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(7)M(-1)s(-1), k(d)=1.1 x 10(2)s(-1)). Stopped-flow experiments, performed at low ionic strength (I=0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that C(P)/C(D)<2.5.

Rationalizing 5000-Fold Differences in Receptor-Binding Rate Constants of Four Cytokines

PubMed Central

Pang, Xiaodong; Qin, Sanbo; Zhou, Huan-Xiang

2011-01-01

The four cytokines erythropoietin (EPO), interleukin-4 (IL4), human growth hormone (hGH), and prolactin (PRL) all form four-helix bundles and bind to type I cytokine receptors. However, their receptor-binding rate constants span a 5000-fold range. Here, we quantitatively rationalize these vast differences in rate constants by our transient-complex theory for protein-protein association. In the transient complex, the two proteins have near-native separation and relative orientation, but have yet to form the short-range specific interactions of the native complex. The theory predicts the association rate constant as ka=ka0exp(−ΔGel∗/kBT) where ka0 is the basal rate constant for reaching the transient complex by random diffusion, and the Boltzmann factor captures the rate enhancement due to electrostatic attraction. We found that the vast differences in receptor-binding rate constants of the four cytokines arise mostly from the differences in charge complementarity among the four cytokine-receptor complexes. The basal rate constants (ka0) of EPO, IL4, hGH, and PRL were similar (5.2 × 105 M−1s−1, 2.4 × 105 M−1s−1, 1.7 × 105 M−1s−1, and 1.7 × 105 M−1s−1, respectively). However, the average electrostatic free energies (ΔGe1∗) were very different (−4.2 kcal/mol, −2.4 kcal/mol, −0.1 kcal/mol, and −0.5 kcal/mol, respectively, at ionic strength = 160 mM). The receptor-binding rate constants predicted without adjusting any parameters, 6.2 × 108 M−1s−1, 1.3 × 107 M−1s−1, 2.0 × 105 M−1s−1, and 7.6 × 104 M−1s−1, respectively, for EPO, IL4, hGH, and PRL agree well with experimental results. We uncover that these diverse rate constants are anticorrelated with the circulation concentrations of the cytokines, with the resulting cytokine-receptor binding rates very close to the limits set by the half-lives of the receptors, suggesting that these binding rates are functionally relevant and perhaps evolutionarily tuned. Our calculations also reproduced well-observed effects of mutations and ionic strength on the rate constants and produced a set of mutations on the complex of hGH with its receptor that putatively enhances the rate constant by nearly 100-fold through increasing charge complementarity. To quantify charge complementarity, we propose a simple index based on the charge distribution within the binding interface, which shows good correlation with ΔGe1∗. Together these results suggest that protein charges can be manipulated to tune ka and control biological function. PMID:21889455

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases.

PubMed

Soya, Naoto; Shoemaker, Glen K; Palcic, Monica M; Klassen, John S

2009-11-01

The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

Magnetic susceptibility and dielectric properties of peat in Central Kalimantan, Indonesia

NASA Astrophysics Data System (ADS)

Budi, Pranitha Septiana; Zulaikah, Siti; Hidayat, Arif; Azzahro, Rosyida

2017-07-01

Peatlands dominate almost all regions of Borneo, yet its utilization has not been developed optimally. Any information in this field could be obtained using soil magnetization methods by determining the magnetic succeptibility in terms of magnetic susceptibility value that could describe the source and type of magnetic minerals which could describe the source and type of magnetic minerals. Moreover, the dielectric properties of peat soil were also investigated to determine the level of water content by using the dielectric constant value. Samples was taken at six different locations along Pulang pisau to Berengbengkel. Magnetic susceptibility mass value at these locations ranged between -0.0009 - 0.712 (×10-6 m3/kg). Based on the average magnetic susceptibility value, samples that were taken from T1, T3 and T5 belonged to the type of paramagnetic mineral, while samples which were taken from T2, T4 and T6 belonged to the group of diamagnetic mineral. The low value of magnetic susceptibility of peat was probably derived from the pedogenic process. The average value of peat soil in six locations has a large dielectric constant value that is 28.2 which indicated that there was considerable moisture content due to the hydrophilic nature of peatland which means that the ability of peat in water binding is considerably high.

Binding of naringin and naringenin with hen egg white lysozyme: A spectroscopic investigation and molecular docking study

NASA Astrophysics Data System (ADS)

Das, Sourav; Ghosh, Pooja; Koley, Sudipta; Singha Roy, Atanu

2018-03-01

The interactions of naringenin (NG) and naringin (NR) with Hen Egg White Lysozyme (HEWL) in aqueous medium have been investigated using UV-vis spectroscopy, steady-state fluorescence, circular dichroism (CD), Fourier Transform infrared spectroscopy (FT-IR) and molecular docking analyses. Both NG and NR can quench the intrinsic fluorescence of HEWL via static quenching mechanism. At 300 K, the value of binding constant (Kb) of HEWL-NG complex (5.596 ± 0.063 × 104 M- 1) was found to be greater than that of HEWL-NR complex (3.404 ± 0.407 × 104 M- 1). The negative ΔG° values in cases of both the complexes specify the spontaneous binding. The binding distance between the donor (HEWL) and acceptor (NG/NR) was estimated using the Försters theory and the possibility of non-radiative energy transfer from HEWL to NG/NR was observed. The presence of metal ions (Ca2 +, Cu2 + and Fe2 +) decreased the binding affinity of NG/NR towards HEWL. Synchronous fluorescence studies indicate the change in Trp micro-environment due to the incorporation of NG/NR into HEWL. CD and FT-IR studies indicated that the α-helicity of the HEWL was slightly enhanced due to ligand binding. NG and NR inhibited the enzymatic activity of HEWL and exhibited their affinity for the active site of HEWL. Molecular docking studies revealed that both NG and NR bind in the close vicinity of Trp 62 and Trp 63 residues which is vital for the catalytic activity.

Formation of the prebiotic molecule NH2CHO on astronomical amorphous solid water surfaces: accurate tunneling rate calculations.

PubMed

Song, Lei; Kästner, Johannes

2016-10-26

Investigating how formamide forms in the interstellar medium is a hot topic in astrochemistry, which can contribute to our understanding of the origin of life on Earth. We have constructed a QM/MM model to simulate the hydrogenation of isocyanic acid on amorphous solid water surfaces to form formamide. The binding energy of HNCO on the ASW surface varies significantly between different binding sites, we found values between ∼0 and 100 kJ mol -1 . The barrier for the hydrogenation reaction is almost independent of the binding energy, though. We calculated tunneling rate constants of H + HNCO → NH 2 CO at temperatures down to 103 K combining QM/MM with instanton theory. Tunneling dominates the reaction at such low temperatures. The tunneling reaction is hardly accelerated by the amorphous solid water surface compared to the gas phase for this system, even though the activation energy of the surface reaction is lower than the one of the gas-phase reaction. Both the height and width of the barrier affect the tunneling rate in practice. Strong kinetic isotope effects were observed by comparing to rate constants of D + HNCO → NHDCO. At 103 K we found a KIE of 231 on the surface and 146 in the gas phase. Furthermore, we investigated the gas-phase reaction NH 2 + H 2 CO → NH 2 CHO + H and found it unlikely to occur at cryogenic temperatures. The data of our tunneling rate constants are expected to significantly influence astrochemical models.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors.

PubMed

Jadey, Snehal; Auerbach, Anthony

2012-07-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes ("catch" and "hold") that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement ("capping"). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

PubMed Central

Jadey, Snehal

2012-01-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation. PMID:22732309

Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise

PubMed Central

1995-01-01

LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes. The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar. The on and off reactions of sugar binding cause an increase of the noise of the current through the channel. The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose. The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site. The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose. The kinetics for sucrose movement was considerably slower. The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures. The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed. PMID:7539481

Copper complexes containing thiosemicarbazones derived from 6-nitropiperonal: Antimicrobial and biophysical properties

NASA Astrophysics Data System (ADS)

Beckford, Floyd A.; Webb, Kelsey R.

2017-08-01

A series of four thiosemicarbazones from 6-nitropiperonal along with the corresponding copper complexes were synthesized. The biophysical characteristics of the complexes were investigated by the binding to DNA and human serum albumin. The binding to DNA is moderate; the binding constants run from (0.49-7.50) × 104 M- 1. In relation to HSA, the complexes interact strongly with binding constants on the order of 105 M- 1. The complexes also display antioxidant behavior as determined by the ability to scavenge diphenylpicrylhydrazyl (dpph) and nitric oxide radicals. The antimicrobial profiles of the compounds, tested against a panel of microbes including five of the ESKAPE pathogens (Staphylococcus aureus, MRSA, Escherichia coli, Klebsiella pneumoniae, MDR, Acinetobacter baumannii, Pseudomonas aeruginosa) and two yeasts (Candida albicans and Cryptococcus neoformans var. grubii), are also described. The compounds contain a core moiety that is similar to oxolinic acid, a quinolone antibiotic that targets DNA gyrase and topoisomerase (IV). The binding interaction between the complexes and these important antibacterial targets were studied by computational methods, chiefly docking studies. The calculated dissociation constants for the interaction with DNA gyrase B (from Staphylococcus aureus) range from 4.32 to 24.65 μM; the binding was much stronger to topoisomerase IV, with dissociation constants ranging from 0.37 to 1.27 μM.

Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA

NASA Astrophysics Data System (ADS)

Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

2016-01-01

A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.

Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA.

PubMed

Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

2016-01-15

A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging. Copyright © 2015 Elsevier B.V. All rights reserved.

Origin of the unusually strong and selective binding of vanadium by polyamidoximes in seawater

DOE PAGES

Ivanov, Alexander S.; Leggett, Christina J.; Parker, Bernard F.; ...

2017-11-16

Amidoxime-functionalized polymeric adsorbents are the current state-of-the-art materials for collecting uranium (U) from seawater. However, marine tests show that vanadium (V) is preferentially extracted over U and many other cations. Here in this paper, we report a complementary and comprehensive investigation integrating ab initio simulations with thermochemical titrations and XAFS spectroscopy to understand the unusually strong and selective binding of V by polyamidoximes. While the open-chain amidoxime functionalities do not bind V, the cyclic imide-dioxime group of the adsorbent forms a peculiar non-oxido V 5+ complex, exhibiting the highest stability constant value ever observed for the V 5+ species. XAFSmore » analysis of adsorbents following deployment in environmental seawater confirms V binding solely by the imide-dioximes. Our fundamental findings offer not only guidance for future optimization of selectivity in amidoxime-based sorbent materials, but may also afford insight to understanding the extensive accumulation of V in some marine organisms.« less

Origin of the unusually strong and selective binding of vanadium by polyamidoximes in seawater

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ivanov, Alexander S.; Leggett, Christina J.; Parker, Bernard F.

Amidoxime-functionalized polymeric adsorbents are the current state-of-the-art materials for collecting uranium (U) from seawater. However, marine tests show that vanadium (V) is preferentially extracted over U and many other cations. Here in this paper, we report a complementary and comprehensive investigation integrating ab initio simulations with thermochemical titrations and XAFS spectroscopy to understand the unusually strong and selective binding of V by polyamidoximes. While the open-chain amidoxime functionalities do not bind V, the cyclic imide-dioxime group of the adsorbent forms a peculiar non-oxido V 5+ complex, exhibiting the highest stability constant value ever observed for the V 5+ species. XAFSmore » analysis of adsorbents following deployment in environmental seawater confirms V binding solely by the imide-dioximes. Our fundamental findings offer not only guidance for future optimization of selectivity in amidoxime-based sorbent materials, but may also afford insight to understanding the extensive accumulation of V in some marine organisms.« less

Binding, stability, and antioxidant activity of quercetin with soy protein isolate particles.

PubMed

Wang, Yufang; Wang, Xiaoyong

2015-12-01

This work is to study the potential of particles fabricated from soy protein isolate (SPI) as a protective carrier for quercetin. When the concentration of SPI particles increases from 0 to 0.35 g/L, quercetin gives a gradually increased fluorescence intensity and fluorescence anisotropy. The addition of quercetin can highly quench the intrinsic fluorescence of SPI particles. These results are explained in terms of the binding of quercetin to the hydrophobic pockets of SPI particles mainly through the hydrophobic force together with the hydrogen bonding. The small difference in the binding constants at 25 and 40 °C suggests the structural stability of SPI particles. The relative changes in values of Gibbs energy, enthalpy, and entropy indicate that the binding of quercetin with SPI particles is spontaneous and hydrophobic interaction is the major force. Furthermore, SPI particles are superior to native SPI for improving the stability and radical scavenging activity of quercetin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Study of interactions of an anticancer drug neratinib with bovine serum albumin: Spectroscopic and molecular docking approach

NASA Astrophysics Data System (ADS)

Wani, Tanveer A.; Bakheit, Ahmed H.; Abounassif, M. A.; Zargar, Seema

2018-03-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.

Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach.

PubMed

Wani, Tanveer A; Bakheit, Ahmed H; Abounassif, M A; Zargar, Seema

2018-01-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes.

Study of Interactions of an Anticancer Drug Neratinib With Bovine Serum Albumin: Spectroscopic and Molecular Docking Approach

PubMed Central

Wani, Tanveer A.; Bakheit, Ahmed H.; Abounassif, M. A.; Zargar, Seema

2018-01-01

Binding of therapeutic agents to plasma proteins, particularly to serum albumin, provides valuable information in the drug development. This study was designed to evaluate the binding interaction of neratinib with bovine serum albumin (BSA). Neratinib blocks HER2 signaling and is effective in trastuzumab-resistant breast cancer treatment. Spectrofluorometric, UV spectrophotometric, and fourier transform infrared (FT-IR) and molecular docking experiments were performed to study this interaction. The fluorescence of BSA is attributed to the presence of tryptophan (Trp) residues. The fluorescence of BSA in presence of neratinib was studied using the excitation wavelength of 280 nm and the emission was measured at 300-500 nm at three different temperatures. Neratinib quenched the BSA intrinsic fluorescence by static mechanism. A complex formation occurred due to the interaction leading to BSA absorption shift. The fluorescence, UV- absorption, three dimensional fluorescence and FT-IR data showed conformational changes occurred in BSA after interaction with neratinib. The binding constant values decreased as the temperature increased suggesting an instable complex formation at high temperature. Site I (sub-domain IIA) was observed as the principal binding site for neratinib. Hydrogen bonding and Van der Waals forces were suggested to be involved in the BSA-neratinib interaction due to the negative values of entropy and enthalpy changes. PMID:29564326

Kinetic analysis of cooperative interactions induced by Mn2+ binding to the chloroplast H(+)-ATPase.

PubMed

Hiller, R; Carmeli, C

1990-07-03

The kinetics of Mn2+ binding to three cooperatively interacting sites in chloroplast H(+)-ATPase (CF1) were measured by EPR following rapid mixing of the enzyme with MnCl2 with a time resolution of 8 ms. Mixing of the enzyme-bound Mn2+ with MgCl2 gave a measure of the rate of exchange. The data could be best fitted to a kinetic model assuming three sequential, positively cooperative binding sites. (1) In the latent CF1, the binding to all three sites had a similar on-rate constants of (1.1 +/- 0.04) X 10(4) M-1s-1. (2) Site segregation was found in the release of ions with off-rate constants of 0.69 +/- 0.04 s-1 for the first two and 0.055 +/- 0.003 s-1 for the third. (3) Addition of one ADP per CF1 caused a decrease in the off-rate constants to 0.31 +/- 0.02 and 0.033 +/- 0.008 s-1 for the first two and the third sites, respectively. (4) Heat activation of CF1 increased the on-rate constant to (4.2 +/- 0.92) X 10(4) M-1s-1 and the off-rate constants of the first two and the third site to 1.34 +/- 0.08 and 0.16 +/- 0.07 s-1, respectively. (5) The calculated thermodynamic dissociation constants were similar to those previously obtained from equilibrium binding studies. These findings were correlated to the rate constants obtained from studies of the catalysis and regulation of the H(+)-ATPase. The data support the suggestion that regulation induces sequential progress of catalysis through the three active sites of the enzyme.

Selectivity and stoichiometry boosting of beta-cyclodextrin in cationic/anionic surfactant systems: when host-guest equilibrium meets biased aggregation equilibrium.

PubMed

Jiang, Lingxiang; Yu, Caifang; Deng, Manli; Jin, Changwen; Wang, Yilin; Yan, Yun; Huang, Jianbin

2010-02-18

Cationic surfactant/anionic surfactant/beta-CD ternary aqueous systems provide a platform for the coexistence of the host-guest (beta-CD/surfactant) equilibrium and the biased aggregation (monomeric/aggregated surfactants) equilibrium. We report here that the interplay between the two equilibria dominates the systems as follows. (1) The biased aggregation equilibrium imposes an apparent selectivity on the host-guest equilibrium, namely, beta-CD has to always selectively bind the major surfactant (molar fraction > 0.5) even if binding constants of beta-CD to the pair of surfactants are quite similar. (2) In return, the host-guest equilibrium amplifies the bias of the aggregation equilibrium, that is, the selective binding partly removes the major surfactant from the aggregates and leaves the aggregate composition approaching the electroneutral mixing stoichiometry. (3) This composition variation enhances electrostatic attractions between oppositely charged surfactant head groups, thus resulting in less-curved aggregates. In particular, the present apparent host-guest selectivity is of remarkably high values, and the selectivity stems from the bias of the aggregation equilibrium rather than the difference in binding constants. Moreover, beta-CD is defined as a "stoichiometry booster" for the whole class of cationic/anionic surfactant systems, which provides an additional degree of freedom to directly adjust aggregate compositions of the systems. The stoichiometry boosting of the compositions can in turn affect or even determine microstructures and macroproperties of the systems.

Binding interaction of atorvastatin with bovine serum albumin: Spectroscopic methods and molecular docking

NASA Astrophysics Data System (ADS)

Wang, Qi; Huang, Chuan-ren; Jiang, Min; Zhu, Ying-yao; Wang, Jing; Chen, Jun; Shi, Jie-hua

2016-03-01

The interaction of atorvastatin with bovine serum albumin (BSA) was investigated using multi-spectroscopic methods and molecular docking technique for providing important insight into further elucidating the store and transport process of atorvastatin in the body and the mechanism of action and pharmacokinetics. The experimental results revealed that the fluorescence quenching mechanism of BSA induced atorvastatin was a combined dynamic and static quenching. The binding constant and number of binding site of atorvastatin with BSA under simulated physiological conditions (pH = 7.4) were 1.41 × 105 M- 1 and about 1 at 310 K, respectively. The values of the enthalpic change (ΔH0), entropic change (ΔS0) and Gibbs free energy (ΔG0) in the binding process of atorvastatin with BSA at 310 K were negative, suggesting that the binding process of atorvastatin and BSA was spontaneous and the main interaction forces were van der Waals force and hydrogen bonding interaction. Moreover, atorvastatin was bound into the subdomain IIA (site I) of BSA, resulting in a slight change of the conformation of BSA.

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycocluster to lectin and tetanus toxin c-fragment

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Minoura, Norihiko

2010-02-01

We have developed a fluorescent ruthenium metalloglycocluster as a powerful molecular probe for evaluating a binding event between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analysis. The fluorescent ruthenium metalloglycoclusters, [Ru(bpy-2Gal)3] and [Ru(bpy-2Glc)3], possess clustered galactose and glucose surrounding the ruthenium center. Changes in FE and FP of these metalloglycoclusters were measured by adding each lectin (Peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), Concanavalin A (ConA), or Wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA, the FE spectrum of [Ru(bpy- 2Gal)3] showed new emission peak and the FP value of [Ru(bpy-2Gal)3] increased. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] showed new emission peak and the FP value increased following the addition of ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were proved by the FE and FP measurement. From nonlinear least-squares fitting, dissociation constants (Kd) of [Ru(bpy-2Gal)3] to PNA was 6.1 μM, while the Kd values of [Ru(bpy)2(bpy-2Gal)] to PNA was ca. 10-4 M. Therefore, the clustered carbohydrates were proved to increase affinity to lectins. Furthermore, the FP measurements proved specific binding of [Ru(bpy-2Gal)3] to TCF.

Widely different luminescence lifetimes of the [Delta]RRR, [Lambda]SSS and the [Delta]RRS, [Lambda]SSR diastereomers of fac-tris[(8-quinolyl)phenylmethylsily] iridium(III): Exciplex formation with solvents by distinct [sigma]-donor and [pi]-acceptor binding mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Djurovich, P.I.; Cook, W.; Joshi, R.

1994-01-13

Luminescence lifetimes ([tau][sub m]) of the [sigma]-bond-to-ligand charge-transfer (SBLCT) excited states of two diastereomers of fac-tris[(8-quinolyl)phenylmethylsilyl]iridium(III) differ by about a factor of 2 and are strongly solvent dependent. The [tau][sub m] values of the more symmetric [Delta]RRR, [Lambda]SSS diastereomer (A) are generally longer than those of the less symmetric [Delta]RRS, [Lambda]SSR diastereomer (B); [tau][sub m]'s of both diastereomers are substantially shortened relative to their values in aliphatic hydrocarbons by exciplex formation with a variety of weakly coordinating solvents including aromatic hydrocarbons, olefins, ethers, ketones, alcohols, and nitriles. Quenching constants (k[sub q]) due to exciplex formation are found to be muchmore » larger for B than they are for A in the [sigma]-donor solvents (cyclic ethers, ketones, alcohols, and nitriles); however, k[sub q] values of B are slightly smaller than those of A in [pi]-acceptor solvents (aromatic hydrocarbons, olefins). The results suggest that [sigma]-donor solvents form exciplexes by binding at the metal center, whereas [pi]-acceptor solvents bind at a quinolyl radical anion ligand site. A and B may prove useful as luminescent environmental probes which can distinguish between [sigma]-donor and [pi]-acceptor binding sites. 19 refs., 1 fig., 1 tab.« less

Toxic metals (Ni2+, Pb2+, Hg2+) binding affinity of dissolved organic matter (DOM) derived from different ages municipal landfill leachate

NASA Astrophysics Data System (ADS)

Rikta, S. Y.; Tareq, Shafi M.; Uddin, M. Khabir

2018-03-01

Solid waste production is rapidly increasing in Bangladesh and landfill leachate is the consequence of the decomposition of this waste. These leachates contain heavy metals and significant amount of dissolved organic matter (DOM). DOM is known to have considerable role in heavy metals speciation. Hence, it is important to characterize DOM/leachate and evaluate toxic metals binding affinity of DOM. The objectives of this study were to characterize the DOM in landfill leachate through physico-chemical and optical analyses and to investigate the toxic metals (Ni2+, Pb2+ and Hg2+) binding affinity of three different ages (fresh sample L-1, young sample L-2 and mature sample L-3) DOM samples. Results suggested that leachate is a potential pollutant which contained very high organic pollutant load. Conditional stability constant (Log K) and percentages of fluorophores that correspond to metal binding (% f) values indicated that young DOM sample (L-2) had the highest binding affinity to all the three metals ions. In general, DOM samples showed the following order affinity to the metal ions; Ni2+ binding affinity: L-2 > L-3 > L-1, Pb2+ binding affinity: L-2 > L-3 > L-1 and Hg2+ binding affinity: L-2 > L-1 > L-3.

The transition state structure for binding between TAZ1 of CBP and the disordered Hif-1α CAD.

PubMed

Lindström, Ida; Andersson, Eva; Dogan, Jakob

2018-05-18

Intrinsically disordered proteins (IDPs) are common in eukaryotes. However, relatively few experimental studies have addressed the nature of the rate-limiting transition state for the coupled binding and folding reactions involving IDPs. By using site-directed mutagenesis in combination with kinetics measurements we have here characterized the transition state for binding between the globular TAZ1 domain of CREB binding protein and the intrinsically disordered C-terminal activation domain of Hif-1α (Hif-1α CAD). A total of 17 Hif-1α CAD point-mutations were generated and a Φ-value binding analysis was carried out. We found that native hydrophobic binding interactions are not formed at the transition state. We also investigated the effect the biologically important Hif-1α CAD Asn-803 hydroxylation has on the binding kinetics, and found that the whole destabilization effect due the hydroxylation is within the dissociation rate constant. Thus, the rate-limiting transition state is "disordered-like", with native hydrophobic binding contacts being formed cooperatively after the rate-limiting barrier, which is clearly shown by linear free energy relationships. The same behavior was observed in a previously characterized TAZ1/IDP interaction, which may suggest common features for the rate-limiting transition state for TAZ1/IDP interactions.

Spectroscopic analyses on interaction of o-Vanillin- D-Phenylalanine, o-Vanillin- L-Tyrosine and o-Vanillin- L-Levodopa Schiff Bases with bovine serum albumin (BSA)

NASA Astrophysics Data System (ADS)

Gao, Jingqun; Guo, Yuwei; Wang, Jun; Wang, Zhiqiu; Jin, Xudong; Cheng, Chunping; Li, Ying; Li, Kai

2011-04-01

In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin- D-Phenylalanine (o-VDP), o-Vanillin- L-Tyrosine (o-VLT) and o-Vanillin- L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant ( Kq), apparent quenching constant ( Ksv), effective binding constant ( KA) and corresponding dissociation constant ( KD) as well as binding site number ( n) were obtained. In addition, the binding distance ( r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA > o-VLT-BSA > o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.

Spectroscopic analyses on interaction of o-Vanillin-D-Phenylalanine, o-Vanillin-L-Tyrosine and o-Vanillin-L-Levodopa Schiff Bases with bovine serum albumin (BSA).

PubMed

Gao, Jingqun; Guo, Yuwei; Wang, Jun; Wang, Zhiqiu; Jin, Xudong; Cheng, Chunping; Li, Ying; Li, Kai

2011-04-01

In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin-D-Phenylalanine (o-VDP), o-Vanillin-L-Tyrosine (o-VLT) and o-Vanillin-L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant (K(q)), apparent quenching constant (K(sv)), effective binding constant (K(A)) and corresponding dissociation constant (K(D)) as well as binding site number (n) were obtained. In addition, the binding distance (r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA>o-VLT-BSA>o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues. Copyright © 2010 Elsevier B.V. All rights reserved.

Isotope Effects Associated with N2O Production By Fungal and Bacterial Nitric Oxide Reductases: Implications for Tracing Microbial Production Pathways

NASA Astrophysics Data System (ADS)

Ostrom, N. E.; Yang, H.; Gandhi, H.; Hegg, E. L.

2014-12-01

Site preference (SP), the difference in δ15N between the central (α) and outer (β) N atoms in N2O, has emerged as a conservative tracer of microbial N2O production. The key advantages of SP relative to bulk isotopes are (1) that it is independent of the isotope composition of the substrates of nitrification and denitrification and (2) has not been shown to exhibit fractionation during production. In pure microbial culture distinct SP values for N2O production from bacterial denitrification, including nitrifier-denitrification (-10 to 0 ‰), relative to hydroxylamine oxidation and fungal denitrification (33-37 ‰) provide a promising basis to resolve production pathways. In this study, we determined the δ15N, δ18O, δ15Nα, and δ15Nβ of N2O generated by purified fungal (P450nor) and bacterial nitric oxide reductases. The isotope values were used to calculate SP values, enrichment factors (e), and kinetic isotope effects (KIEs). Both O and Nα displayed normal isotope effects during enzymatic NO reduction by the P450nor with e values of -25.7‰ (KIE = 1.0264) and -12.6‰ (KIE = 1.0127), respectively. However, bulk nitrogen (average δ15N of Nα and Nβ) and Nβ exhibited inverse isotope effects with e values of 14.0‰ (KIE = 0.9862) and 36.1‰ (KIE = 0.9651), respectively. The observed inverse isotope effect in δ15Nβ is consistent with reversible binding of the first NO in the P450nor reaction mechanism. Experiments with bacterial nitric oxide reductase are ongoing, however, preliminary data indicates a inverse isotope effect in the α and β positions and a normal isotope effect in δ18O. In contrast to the constant SP observed during N2O production observed in microbial cultures, the SP measured for purified P450nor was not constant, increasing from ~15‰ to ~29‰ during the course of the reaction. Our results clearly indicate that fractionation of SP during N2O production by P450nor is not zero, and that SP values higher and lower than the proposed end member value of 37‰ can be expected during fungal denitrification. The observation in pure microbial culture of constant SP can only be reconciled if the rate of nitrite and NO reduction are the same (thereby maintaining a steady NO concentration in the cell), and, further, that the magnitude of the P450nor NO binding constant (Kd) maintains the extent of the reaction (1-f) at 65%.

Exploring 3D structural influences of aliphatic and aromatic chemicals on α-cyclodextrin binding.

PubMed

Linden, Lukas; Goss, Kai-Uwe; Endo, Satoshi

2016-04-15

Binding of solutes to macromolecules is often influenced by steric effects caused by the 3D structures of both binding partners. In this study, the 1:1 α-cyclodextrin (αCD) binding constants (Ka1) for 70 organic chemicals were determined to explore the solute-structural effects on the αCD binding. Ka1 was measured using a three-part partitioning system with either a headspace or a passive sampler serving as the reference phase. The Ka1 values ranged from 1.08 to 4.97 log units. The results show that longer linear aliphatic chemicals form more stable complexes than shorter ones, and that the position of the functional group has a strong influence on Ka1, even stronger than the type of the functional group. Comparison of linear and variously branched aliphatic chemicals indicates that having a sterically unhindered alkyl chain is favorable for binding. These results suggest that only one alkyl chain can enter the binding cavity. Relatively small aromatic chemicals such as 1,3-dichlorobenzene bind to αCD well, while larger ones like tetrachlorobenzene and 3-ring aromatic chemicals show only a weak interaction with αCD, which can be explained by cavity exclusion. The findings of this study help interpret cyclodextrin binding data and facilitate the understanding of binding processes to macromolecules. Copyright © 2016 Elsevier Inc. All rights reserved.

"Hook"-calibration of GeneChip-microarrays: theory and algorithm.

PubMed

Binder, Hans; Preibisch, Stephan

2008-08-29

: The improvement of microarray calibration methods is an essential prerequisite for quantitative expression analysis. This issue requires the formulation of an appropriate model describing the basic relationship between the probe intensity and the specific transcript concentration in a complex environment of competing interactions, the estimation of the magnitude these effects and their correction using the intensity information of a given chip and, finally the development of practicable algorithms which judge the quality of a particular hybridization and estimate the expression degree from the intensity values. : We present the so-called hook-calibration method which co-processes the log-difference (delta) and -sum (sigma) of the perfect match (PM) and mismatch (MM) probe-intensities. The MM probes are utilized as an internal reference which is subjected to the same hybridization law as the PM, however with modified characteristics. After sequence-specific affinity correction the method fits the Langmuir-adsorption model to the smoothed delta-versus-sigma plot. The geometrical dimensions of this so-called hook-curve characterize the particular hybridization in terms of simple geometric parameters which provide information about the mean non-specific background intensity, the saturation value, the mean PM/MM-sensitivity gain and the fraction of absent probes. This graphical summary spans a metrics system for expression estimates in natural units such as the mean binding constants and the occupancy of the probe spots. The method is single-chip based, i.e. it separately uses the intensities for each selected chip. : The hook-method corrects the raw intensities for the non-specific background hybridization in a sequence-specific manner, for the potential saturation of the probe-spots with bound transcripts and for the sequence-specific binding of specific transcripts. The obtained chip characteristics in combination with the sensitivity corrected probe-intensity values provide expression estimates scaled in natural units which are given by the binding constants of the particular hybridization.

The structure of binding curves and practical identifiability of equilibrium ligand-binding parameters

PubMed Central

Middendorf, Thomas R.

2017-01-01

A critical but often overlooked question in the study of ligands binding to proteins is whether the parameters obtained from analyzing binding data are practically identifiable (PI), i.e., whether the estimates obtained from fitting models to noisy data are accurate and unique. Here we report a general approach to assess and understand binding parameter identifiability, which provides a toolkit to assist experimentalists in the design of binding studies and in the analysis of binding data. The partial fraction (PF) expansion technique is used to decompose binding curves for proteins with n ligand-binding sites exactly and uniquely into n components, each of which has the form of a one-site binding curve. The association constants of the PF component curves, being the roots of an n-th order polynomial, may be real or complex. We demonstrate a fundamental connection between binding parameter identifiability and the nature of these one-site association constants: all binding parameters are identifiable if the constants are all real and distinct; otherwise, at least some of the parameters are not identifiable. The theory is used to construct identifiability maps from which the practical identifiability of binding parameters for any two-, three-, or four-site binding curve can be assessed. Instructions for extending the method to generate identifiability maps for proteins with more than four binding sites are also given. Further analysis of the identifiability maps leads to the simple rule that the maximum number of structurally identifiable binding parameters (shown in the previous paper to be equal to n) will also be PI only if the binding curve line shape contains n resolved components. PMID:27993951

Studies on the structural, optical and dielectric properties of samarium coordinated with salicylic acid single crystal

NASA Astrophysics Data System (ADS)

Singh, Harjinder; Slathia, Goldy; Gupta, Rashmi; Bamzai, K. K.

2018-04-01

Samarium coordinated with salicylic acid was successfully grown as a single crystal by low temperature solution technique using mixed solvent of methanol and water in equal ratio. Structural characterization was carried out by single crystal X-ray diffraction analysis and it crystallizes in centrosymmetric space group P121/c1. FTIR and UV-Vis-NIR spectroscopy confirmed the compound formation and help to determine the mode of binding of the ligand to the rare earth-metal ion. Dielectric constant and dielectric loss have been measured over the frequency range 100 Hz - 30MHz. The decrease in dielectric constant with increases in frequency is due to the transition from interfacial polarization to dipolar polarization. The small value of dielectric constant at higher frequency ensures that the crystal is good candidate for NLO devices. Dielectric loss represents the resistive nature of the material.

Quantitative structure activity relationships from optimised ab initio bond lengths: steroid binding affinity and antibacterial activity of nitrofuran derivatives

NASA Astrophysics Data System (ADS)

Smith, P. J.; Popelier, P. L. A.

2004-02-01

The present day abundance of cheap computing power enables the use of quantum chemical ab initio data in Quantitative Structure-Activity Relationships (QSARs). Optimised bond lengths are a new such class of descriptors, which we have successfully used previously in representing electronic effects in medicinal and ecological QSARs (enzyme inhibitory activity, hydrolysis rate constants and pKas). Here we use AM1 and HF/3-21G* bond lengths in conjunction with Partial Least Squares (PLS) and a Genetic Algorithm (GA) to predict the Corticosteroid-Binding Globulin (CBG) binding activity of the classic steroid data set, and the antibacterial activity of nitrofuran derivatives. The current procedure, which does not require molecular alignment, produces good r2 and q2 values. Moreover, it highlights regions in the common steroid skeleton deemed relevant to the active regions of the steroids and nitrofuran derivatives.

Prostate cell membrane chromatography-liquid chromatography-mass spectrometry for screening of active constituents from Uncaria rhynchophylla.

PubMed

He, Jianyu; Han, Shengli; Yang, Fangfang; Zhou, Nan; Wang, Sicen

2013-01-01

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography-mass spectrometry (LC-MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC-MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors.

CONSTANTS FOR MERCURY BINDING BY DISSOLVED ORGANIC MATTER ISOLATES FROM THE FLORIDA EVERGLADES. (R827653)

EPA Science Inventory

Dissolved organic matter (DOM) has been implicated as an important complexing agent for Hg that can affect its mobility and bioavailability in aquatic ecosystems. However, binding constants for natural Hg-DOM complexes are not well known. We employed a competitive ligand appro...

Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α.

PubMed

Winiewska, Maria; Kucińska, Katarzyna; Makowska, Małgorzata; Poznański, Jarosław; Shugar, David

2015-10-01

The interaction of human CK2α (hCK2α) with nine halogenated benzotriazoles, TBBt and its analogues representing all possible patterns of halogenation on the benzene ring of benzotriazole, was studied by biophysical methods. Thermal stability of protein-ligand complexes, monitored by calorimetric (DSC) and optical (DSF) methods, showed that the increase in the mid-point temperature for unfolding of protein-ligand complexes (i.e. potency of ligand binding to hCK2α) follow the inhibitory activities determined by biochemical assays. The dissociation constant for the ATP-hCK2α complex was estimated with the aid of microscale thermophoresis (MST) as 4.3±1.8 μM, and MST-derived dissociation constants determined for halogenated benzotriazoles, when converted according to known ATP concentrations, perfectly reconstruct IC50 values determined by the biochemical assays. Ligand-dependent quenching of tyrosine fluorescence, together with molecular modeling and DSC-derived heats of unfolding, support the hypothesis that halogenated benzotriazoles bind in at least two alternative orientations, and those that are efficient hCK2α inhibitors bind in the orientation which TBBt adopts in its complex with maize CK2α. DSC-derived apparent heat for ligand binding (ΔΔHbind) is driven by intermolecular electrostatic interactions between Lys68 and the triazole ring of the ligand, as indicated by a good correlation between ΔΔHbind and ligand pKa. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly (~40 kJ/mol), relative to possible intermolecular halogen/hydrogen bonding (less than 10 kJ/mol), in binding of halogenated benzotriazoles to the ATP-binding site of hCK2α. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

Photolabeling of Tonoplast from Sugar Beet Cell Suspensions by [3H]5-(N-Methyl-N-Isobutyl)-Amiloride, an Inhibitor of the Vacuolar Na+/H+ Antiport 1

PubMed Central

Barkla, Bronwyn J.; Charuk, Jeffrey H. M.; Cragoe, Edward J.; Blumwald, Eduardo

1990-01-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na+/H+ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na+/H+ exchange in a competitive manner with a Ki of 2.5 and 5.9 micromolar for ΔpH-dependent 22Na+ influx in tonoplast vesicles and Na+-dependent H+ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [3H]MIA to tonoplast membranes revealed a high affinity binding component with a Kd of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na+/H+ antiport. Photolabeling of the tonoplast with [3H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog. Images Figure 7 PMID:16667602

Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport.

PubMed

Barkla, B J; Charuk, J H; Cragoe, E J; Blumwald, E

1990-07-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.

Measurement of radon and xenon binding to a cryptophane molecular host

PubMed Central

Jacobson, David R.; Khan, Najat S.; Collé, Ronald; Fitzgerald, Ryan; Laureano-Pérez, Lizbeth; Bai, Yubin; Dmochowski, Ivan J.

2011-01-01

Xenon and radon have many similar properties, a difference being that all 35 isotopes of radon (195Rn–229Rn) are radioactive. Radon is a pervasive indoor air pollutant believed to cause significant incidence of lung cancer in many geographic regions, yet radon affinity for a discrete molecular species has never been determined. By comparison, the chemistry of xenon has been widely studied and applied in science and technology. Here, both noble gases were found to bind with exceptional affinity to tris-(triazole ethylamine) cryptophane, a previously unsynthesized water-soluble organic host molecule. The cryptophane–xenon association constant, Ka = 42,000 ± 2,000 M-1 at 293 K, was determined by isothermal titration calorimetry. This value represents the highest measured xenon affinity for a host molecule. The partitioning of radon between air and aqueous cryptophane solutions of varying concentration was determined radiometrically to give the cryptophane–radon association constant Ka = 49,000 ± 12,000 M-1 at 293 K. PMID:21690357

Different kinetic pathways of the binding of two biphenyl analogues of colchicine to tubulin.

PubMed

Dumortier, C; Gorbunoff, M J; Andreu, J M; Engelborghs, Y

1996-04-09

The kinetics of the interaction of tubulin with two biphenyl analogues of colchicine were measured by fluorescence stopped flow. The ligands were 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB) and 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB). The binding of both analogues is accompanied by a fluorescence increase with monophasic kinetics, which indicates that these drugs, unlike colchicine, do not discriminate between the isoforms of tubulin. The observed pseudo-first-order rate constant increases in a nonlinear way with the drug concentration, indicating that the binding of the biphenyl analogues to tubulin occurs, like colchicine, in two steps: a fast reversible equilibrium followed by an isomerization of the initial complex. Kinetic analysis shows that TCB and TKB exhibit differences in their K1 values. At 25 degrees C, these are 114,000 +/- 15,000 M(-1) for TCB and 8,300 +/- 900 M(-1) for TKB. Both molecules show a much higher affinity than colchicine for the initial binding site. Also at 25 degrees C, the k2 value is 0.66 +/- 0.04 s(-1) for TCB and 3.0 +/- 0.2 s(-1) for TKB. From the temperature dependence, a reaction enthalpy change for the initial binding (deltaH(zero)1) of 44 +/- 9 kJ x mol(-1) (TCB) and -40 +/- 14 kJ x mol(-1) (TKB) and an activation energy for the second forward step of 64 +/- 2 kJ x mol(-1) (TCB) and 101 +/- 10 kJ x mol(-1) (TKB) were calculated. The dissociation kinetics were studied by displacement experiments, in which podophyllotoxin was used as a displacing ligand. The rate constant for the second step in the off direction (k(-2)) is 0.25 +/- 0.05 s(-1) for TCB and 0.093 +/- 0.009 s(-1) for TKB at 25 degrees C. The activation energies for the backward isomerization of the complexes were found to be 86 +/- 20 kJ x mol(-1) (TCB) and 79 +/- 5 kJ x mol(-1) (TKB). Combination of these results with the kinetic parameters for association gives a full characterization of the enthalpy pathway for the binding of TCB and TKB. The pathway of TCB binding is shown to differ considerably from that of TKB binding. Since their structural difference is located in ring C', this result points to their use of the ring C' in the first binding step. The competitiveness of the binding of TCB and TKB with those of podophyllotoxin, MTC, and MDL 27048 indicates that the two biphenyls interact as well with the trimethoxyphenyl-specific subsite.

Quantum chemical calculations and molecular docking studies of 5-(4-chlorobenzylidene)thiazolidine-2,4-dione(CTD) and its mannich product 5-(4-chlorobenzylidene)-3-(morpholinomethyl)thiazolidine-2,4-dione (CMTD)

NASA Astrophysics Data System (ADS)

Fatma, Shaheen; Bishnoi, Abha; Verma, Anil Kumar; Singh, Vineeta; Srivastava, Krishna

2018-04-01

This work presents the synthesis of 5-(4-chlorobenzylidene)thiazolidine-2,4-dione (CTD) by Claisen condensation of thiazolidine-2,4-dione and mannich product of CTD, 5-(4-chlorobenzylidene)-3-(morpholinomethyl)thiazolidine-2,4-dione (CMTD). The static first hyperpolarizability values for thiazolidine-2,4-dione derivatives have been calculated as 10.28 × 10-30 esu for CTD and 19.42 × 10-30 esu for CMTD. The gradual increase in hyperpolarizability values of synthesized thiazolidine-2,4-dione derivatives from CTD to CMTD is due to the blockage of sbnd NH group on CTD by mannich reaction. The structures of these compounds have been derived by spectroscopic(IR, UV, Mass, 1H and 13C NMR) analysis as well as with the help of theoretical studies. The high values of first static hyperpolarizability indicate that the synthesized derivatives are suitable as non-linear optical (NLO) material. CTD with MIC value of 12.5 μg/mL can be developed as an alternative drug for the treatment of enteric fever. Calculated frontier orbital gap values suggest that the CMTD is a soft molecule with high chemical reactivity and is more polarizable as compared to the CTD. Molecular electrostatic potential is calculated for the optimized geometry of the molecules to estimate their chemical reactivity. The inhibitor CTD forms a stable complex with 3-dehydroquinase enzyme of Salmonella typhi. It is evident from the ligand receptor interactions and a binding affinity value of -5.88 kcal/mol and an inhibition constant of 49.22 μM. This is further confirmed by the experimental biological data. The molecular docking studies are supportive of the antibacterial activity of CTD exhibiting high inhibition constant and binding energy.

Human lactoferrin binding to Porphyromonas gingivalis, Prevotella intermedia and Prevotella melaninogenica.

PubMed

Kalfas, S; Andersson, M; Edwardsson, S; Forsgren, A; Naidu, A S

1991-12-01

Human isolates of Porphyromonas gingivalis (n = 16), Prevotella intermedia n = 82) and Prevotella melaninogenica (n = 18) from diseased periodontal pockets were examined for interaction with human lactoferrin (HLf) in a standardized 125I-labeled protein binding assay. The highest HLf binding was found in P. intermedia strains, followed by P. gingivalis and P. melaninogenica. Further characterization of the interaction was performed with 1 representative strain from each species. HLf binding to P. gingivalis reached a saturation instantly and was optimal at pH 5.0-6.5. The corresponding values for P. melaninogenica were 90 min and pH 3.0-5.5. The HLf binding to the 2 strains seem to be nonspecific. In contrast, P. intermedia demonstrated specific binding, and a time-saturability within 60 min with an optimal uptake at pH 6.0-7.5. Scatchard analysis implied 45,000 receptors per cell with an affinity constant of 5.5 x 10(-7) M on P. intermedia strain 4H. The binding capacity in all 3 strains was affected by the culture medium. HLf binding components in these strains were susceptible to heat or proteases. Binding was eliminated in P. gingivalis and was enhanced in P. intermedia and P. melaninogenica by periodate treatment. Unlabeled HLf or bovine lactoferrin effectively displaced labeled HLf binding. Various proteins and carbohydrates did not inhibit HLf binding. Our data suggest that HLf binds to these periodontitis-associated species and that this mechanism is distinct from the previously known ligand interactions in oral bacteria.

Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

PubMed

Weikl, Thomas R; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

2016-09-02

The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

PubMed Central

Weikl, Thomas R.; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

2016-01-01

ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin.

PubMed

Dobreva, Marina A; Green, Rebecca J; Mueller-Harvey, Irene; Salminen, Juha-Pekka; Howlin, Brendan J; Frazier, Richard A

2014-09-17

Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 10(4)-10(6) M(-1) and stoichiometries, n1, of 2-13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 10(3)-10(5) M(-1) and stoichiometries, n2, of 16-30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern-Volmer quenching constant, K(SV).

Imino proton exchange and base-pair kinetics in the AMP-RNA aptamer complex.

PubMed

Nonin, S; Jiang, F; Patel, D J

1997-05-02

We report on the dynamics of base-pair opening in the ATP-binding asymmetric internal loop and flanking base-pairs of the AMP-RNA aptamer complex by monitoring the exchange characteristics of the extremely well resolved imino protons in the NMR spectrum of the complex. The kinetics of imino proton exchange as a function of basic pH or added ammonia catalyst are used to measure the apparent base-pair dissociation constants and lifetimes of Watson-Crick and mismatched base-pairs, as well as the solvent accessibility of the unpaired imino protons in the complex. The exchange characteristics of the imino protons identify the existence of four additional hydrogen bonds stabilizing the conformation of the asymmetric ATP-binding internal loop that were not detected by NOEs and coupling constants alone, but are readily accommodated in the previously reported solution structure of the AMP-RNA aptamer complex published from our laboratory. The hydrogen exchange kinetics of the non-Watson-Crick pairs in the asymmetric internal loop of the AMP-RNA aptamer complex have been characterized and yield apparent dissociation constants (alphaKd) that range from 10(-2) to 10(-7). Surprisingly, three of these alphaKd values are amongst the lowest measured for all base-pairs in the AMP-RNA aptamer complex. Comparative studies of hydrogen exchange of the imino protons in the free RNA aptamer and the AMP-RNA aptamer complex establish that complexation stabilizes not only the bases within the ATP-binding asymmetric internal loop, but also the flanking stem base-pairs (two pairs on either side) of the binding site. We also outline some preliminary results related to the exchange properties of a sugar 2'-hydroxyl proton of a guanosine residue involved in a novel hydrogen bond that has been shown to contribute to the immobilization of the bound AMP by the RNA aptamer, and whose resonance is narrow and downfield shifted in the spectrum.

Influence of pH for the determination of serum albumin by a dye-binding method in the presence of a detergent.

PubMed

Suzuki, Yuji

2008-08-01

In the dye-binding method, the absorbance increase caused by a protein error of a pH indicator is observed only in a restricted pH range. However, this pH range in the presence of a detergent has not yet been examined. Thus, the author investigated the pH (pH(UL)) where the absorbance increase becomes zero by a calculation based on the chemical equilibrium of a protein error of a pH indicator, and by experiments using four sulfonephthalein dyes. The pH(UL) value changed only with the detergent concentration, but did not change at all due to the dye, buffer solution or protein concentrations. Although the pH(UL) value was different according to the kind of dye used, it correlated well with the pK(D) values (dissociation constant) of BPB, BCG, BCP and BTB. The characteristics of pH(UL) in the reactions of the four dyes indicated good agreement with that obtained by a calculation.

Kinetics of binding of chicken cystatin to papain.

PubMed

Björk, I; Alriksson, E; Ylinenjärvi, K

1989-02-21

The kinetics of binding of chicken cystatin to papain were studied by stopped-flow fluorometry under pseudo-first-order conditions, i.e., with an excess of inhibitor. All reactions showed first-order behavior, and the observed pseudo-first-order rate constant increased linearly with the cystatin concentration up to the highest concentration that could be studied, 35 microM. The analyses thus provided no evidence for a limiting rate resulting from a conformational change stabilizing an initial encounter complex, in contrast with previous studies of reactions between serine proteinases and their protein inhibitors. The second-order association rate constant for complex formation was 9.9 X 10(6) M-1 s-1 at 25 degrees C, pH 7.4, I = 0.15, for both forms of cystatin, 1 and 2. This value approaches that expected for a diffusion-controlled rate. The temperature dependence of the association rate constant gave an enthalpy of activation at 25 degrees C of 31.5 kJ mol-1 and an entropy of activation at 25 degrees C of -7 J K-1 mol-1, compatible with no appreciable conformational change during the reaction. The association rate constant was independent of pH between pH 6 and 8 but decreased at lower and higher pH in a manner consistent with involvement of an unprotonated acid group with a pKa of 4-4.5 and a protonated basic group with a pKa of 9-9.5 in the interaction. The association rate constant was unaffected by ionic strengths between 0.15 and 1.0 but decreased somewhat at lower ionic strengths. Incubation of the complex between cystatin 2 and papain with an excess of cystatin 1 resulted in slow displacement of cystatin 2 from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of monomolecular binding constants of neutral phenols into the beta-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay.

PubMed

Le Saux, Thomas; Hisamoto, Hideaki; Terabe, Shigeru

2006-02-03

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into beta-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.

Circular dichroism spectroscopic investigation of double-decker phthalocyanine with G-Quadruplex as promising telomerase inhibitor

NASA Astrophysics Data System (ADS)

Baǧda, Efkan; Baǧda, Esra; Yabaş, Ebru

2017-01-01

In the present study, interaction of a double-decker phthalocyanine with two G-quadruplex DNA, Tel 21 and cMYC, was investigated. To the best of our knowledge, this is the first study about G-quadruplex-double decker phthalocyanine interaction. The spectrophotometric titration method was used for binding constant calculations. From the binding constants, it can be said that double-decker phthalocyanine more likely to bind Tel 21 rather than cMYC. The conformational changes upon binding were monitored via circular dichroism spectroscopy. The ethidium bromide replacement assay was investigated spectrofluorometrically.

Affinity interactions between natural pigments and human whole saliva.

PubMed

Yao, Jiang-Wu; Lin, Feng; Tao, Tao; Lin, Chang-Jian

2011-03-01

The aim of the present study was to assess the null hypothesis that there are no differences of affinity between pigments and human whole saliva (WS), and the affinity is not influenced by the functional groups of pigments, temperatures, pH values, and salt concentrations. The affinity constants of interactions between WS and theaflavin (TF)/curcumin (Cur)/cyanidin (Cy) were determined by surface plasmon resonance (SPR) and fluorescence quenching. Mass-uptake at various temperatures, pH values, and salt concentrations was also carried out. The order of affinity of the pigments binding to WS is TF>Cur>Cy. A large number of complexes and precipitations of pigments/proteins were formed through a quick, strong, and almost irreversible binding process. The mass-uptake of pigments was affected not only by the functional groups, but also by molecular weight of pigments, temperatures, pH values, and salt concentrations. The complex of pigments may easily and rapidly deposit onto the WS film, and are difficult to remove from the WS surface. However, the complex of pigments can be reduced by properly regulating the physicochemical conditions, such as temperatures, pH values, and salt concentrations. Copyright © 2010 Elsevier Ltd. All rights reserved.

Van’t Hoff global analyses of variable temperature isothermal titration calorimetry data

PubMed Central

Freiburger, Lee A.; Auclair, Karine; Mittermaier, Anthony K.

2016-01-01

Isothermal titration calorimetry (ITC) can provide detailed information on the thermodynamics of biomolecular interactions in the form of equilibrium constants, KA, and enthalpy changes, ΔHA. A powerful application of this technique involves analyzing the temperature dependences of ITC-derived KA and ΔHA values to gain insight into thermodynamic linkage between binding and additional equilibria, such as protein folding. We recently developed a general method for global analysis of variable temperature ITC data that significantly improves the accuracy of extracted thermodynamic parameters and requires no prior knowledge of the coupled equilibria. Here we report detailed validation of this method using Monte Carlo simulations and an application to study coupled folding and binding in an aminoglycoside acetyltransferase enzyme. PMID:28018008

Kinetic analysis of central ( sup 11 C)raclopride binding to D2-dopamine receptors studied by PET--a comparison to the equilibrium analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farde, L.; Eriksson, L.; Blomquist, G.

1989-10-01

(11C)Raclopride binding to central D2-dopamine receptors in humans has previously been examined by positron emission tomography (PET). Based on the rapid occurrence of binding equilibrium, a saturation analysis has been developed for the determination of receptor density (Bmax) and affinity (Kd). For analysis of PET measurements obtained with other ligands, a kinetic three-compartment model has been used. In the present study, the brain uptake of (11C)raclopride was analyzed further by applying both a kinetic and an equilibrium analysis to data obtained from four PET experiments in each of three healthy subjects. First regional CBV was determined. In the second andmore » third experiment, (11C)-raclopride with high and low specific activity was used. In a fourth experiment, the (11C)raclopride enantiomer (11C)FLB472 was used to examine the concentration of free radioligand and nonspecific binding in brain. Radio-activity in arterial blood was measured using an automated blood sampling system. Bmax and Kd values for (11C)raclopride binding could be determined also with the kinetic analysis. As expected theoretically, those values were similar to those obtained with the equilibrium analysis. In addition, the kinetic analysis allowed separate determination of the association and dissociation rate constants, kon and koff, respectively. Examination of (11C)raclopride and (11C)FLB472 uptake in brain regions devoid of specific D2-dopamine receptor binding indicated a fourth compartment in which uptake was reversible, nonstereoselective, and nonsaturable in the dose range studied.« less

Free energy landscape for the binding process of Huperzine A to acetylcholinesterase

PubMed Central

Bai, Fang; Xu, Yechun; Chen, Jing; Liu, Qiufeng; Gu, Junfeng; Wang, Xicheng; Ma, Jianpeng; Li, Honglin; Onuchic, José N.; Jiang, Hualiang

2013-01-01

Drug-target residence time (t = 1/koff, where koff is the dissociation rate constant) has become an important index in discovering better- or best-in-class drugs. However, little effort has been dedicated to developing computational methods that can accurately predict this kinetic parameter or related parameters, koff and activation free energy of dissociation (). In this paper, energy landscape theory that has been developed to understand protein folding and function is extended to develop a generally applicable computational framework that is able to construct a complete ligand-target binding free energy landscape. This enables both the binding affinity and the binding kinetics to be accurately estimated. We applied this method to simulate the binding event of the anti-Alzheimer’s disease drug (−)−Huperzine A to its target acetylcholinesterase (AChE). The computational results are in excellent agreement with our concurrent experimental measurements. All of the predicted values of binding free energy and activation free energies of association and dissociation deviate from the experimental data only by less than 1 kcal/mol. The method also provides atomic resolution information for the (−)−Huperzine A binding pathway, which may be useful in designing more potent AChE inhibitors. We expect this methodology to be widely applicable to drug discovery and development. PMID:23440190

Free energy landscape for the binding process of Huperzine A to acetylcholinesterase.

PubMed

Bai, Fang; Xu, Yechun; Chen, Jing; Liu, Qiufeng; Gu, Junfeng; Wang, Xicheng; Ma, Jianpeng; Li, Honglin; Onuchic, José N; Jiang, Hualiang

2013-03-12

Drug-target residence time (t = 1/k(off), where k(off) is the dissociation rate constant) has become an important index in discovering better- or best-in-class drugs. However, little effort has been dedicated to developing computational methods that can accurately predict this kinetic parameter or related parameters, k(off) and activation free energy of dissociation (ΔG(off)≠). In this paper, energy landscape theory that has been developed to understand protein folding and function is extended to develop a generally applicable computational framework that is able to construct a complete ligand-target binding free energy landscape. This enables both the binding affinity and the binding kinetics to be accurately estimated. We applied this method to simulate the binding event of the anti-Alzheimer's disease drug (-)-Huperzine A to its target acetylcholinesterase (AChE). The computational results are in excellent agreement with our concurrent experimental measurements. All of the predicted values of binding free energy and activation free energies of association and dissociation deviate from the experimental data only by less than 1 kcal/mol. The method also provides atomic resolution information for the (-)-Huperzine A binding pathway, which may be useful in designing more potent AChE inhibitors. We expect this methodology to be widely applicable to drug discovery and development.

Probing into the binding interaction between medroxyprogesterone acetate and bovine serum albumin (BSA): spectroscopic and molecular docking methods.

PubMed

Fang, Fang; Pan, Dong-Qi; Qiu, Min-Jie; Liu, Ting-Ting; Jiang, Min; Wang, Qi; Shi, Jie-Hua

2016-09-01

To further understand the mechanism of action and pharmacokinetics of medroxyprogesterone acetate (MPA), the binding interaction of MPA with bovine serum albumin (BSA) under simulated physiological conditions (pH 7.4) was studied using fluorescence emission spectroscopy, synchronous fluorescence spectroscopy, circular dichroism and molecular docking methods. The experimental results reveal that the fluorescence of BSA quenches due to the formation of MPA-BSA complex. The number of binding sites (n) and the binding constant for MPA-BSA complex are ~1 and 4.6 × 10(3)  M(-1) at 310 K, respectively. However, it can be concluded that the binding process of MPA with BSA is spontaneous and the main interaction forces between MPA and BSA are van der Waals force and hydrogen bonding interaction due to the negative values of ΔG(0) , ΔH(0) and ΔS(0) in the binding process of MPA with BSA. MPA prefers binding on the hydrophobic cavity in subdomain IIIA (site II'') of BSA resulting in a slight change in the conformation of BSA, but BSA retaining the α-helix structure. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Binding of a cyclic organoselenium compound with gold nanoparticles (GNP) and its effect on electron transfer properties.

PubMed

Kumar, Pavitra V; Singh, Beena G; Maiti, Nandita; Iwaoka, Michio; Priyadarsini, K Indira

2014-12-15

Binding of a cyclic organoselenium compound, DL-trans-3,4-dihydroxy-1-selenolane (DHSred) with gold nanoparticles (GNP) of different sizes was studied by absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), surface enhanced Raman spectroscopy (SERS) and zeta-potential (ζ) measurements. GNP of different size were synthesized by varying the reaction conditions and their size was determined by DLS and TEM techniques. The absorption spectral data showed red shift in the surface plasmon resonance (SPR) band indicating increase in the size of GNP on binding to DHSred. SERS studies confirmed that the binding of DHSred with GNP is through selenium center with planar orientation of DHSred on the GNP surface. The product of the number of binding sites (n) in GNP and the binding constant (K) was estimated for GNP of different particle size. The zeta potential (ζ) value of GNP decreased marginally in the presence of DHSred. Further, the binding of DHSred with GNP was found to enhance its reactivity with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS(·-)) and the reactivity increased with decrease in the GNP size. Such enhancement in the reducing ability may have a greater impact on the antioxidant activity of DHSred. Copyright © 2014 Elsevier Inc. All rights reserved.

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

PubMed Central

Sauvé, K; Nachman, M; Spence, C; Bailon, P; Campbell, E; Tsien, W H; Kondas, J A; Hakimi, J; Ju, G

1991-01-01

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction. PMID:2052547

Kinetic studies of amino acid-based surfactant binding to DNA.

PubMed

Santhiya, Deenan; Dias, Rita S; Dutta, Sounak; Das, Prasanta Kumar; Miguel, Maria G; Lindman, Björn; Maiti, Souvik

2012-05-24

In this work, the binding kinetics of amino acid-based surfactants, presenting different linkers and head groups, with calf thymus (CT)-DNA was studied using stopped-flow fluorescence spectroscopy. The kinetic studies were carried out as a function of Na(+) concentration and surfactant-to-DNA charge ratio. The surfactant binding on DNA took place in two consecutive steps, for which the corresponding first and second relative rate constants (k(1) and k(2)) were determined. The fast step was attributed to the surfactant binding to DNA and micelle formation in its vicinity, the slower step to DNA condensation and possible rearrangement of the surfactant aggregates. In general, both relative rate constants increase with surfactant concentration and decrease with the ionic strength of the medium. The architecture of the surfactant was found to have a significant impact on the kinetics of the DNA-surfactant complexation. Surfactants with amide linkers showed larger relative rate constants than those with ester linkers. The variation of the relative rate constants with the head groups of the surfactants, alanine and proline, was found to be less obvious, being partially dependent on the surfactant concentration.

Fluorescence spectroscopic study on the interaction of resveratrol with lipoxygenase

NASA Astrophysics Data System (ADS)

Pinto, María del Carmen; Duque, Antonio Luis; Macías, Pedro

2010-09-01

The interaction of lipoxygenase with (E)-resveratrol was investigated by fluorescence spectroscopy. The data obtained revealed that the quenching of intrinsic fluorescence of lipoxygenase is produced by the formation of a complex lipoxygenase-(E)-resveratrol. From the value obtained for the binding constant, according to the Stern-Volmer modified equation, was deduced the existence of static quenching mechanism and, as consequence, the existence of a strong interaction between (E)-resveratrol and lipoxygenase. The values obtained for the thermodynamic parameter Δ H (-3.58 kJ mol -1) and Δ S (87.97 J mol -1K -1) suggested the participation of hydrophobic interactions and hydrogen bonds in the stabilization of the complex ligand-protein. From the static quenching we determined that only exist one independent binding site. Based on the Förster energy transfer theory, the distance between the acceptor ((E)-resveratrol) and the donor (Trp residues of lipoxygenase) was calculated to be 3.42 nm. Finally, based on the information obtained from the evaluation of synchronous and three-dimensional fluorescence spectroscopy, we deduced that the interaction of (E)-resveratrol with lipoxygenase produces micro-environmental and conformational alterations of protein in the binding region.

Binding of curcumin with bovine serum albumin in the presence of ι-carrageenan and implications on the stability and antioxidant activity of curcumin.

PubMed

Yang, Mingling; Wu, Yue; Li, Jinbing; Zhou, Haibo; Wang, Xiaoyong

2013-07-24

This work studied the influences of formation of BSA/ι-carrageenan complexes on the binding, stability, and antioxidant activity of curcumin. In the presence of BSA and ι-carrageenan, curcumin gives higher intensities of absorption and fluorescence than free curcumin and curcumin only combined with BSA. The added ι-carrageenan is observed to promote curcumin for quenching the instrinsic fluorescence of BSA. These results are explained in terms of the formation of BSA/ι-carrageenan complexes, which help to stabilize the folded structure of BSA for providing curcumin with a more hydrophobic microenvironment. The small difference in anisotropy values of curcumin with BSA alone and of BSA/ι-carrageenan complexes suggests that ι-carrageenan acts as outer stretch conformation in BSA/ι-carrageenan complexes but does not directly disturb the hydrophobic pockets inside BSA, where curcumin is hydrophobically located. The determined values of the binding constant are higher for curcumin with BSA/ι-carrageenan complexes than with BSA alone. Moreover, BSA/ι-carrageenan complexes are found to be superior to single BSA for enhancing the stability and DPPH radical-scavenging ability of curcumin.

Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.

PubMed

Pantoja-Uceda, David; Neira, José L; Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel; Santoro, Jorge

2016-06-21

Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited.

Calculations of binding affinity between C8-substituted GTP analogs and the bacterial cell-division protein FtsZ

PubMed Central

Hritz, Jozef; Läppchen, Tilman

2010-01-01

The FtsZ protein is a self-polymerizing GTPase that plays a central role in bacterial cell division. Several C8-substituted GTP analogs are known to inhibit the polymerization of FtsZ by competing for the same binding site as its endogenous activating ligand GTP. Free energy calculations of the relative binding affinities to FtsZ for a set of five C8-substituted GTP analogs were performed. The calculated values agree well with the available experimental data, and the main contribution to the free energy differences is determined to be the conformational restriction of the ligands. The dihedral angle distributions around the glycosidic bond of these compounds in water are known to vary considerably depending on the physicochemical properties of the substituent at C8. However, within the FtsZ protein, this substitution has a negligible influence on the dihedral angle distributions, which fall within the narrow range of −140° to −90° for all investigated compounds. The corresponding ensemble average of the coupling constants 3J(C4,H1′) is calculated to be 2.95 ± 0.1 Hz. The contribution of the conformational selection of the GTP analogs upon binding was quantified from the corresponding populations. The obtained restraining free energy values follow the same trend as the relative binding affinities to FtsZ, indicating their dominant contribution. PMID:20559630

Discovery of non-peptidic small molecule inhibitors of cyclophilin D as neuroprotective agents in Aβ-induced mitochondrial dysfunction

NASA Astrophysics Data System (ADS)

Park, Insun; Londhe, Ashwini M.; Lim, Ji Woong; Park, Beoung-Geon; Jung, Seo Yun; Lee, Jae Yeol; Lim, Sang Min; No, Kyoung Tai; Lee, Jiyoun; Pae, Ae Nim

2017-10-01

Cyclophilin D (CypD) is a mitochondria-specific cyclophilin that is known to play a pivotal role in the formation of the mitochondrial permeability transition pore (mPTP).The formation and opening of the mPTP disrupt mitochondrial homeostasis, cause mitochondrial dysfunction and eventually lead to cell death. Several recent studies have found that CypD promotes the formation of the mPTP upon binding to β amyloid (Aβ) peptides inside brain mitochondria, suggesting that neuronal CypD has a potential to be a promising therapeutic target for Alzheimer's disease (AD). In this study, we generated an energy-based pharmacophore model by using the crystal structure of CypD—cyclosporine A (CsA) complex and performed virtual screening of ChemDiv database, which yielded forty-five potential hit compounds with novel scaffolds. We further tested those compounds using mitochondrial functional assays in neuronal cells and identified fifteen compounds with excellent protective effects against Aβ-induced mitochondrial dysfunction. To validate whether these effects derived from binding to CypD, we performed surface plasmon resonance (SPR)—based direct binding assays with selected compounds and discovered compound 29 was found to have the equilibrium dissociation constants (KD) value of 88.2 nM. This binding affinity value and biological activity correspond well with our predicted binding mode. We believe that this study offers new insights into the rational design of small molecule CypD inhibitors, and provides a promising lead for future therapeutic development.

Spectroscopic, molecular docking and structural activity studies of (E)-N‧-(substituted benzylidene/methylene) isonicotinohydrazide derivatives for DNA binding and their biological screening

NASA Astrophysics Data System (ADS)

Arshad, Nasima; Perveen, Fouzia; Saeed, Aamer; Channar, Pervaiz Ali; Farooqi, Shahid Iqbal; Larik, Fayaz Ali; Ismail, Hammad; Mirza, Bushra

2017-07-01

Acid catalyzed condensation of isoniazid with a number of suitably substituted aromatic and heterocyclic aldehydes was carried out in dry ethanol to afford the title (E)-N‧-(substituted benzylidene/methylene) isonicotinohydrazides (SF 1 - SF 4) in good yields. These compounds were characterized and further investigated for their binding with ds.DNA using UV- spectroscopy and molecular docking and for antitumor and antimicrobial potentials. A good correlation was found among spectroscopic, theoretical and biological results. UV- spectra in the presence of DNA concentrations and their data interpretation in terms binding constant "Kb" and free energy change (ΔG) provided evidences for the significant and spontaneous binding of the compounds with DNA. Molecular docking studies and structural analysis further supported the UV-findings and indicated that the modes of interactions between bromo- (SF 1) and flouro- (SF 4) substituted isonicotinohydrazides is intercalation while methoxy- (SF 2) and hydroxy- (SF 3) substituted isonicotinohydrazides interact with DNA helix via groove binding. SF 1 exhibited comparatively higher Kb value (UV-; 8.07 × 103 M-1, docking; 8.11 × 103 M-1) which inferred that the respective compound muddles to DNA most powerfully. SF 1 has shown the lowest IC50 (345.3 μg/mL) value among all the compounds indicating its comparatively highest activity towards tumor inhibition. None of the compound has shown perceptible antibacterial and antifungal activities.

High-affinity binding of laurate to naturally occurring mutants of human serum albumin and proalbumin.

PubMed

Kragh-Hansen, U; Pedersen, A O; Galliano, M; Minchiotti, L; Brennan, S O; Tárnoky, A L; Franco, M H; Salzano, F M

1996-12-15

Binding of laurate (n-dodecanoate) to genetic variants of albumin or its proprotein and to normal albumin isolated from the same heterozygous carriers was studied by a kinetic dialysis technique at physiological pH. The first stoichiometric association constant for binding to proalbumin Lille (Arg-2-->His) and albumin (Alb) Roma (Glu321-->Lys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys541-->Glu) was decreased to 80%. In contrast, normal laurate-binding properties were found for as many as nine other albumin variants with single amino acid substitutions. Because the net charges of all these mutants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long-range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. The most pronounced effect was observed for the glycosylated Alb Casebrook, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total blocking of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes induced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using different preparations of normal albumin as controls in the binding experiments, it was also possible to compare the effect of various methods for isolation and defatting on laurate binding.

Bioinformatic Analysis of the Contribution of Primer Sequences to Aptamer Structures

PubMed Central

Ellington, Andrew D.

2009-01-01

Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust. PMID:18594898

Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes.

PubMed Central

Lowe, D J; Eady, R R; Thorneley, N F

1978-01-01

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo--Fe protein showed that they arose from Fe--S clusters in the Mo--Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the --1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme--product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe--S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO. PMID:210766

Cobalt porphyrin-mediated oxygen transport in a polymer membrane. Effect of the cobalt porphyrin structure on the oxygen-binding reaction, oxygen-diffusion constants, and oxygen-transport efficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishide, Hiroyuki; Suzuki, Takayuki; Kawakami, Hiroyoshi

1994-05-12

New derivatives of (meso-[alpha],[alpha],[alpha],[alpha]-tetrakis(o-pivalamidophenyl)porphinato)cobalt (CoPs) were characterized by oxygen-binding equilibrium and rate constants of the cobalt centered in the porphyrins. They depended on the structure of the porphyrin; for example, the rate constants of oxygen binding and dissociation (k[sub on] and k[sub off]) for [alpha][sup 3][beta]-CoP[sub 4]P were 3 and 20 times as large as those for [alpha][sup 4]-CoB[sub 4]P, respectively. Oxygen transport through the polymer membranes containing CoPs as the fixed oxygen carriers was facilitated and was affected by the oxygen-binding character or the structure of CoPs. The logarithmically linear correlation of the oxygen-dissociation rate constant of CoPs (k[submore » off] = (3-66) x 10[sup 3] S[sup [minus]1]) with the diffusion constant of oxygen via CoPs fixed in the membranes (D[sub cc] = (3-140) x 10[sup [minus]9] cm[sup 2] s[sup [minus]1]) was given for those six CoP derivatives. 26 refs., 5 figs., 2 tabs.« less

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry.

PubMed

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-01-15

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu(2+) with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15K in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu(2+) ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu(2+) ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu(2+) ions are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry

NASA Astrophysics Data System (ADS)

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-01-01

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu2 + with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15 K in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu2 + ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu2 + ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu2 + ions are discussed.

A universal surface complexation framework for modeling proton binding onto bacterial surfaces in geologic settings

USGS Publications Warehouse

Borrok, D.; Turner, B.F.; Fein, J.B.

2005-01-01

Adsorption onto bacterial cell walls can significantly affect the speciation and mobility of aqueous metal cations in many geologic settings. However, a unified thermodynamic framework for describing bacterial adsorption reactions does not exist. This problem originates from the numerous approaches that have been chosen for modeling bacterial surface protonation reactions. In this study, we compile all currently available potentiometric titration datasets for individual bacterial species, bacterial consortia, and bacterial cell wall components. Using a consistent, four discrete site, non-electrostatic surface complexation model, we determine total functional group site densities for all suitable datasets, and present an averaged set of 'universal' thermodynamic proton binding and site density parameters for modeling bacterial adsorption reactions in geologic systems. Modeling results demonstrate that the total concentrations of proton-active functional group sites for the 36 bacterial species and consortia tested are remarkably similar, averaging 3.2 ?? 1.0 (1??) ?? 10-4 moles/wet gram. Examination of the uncertainties involved in the development of proton-binding modeling parameters suggests that ignoring factors such as bacterial species, ionic strength, temperature, and growth conditions introduces relatively small error compared to the unavoidable uncertainty associated with the determination of cell abundances in realistic geologic systems. Hence, we propose that reasonable estimates of the extent of bacterial cell wall deprotonation can be made using averaged thermodynamic modeling parameters from all of the experiments that are considered in this study, regardless of bacterial species used, ionic strength, temperature, or growth condition of the experiment. The average site densities for the four discrete sites are 1.1 ?? 0.7 ?? 10-4, 9.1 ?? 3.8 ?? 10-5, 5.3 ?? 2.1 ?? 10-5, and 6.6 ?? 3.0 ?? 10-5 moles/wet gram bacteria for the sites with pKa values of 3.1, 4.7, 6.6, and 9.0, respectively. It is our hope that this thermodynamic framework for modeling bacteria-proton binding reactions will also provide the basis for the development of an internally consistent set of bacteria-metal binding constants. 'Universal' constants for bacteria-metal binding reactions can then be used in conjunction with equilibrium constants for other important metal adsorption and complexation reactions to calculate the overall distribution of metals in realistic geologic systems.

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

PubMed Central

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki

2014-01-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)–Ras–extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. PMID:24958104

Investigation of interactions of Comtan with human serum albumin by mathematically modeled voltammetric data: A study from bio-interaction to biosensing.

PubMed

Jalalvand, Ali R; Ghobadi, Sirous; Goicoechea, Hector C; Gu, Hui-Wen; Sanchooli, Esmael

2018-05-16

In this work, voltammetric data recorded at a glassy carbon electrode (GCE) were separately used to investigate the interactions of entacapone (Comtan, CAT) with human serum albumin (HSA). Then, an augmented data matrix was constructed by the combination of voltammetric and spectroscopic data and simultaneously analysed by multivariate curve resolution-alternating least squares (MCR-ALS) to obtain more information about CAT-HSA interactions. The absence of rotational ambiguities in results obtained by MCR-ALS was verified with the help of MCR-BANDS and we confirmed that the results were unambiguous and reliable. Binding of CAT to HSA was also modeled by molecular docking and the results were compatible with those of obtained by recording experimental data. Hard-modeling of combined voltammetric and spectroscopic data by EQUISPEC as an efficient chemometric algorithm helped us to compute binding constant of CAT-HSA complex specie which was in a good agreement with the binding constant value obtained by direct analysis of experimental data. For electrochemical sensing of serum albumin two amperometric measurements were performed to determine HSA in 2-27 nM and 27-70 nM with a limit of detection of 0.51 nM and a sensitivity of 1.84 μA nM -1 . Copyright © 2017 Elsevier B.V. All rights reserved.

Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it; Ciaccio, Chiara; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, I-70126 Bari

2011-01-07

Research highlights: {yields} Cardiolipin binding to cytochrome c. {yields} Cardiolipin-dependent peroxynitrite isomerization by cytochrome c. {yields} Cardiolipin-cytochrome c complex plays pro-apoptotic effects. {yields} Cardiolipin-cytochrome c complex plays anti-apoptotic effects. -- Abstract: Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does notmore » catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k{sub on}) is (3.2 {+-} 0.4) x 10{sup 5} M{sup -1} s{sup -1}. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 {+-} 0.8) x 10{sup -5} M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.« less

Nanofiber Ion-Exchange Membranes for the Rapid Uptake and Recovery of Heavy Metals from Water

PubMed Central

Chitpong, Nithinart; Husson, Scott M.

2016-01-01

An evaluation of the performance of polyelectrolyte-modified nanofiber membranes was undertaken to determine their efficacy in the rapid uptake and recovery of heavy metals from impaired waters. The membranes were prepared by grafting poly(acrylic acid) (PAA) and poly(itaconic acid) (PIA) to cellulose nanofiber mats. Performance measurements quantified the dynamic ion-exchange capacity for cadmium (Cd), productivity, and recovery of Cd(II) from the membranes by regeneration. The dynamic binding capacities of Cd(II) on both types of nanofiber membrane were independent of the linear flow velocity, with a residence time of as low as 2 s. Analysis of breakthrough curves indicated that the mass flow rate increased rapidly at constant applied pressure after membranes approached equilibrium load capacity for Cd(II), apparently due to a collapse of the polymer chains on the membrane surface, leading to an increased porosity. This mechanism is supported by hydrodynamic radius (Rh) measurements for PAA and PIA obtained from dynamic light scattering, which show that Rh values decrease upon Cd(II) binding. Volumetric productivity was high for the nanofiber membranes, and reached 0.55 mg Cd/g/min. The use of ethylenediaminetetraacetic acid as regeneration reagent was effective in fully recovering Cd(II) from the membranes. Ion-exchange capacities were constant over five cycles of binding-regeneration. PMID:27999394

Nanofiber Ion-Exchange Membranes for the Rapid Uptake and Recovery of Heavy Metals from Water.

PubMed

Chitpong, Nithinart; Husson, Scott M

2016-12-20

An evaluation of the performance of polyelectrolyte-modified nanofiber membranes was undertaken to determine their efficacy in the rapid uptake and recovery of heavy metals from impaired waters. The membranes were prepared by grafting poly(acrylic acid) (PAA) and poly(itaconic acid) (PIA) to cellulose nanofiber mats. Performance measurements quantified the dynamic ion-exchange capacity for cadmium (Cd), productivity, and recovery of Cd(II) from the membranes by regeneration. The dynamic binding capacities of Cd(II) on both types of nanofiber membrane were independent of the linear flow velocity, with a residence time of as low as 2 s. Analysis of breakthrough curves indicated that the mass flow rate increased rapidly at constant applied pressure after membranes approached equilibrium load capacity for Cd(II), apparently due to a collapse of the polymer chains on the membrane surface, leading to an increased porosity. This mechanism is supported by hydrodynamic radius (R h ) measurements for PAA and PIA obtained from dynamic light scattering, which show that R h values decrease upon Cd(II) binding. Volumetric productivity was high for the nanofiber membranes, and reached 0.55 mg Cd/g/min. The use of ethylenediaminetetraacetic acid as regeneration reagent was effective in fully recovering Cd(II) from the membranes. Ion-exchange capacities were constant over five cycles of binding-regeneration.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques.

PubMed

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R; Abyar, Fatemeh

2018-06-05

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H 2 O] (1) and [Zn (L)] (2) [L=N-2-hydroxyacetophenon-N'-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1 H and 13 C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities. Copyright © 2018 Elsevier B.V. All rights reserved.

Thermodynamic analysis of the interaction of factor VIII with von Willebrand factor.

PubMed

Dimitrov, Jordan D; Christophe, Olivier D; Kang, Jonghoon; Repessé, Yohann; Delignat, Sandrine; Kaveri, Srinivas V; Lacroix-Desmazes, Sébastien

2012-05-22

Factor VIII (FVIII) is a glycoprotein that plays an important role in the intrinsic pathway of coagulation. In circulation, FVIII is protected upon binding to von Willebrand factor (VWF), a chaperone molecule that regulates its half-life, distribution, and activity. Despite the biological significance of this interaction, its molecular mechanisms are not fully characterized. We determined the equilibrium and activation thermodynamics of the interaction between FVIII and VWF. The equilibrium affinity determined by surface plasmon resonance was temperature-dependent with a value of 0.8 nM at 35 °C. The FVIII-VWF interaction was characterized by very fast association (8.56 × 10(6) M(-1) s(-1)) and fast dissociation (6.89 × 10(-3) s(-1)) rates. Both the equilibrium association and association rate constants, but not the dissociation rate constant, were dependent on temperature. Binding of FVIII to VWF was characterized by favorable changes in the equilibrium and activation entropy (TΔS° = 89.4 kJ/mol, and -TΔS(++) = -8.9 kJ/mol) and unfavorable changes in the equilibrium and activation enthalpy (ΔH° = 39.1 kJ/mol, and ΔH(++) = 44.1 kJ/mol), yielding a negative change in the equilibrium Gibbs energy. Binding of FVIII to VWF in solid-phase assays demonstrated a high sensitivity to acidic pH and a sensitivity to ionic strength. Our data indicate that the interaction between FVIII and VWF is mediated mainly by electrostatic forces, and that it is not accompanied by entropic constraints, suggesting the absence of conformational adaptation but the presence of rigid "pre-optimized" binding surfaces.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R.; Abyar, Fatemeh

2018-06-01

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H2O] (1) and [Zn (L)] (2) [L = N-2-hydroxyacetophenon-N‧-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1H and 13C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities.

Insight into magnesium coordination environments in benzoate and salicylate complexes through 25Mg solid-state NMR spectroscopy.

PubMed

Burgess, Kevin M N; Xu, Yang; Leclerc, Matthew C; Bryce, David L

2013-08-01

We report on the (25)Mg solid-state nuclear magnetic resonance (NMR) characterization of a series of magnesium complexes featuring Mg(2+) ions in organic coordination environments. Six compounds have been synthesized with benzoate and salicylate ligands, which are typically used as linkers in metal organic frameworks (MOFs). The use of ultrahigh-field solid-state NMR has revealed a relatively large range of values for the (25)Mg quadrupolar coupling constant, CQ((25)Mg), in these compounds. In contrast to some previously studied inorganic Mg(2+) complexes, the values of CQ((25)Mg) in organic Mg(2+) complexes are well rationalized by the degree of octahedral strain of the "MgO6" coordination polyhedra. (13)C and (25)Mg isotropic chemical shifts were also found to be sensitive to the binding mode of the carboxylate ligands. The experimental findings are corroborated by gauge-including projector-augmented-wave (GIPAW) density functional theory (DFT) computations, and these have allowed for an interpretation of the experimentally observed trend in the CQ((25)Mg) values and for the visualization of the EFG tensor principal components with respect to the molecular structure. These new insights may prove to be valuable for the understanding and interpretation of (25)Mg NMR data for Mg(2+) ions in organic binding environments such as those found in MOFs and protein-divalent metal binding sites.

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes.

PubMed

Verly, Rodrigo M; Rodrigues, Magali A; Daghastanli, Katia Regina P; Denadai, Angelo Márcio L; Cuccovia, Iolanda M; Bloch, Carlos; Frézard, Frédéric; Santoro, Marcelo M; Piló-Veloso, Dorila; Bemquerer, Marcelo P

2008-01-01

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.

Pharmacological activities of Vitex agnus-castus extracts in vitro.

PubMed

Meier, B; Berger, D; Hoberg, E; Sticher, O; Schaffner, W

2000-10-01

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver.

PubMed

Yachi, K; Sugiyama, Y; Sawada, Y; Iga, T; Ikeda, Y; Toda, G; Hanano, M

1989-01-16

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Synthesis, characterization, and binding assessment with human serum albumin of three bipyridine lanthanide(III) complexes.

PubMed

Aramesh-Boroujeni, Zahra; Bordbar, Abdol-Khalegh; Khorasani-Motlagh, Mozhgan; Sattarinezhad, Elham; Fani, Najme; Noroozifar, Meissam

2018-05-18

In this work, the terbium(III), dysprosium(III), and ytterbium(III) complexes containing 2, 2'-bipyridine (bpy) ligand have been synthesized and characterized using CHN elemental analysis, FT-IR, UV-Vis and 1 H-NMR techniques and their binding behavior with human serum albumin (HSA) was studied by UV-Vis, fluorescence and molecular docking examinations. The experimental data indicated that all three lanthanide complexes have high binding affinity to HSA with effective quenching of HSA fluorescence via static mechanism. The binding parameters, the type of interaction, the value of resonance energy transfer, and the binding distance between complexes and HSA were estimated from the analysis of fluorescence measurements and Förster theory. The thermodynamic parameters suggested that van der Waals interactions and hydrogen bonds play an important role in the binding mechanism. While, the energy transfer from HSA molecules to all these complexes occurs with high probability, the order of binding constants (BpyTb > BpyDy > BpyYb) represents the importance of radius of Ln 3+ ion in the complex-HSA interaction. The results of molecular docking calculation and competitive experiments assessed site 3 of HSA, located in subdomain IB, as the most probable binding site for these ligands and also indicated the microenvironment residues around the bound mentioned complexes. The computational results kept in good agreement with experimental data.

Spectroscopic study of binding of chlorogenic acid with the surface of ZnO nanoparticles

NASA Astrophysics Data System (ADS)

Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

2017-09-01

Understanding the interaction properties of biological materials with ZnO NPs is fundamental interest in the field of biotechnological applications as well as in the formation of optoelectronic devices. In this research, the binding of ZnO NPs and chlorogenic acid (CGA) were investigated using fluorescence quenching, UV-Vis absorption spectroscopy, Fourier transform infrared (FTIR), Raman spectroscopy, scanning electron microscopy (TEM), and dynamic light scattering (DLS) techniques. The study results indicated the fluorescence quenching between ZnO NPs and CGA rationalized in terms of static quenching mechanism or the formation of nonfluorescent CGA-ZnO. From fluorescence quenching spectral analysis the binding constant ( K a ), number of binding sites ( n), and thermodynamic properties, were determined. The quenching constants ( K sv) and binding constant ( K a ), decrease with increasing the temperature and their binding sites n are 2. The thermodynamic parameters determined using Van't Hoff equation indicated binding occurs spontaneously involving the hydrogen bond and van der Walls forces played the major role in the reaction of ZnO NPs with CGA. The Raman, SEM, DLS, and Zeta potential measurements were also indicated the differences in the structure, morphology and sizes of CGA, ZnO NPs, and their corresponding CGA-ZnO due to adsorption of CGA on the surface of ZnO NPs

Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins

PubMed Central

Belatik, Ahmed; Hotchandani, Surat; Carpentier, Robert; Tajmir-Riahi, Heidar-Ali

2012-01-01

Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization. PMID:22574219

The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

PubMed

Ganesan, Lakshmi; Buchwald, Peter

2013-04-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

PubMed Central

Ganesan, Lakshmi; Buchwald, Peter

2013-01-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

Influences of urea, pH and metal ions on the interaction between cepharanthine and lysozyme by steady state fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Yumin; Li, Daojin; Xu, Chen

2015-03-01

The study on the binding mode of drug with protein is important to understand the pharmacokinetics and toxicity of the drug as well as the relationship of structure and function of the protein. In the study, the interaction between cepharanthine and lysozyme (Lys) in aqueous solution was first investigated by fluorescence spectroscopic techniques at pH 7.4. The obtained quenching rate constant and binding constant indicated the static quenching mechanism and medium binding force. The effect of cepharanthine on the conformation of Lys was analyzed using synchronous fluorescence and three-dimensional (3D) fluorescence. In addition, the effect of urea on the interaction of cepharanthine with Lys was studied and the binding capacity of cepharanthine to the denatured Lys deceases dramatically, as compared with that of cepharanthine to native Lys. Moreover, influence of pH on the interaction of cepharanthine with Lys was investigated. As compared with that at pH 7.4, the binding abilities of the drug to Lys under other pH conditions (pH 9.0, 5.5, 3.5, and 1.9) deceased. Furthermore, the effect of metal ions on the binding constant of cepharanthine with Lys was investigated.

dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35.

PubMed

Zinzula, Luca; Esposito, Francesca; Pala, Daniela; Tramontano, Enzo

2012-03-01

The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions. Copyright © 2012 Elsevier B.V. All rights reserved.

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue.

PubMed

Bertsch, M; Mayburd, A L; Kassner, R J

2003-02-15

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.

A spectroscopic study on the interaction between gold nanoparticles and hemoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garabagiu, Sorina, E-mail: sgarabagiu@itim-cj.ro

2011-12-15

Highlights: Black-Right-Pointing-Pointer The interaction was studied using UV-vis and fluorescence spectroscopy. Black-Right-Pointing-Pointer Gold nanoparticles quench the fluorescence emission of hemoglobin solution. Black-Right-Pointing-Pointer The binding and thermodynamic constants were calculated. Black-Right-Pointing-Pointer Major impact: electrochemical applications of the complex onto a substrate. -- Abstract: The interaction between horse hemoglobin and gold nanoparticles was studied using optical spectroscopy. UV-vis and fluorescence spectra show that a spontaneous binding process occurred between hemoglobin and gold nanoparticles. The Soret band of hemoglobin in the presence of gold nanoparticles does not show significant changes, which proves that the protein retained its biological function. A shift to longermore » wavelengths appears in the plasmonic band of gold nanoparticles upon the attachment of hemoglobin molecules. Gold nanoparticles quench the fluorescence emission of tryptophan residues in the structure of hemoglobin. The Stern-Volmer quenching constant, the binding constant and the number of binding sites were also calculated. Thermodynamic parameters indicate that the binding was mainly due to hydrophobic interactions.« less

Binding analysis for interaction of diacetylcurcumin with β-casein nanoparticles by using fluorescence spectroscopy and molecular docking calculations

NASA Astrophysics Data System (ADS)

Mehranfar, Fahimeh; Bordbar, Abdol-Khalegh; Fani, Najme; Keyhanfar, Mehrnaz

2013-11-01

The interaction of diacetylcurcumin (DAC), as a novel synthetic derivative of curcumin, with bovine β-casein (an abundant milk protein that is highly amphiphilic and self assembles into stable micellar nanoparticles in aqueous solution) was investigated using fluorescence quenching experiments, Forster energy transfer measurements and molecular docking calculations. The fluorescence quenching measurements revealed the presence of a single binding site on β-casein for DAC with the binding constant value equals to (4.40 ± 0.03) × 104 M-1. Forster energy transfer measurements suggested that the distance between bound DAC and Trp143 residue is higher than the respective critical distance, hence, the static quenching is more likely responsible for fluorescence quenching other than the mechanism of non-radiative energy transfer. Our results from molecular docking calculations indicated that binding of DAC to β-casein predominantly occurred through hydrophobic contacts in the hydrophobic core of protein. Additionally, in vitro investigation of the cytotoxicity of free DAC and DAC-β-casein complex in human breast cancer cell line MCF7 revealed the higher cytotoxic effect of DAC-β-casein complex.

Spectroscopic studies on the interaction of bovine serum albumin with surfactants and apigenin

NASA Astrophysics Data System (ADS)

Zhao, Xu-Na; Liu, Yi; Niu, Li-Yuan; Zhao, Chen-Ping

The binding of apigenin (Ap) to bovine serum albumin (BSA) has been studied using the methods of fluorescence spectroscopy and UV-vis absorption spectroscopy. The spectroscopic analysis of the quenching mechanism indicates that the quenching constants are inversely correlated with the temperatures and the quenching process could result from a static interaction. The type of interaction force was discussed and the binding site of Ap was in site I (subdomain IIA) of BSA. The thermodynamic parameters ΔH and ΔS are -42.02 kJ mol-1 and -48.31 J mol-1 K-1, respectively and the negative ΔG implying that the binding interaction was spontaneous. The distance r between BSA and Ap was calculated according to Förster's theory and the value is 3.44 nm. The synchronous and three-dimensional fluorescence spectra show that the binding of Ap to BSA could lead to the changes in the conformation and microenvironment of BSA. At the same time, the effects of ionic surfactants on the interaction of Ap and BSA have also been investigated.

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Determinants of the Differential Antizyme-Binding Affinity of Ornithine Decarboxylase

PubMed Central

Liu, Yen-Chin; Hsu, Den-Hua; Huang, Chi-Liang; Liu, Yi-Liang; Liu, Guang-Yaw; Hung, Hui-Chih

2011-01-01

Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K d value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the K d value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K d was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC K d, which suggests that residues 119 and 137 play a role in AZ binding. PMID:22073206

Thermometric sensing of nitrofurantoin by noncovalently imprinted polymers containing two complementary functional monomers.

PubMed

Athikomrattanakul, Umporn; Gajovic-Eichelmann, Nenad; Scheller, Frieder W

2011-10-15

Molecularly imprinted polymers (MIPs) for nitrofurantoin (NFT) recognition addressing in parallel of two complementary functional groups were created using a noncovalent imprinting approach. Specific tailor-made functional monomers were synthesized: a diaminopyridine derivative as the receptor for the imide residue and three (thio)urea derivatives for the interaction with the nitro group of NFT. A significantly improved binding of NFT to the new MIPs was revealed from the imprinting factor, efficiency of binding, affinity constants and maximum binding number as compared to previously reported MIPs, which addressed either the imide or the nitro residue. Substances possessing only one functionality (either the imide group or nitro group) showed significantly weaker binding to the new imprinted polymers than NFT. However, the compounds lacking both functionalities binds extremely weak to all imprinted polymers. The new imprinted polymers were applied in a flow-through thermistor in organic solvent for the first time. The MIP-thermistor allows the detection of NFT down to a concentration of 5 μM in acetonitrile + 0.2% dimethyl sulfoxide (DMSO). The imprinting factor of 3.91 at 0.1 mM of NFT as obtained by thermistor measurements is well comparable to the value obtained by batch binding experiments. © 2011 American Chemical Society

Modeling of the solution interaction properties of plastic materials used in pharmaceutical product container systems.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy; Sadain, Salma

2009-01-01

Material/water equilibrium binding constants (Eb) were determined for 14 organic solutes and 17 plastic raw materials that could be used in pharmaceutical product container systems. Correlations between the measured binding constants and the organic solute's octanol/water and hexane/water partition coefficients were obtained. In general, while the materials examined exhibited a wide range of binding characteristics, the tested materials by and large fell within two broad classes: (1) those that were octanol-like in their binding characteristics, and (2) those that were hexane-like. Materials of the same class (e.g., polypropylenes) generally had binding models that were very similar. Rank ordering of the materials in terms of their magnitude of drug binding (least binding to most binding) was as follows: polypropylene < polyethylene < polyamide < styrene-ethylene-butylene-styrene < copolyester ether elastomer approximately equal to amine-terminated poly fatty acid amide polymer. The utilization of the developed models to estimate drug loss via sorption by the container is discussed.

The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

PubMed

Shrestha, Stal S; Liow, Jeih-San; Jenko, Kimberly; Ikawa, Masamichi; Zoghbi, Sami S; Innis, Robert B

2016-12-01

Prazosin, a potent and selective α 1 -adrenoceptor antagonist, displaces 25% of 11 C-CUMI-101 ([O-methyl- 11 C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11 C-CUMI-101 binding to α 1 -adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3 H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3 H-prazosin and the inhibition constant of CUMI-101. α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. Reasoning by analogy, 25% of 11 C-CUMI-101 uptake in human cerebellum reflects binding to α 1 -adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

The Universal Statistical Distributions of the Affinity, Equilibrium Constants, Kinetics and Specificity in Biomolecular Recognition

PubMed Central

Zheng, Xiliang; Wang, Jin

2015-01-01

We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity), the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics. PMID:25885453

Precision Spectroscopy of Atomic Hydrogen

NASA Astrophysics Data System (ADS)

Hänsch, Theodor W.

1994-08-01

The simple hydrogen atom permits unique confrontations between spectroscopic experiment and fundamental theory. The experimental resolution and measurement accuracy continue to improve exponentially. Recent advances include a new measurement of the Lamb shift of the 1S ground state which provides now the most stringent test of QED for an atom and reveals unexpectedly large two-loop binding corrections. The H-D isotope shift of the extremely narrow 1S-2S two-photon resonance is yielding a new value for the structure radius of the deuteron, in agreement with nuclear theory. The Rydberg constant as determined within 3 parts in 1011 by two independent groups has become the most accurately known of any fundamental constant. Advances in the art of absolute optical frequency measurements will permit still more precise experiments in the near future.

Molecular Approach to the Synergistic Effect on Astringency Elicited by Mixtures of Flavanols.

PubMed

Ramos-Pineda, Alba María; García-Estévez, Ignacio; Brás, Natércia F; Martín Del Valle, Eva M; Dueñas, Montserrat; Escribano Bailón, María Teresa

2017-08-09

The interactions between salivary proteins and wine flavanols (catechin, epicatechin, and mixtures thereof) have been studied by HPLC-DAD, isothermal titration microcalorimetry, and molecular dynamics simulations. Chromatographic results suggest that the presence of these flavanol mixtures could facilitate the formation of precipitates to the detriment of soluble aggregates. Comparison between the thermodynamic parameters obtained showed remarkably higher negative values of ΔG in the system containing the mixture of both flavanols in comparison to the systems containing individual flavanols, indicating a more favorable scenario in the mixing system. Also, the apparent binding constants were higher in this system. Furthermore, molecular dynamics simulations suggested a faster and greater cooperative binding of catechin and epicatechin to IB7 14 peptides when both types of flavanols are present simultaneously in solution.

Study on interaction of mangiferin to insulin and glucagon in ternary system

NASA Astrophysics Data System (ADS)

Lin, Hui; Chen, Rui; Liu, Xiaoyan; Sheng, Fenling; Zhang, Haixia

2010-05-01

The binding of mangiferin to insulin and glucagon was investigated in the presence and absence of another Peptide by optical spectroscopy. Fluorescence titration experiments revealed that mangiferin quenched the intrinsic fluorescence of insulin and glucagon by static quenching. The ratios of binding constants of glucagon-mangiferin to insulin-mangiferin at different temperatures were calculated in "pure" and ternary system, respectively. The results indicated that the Peptides were competitive with each other to act on mangiferin. Values of the thermodynamic parameters and the experiments of pH effect proved that the key interacting forces between mangiferin and the Peptides were hydrophobic interaction. In addition, UV-vis absorption, synchronous fluorescence and Fourier transform infrared measurements showed that the conformation of insulin and glucagon were changed after adding mangiferin.

Investigation of glucose binding sites on insulin.

PubMed

Zoete, Vincent; Meuwly, Markus; Karplus, Martin

2004-05-15

Possible insulin binding sites for D-glucose have been investigated theoretically by docking and molecular dynamics (MD) simulations. Two different docking programs for small molecules were used; Multiple Copy Simultaneous Search (MCSS) and Solvation Energy for Exhaustive Docking (SEED) programs. The configurations resulting from the MCSS search were evaluated with a scoring function developed to estimate the binding free energy. SEED calculations were performed using various values for the dielectric constant of the solute. It is found that scores emphasizing non-polar interactions gave a preferential binding site in agreement with that inferred from recent fluorescence and NMR NOESY experiments. The calculated binding affinity of -1.4 to -3.5 kcal/mol is within the measured range of -2.0 +/- 0.5 kcal/mol. The validity of the binding site is suggested by the dynamical stability of the bound glucose when examined with MD simulations with explicit solvent. Alternative binding sites were found in the simulations and their relative stabilities were estimated. The motions of the bound glucose during molecular dynamics simulations are correlated with the motions of the insulin side chains that are in contact with it and with larger scale insulin motions. These results raise the question of whether glucose binding to insulin could play a role in its activity. The results establish the complementarity of molecular dynamics simulations and normal mode analyses with the search for binding sites proposed with small molecule docking programs. Copyright 2004 Wiley-Liss, Inc.

Interaction of Gramicidin S and its Aromatic Amino-Acid Analog with Phospholipid Membranes

PubMed Central

Jelokhani-Niaraki, Masoud; Hodges, Robert S.; Meissner, Joseph E.; Hassenstein, Una E.; Wheaton, Laura

2008-01-01

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (Kb). The Kb1 and Kb2 values for endothermic two-step binding processes corresponded to high and low binding affinities (Kb1 ≥ 100 Kb2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis. PMID:18621820

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tiwari, Manisha; Mikuni, Shintaro; Muto, Hideki

Highlights: •We used two-laser-beam FCCS to determine the dissociation constant (K{sub d}) of IPT domain of p50/p65 heterodimer in living cell. •Interaction of p50 and p65 was analyzed in the cytoplasm and nucleus of single living cell. •Binding affinity of p50/p65 heterodimer is higher in cytoplasm than that of nucleus. -- Abstract: Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K{sub d}, for the heterodimer inmore » living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K{sub d} values of mCherry{sub 2}- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.« less

Ti(IV) and the Siderophore Desferrioxamine B: A Tight Complex Has Biological and Environmental Implications.

PubMed

Jones, Kayleigh E; Batchler, Kathleen L; Zalouk, Célia; Valentine, Ann M

2017-02-06

The siderophore desferrioxamine B (DFOB) binds Ti(IV) tightly and precludes its hydrolytic precipitation under biologically and environmentally relevant conditions. This interaction of DFOB with Ti(IV) is investigated by using spectro-potentiometric and spectro-photometric titrations, mass spectrometry, isothermal titration calorimetry (ITC), and computational modeling. The data from pH 2-10 suggest two one-proton equilibria among three species, with one species predominating below pH 3.5, a second from pH 3.5 to 8, and a third above pH 8. The latter species is prone to slow hydrolytic precipitation. Electrospray mass spectrometry allowed the detection of [Ti(IV) (HDFOB)] 2+ and [Ti(DFOB)] + ; these species were assigned as the pH < 3.5 and the 3.5 < pH < 8 species, respectively. The stability constant for Ti(IV)-DFOB was determined by using UV/vis-monitored competition with ethylenediaminetetraacetic acid (EDTA). Taking into consideration the available binding constant of Ti(IV) and EDTA, the data reveal values of log β 111 = 41.7, log β 110 = 38.1, and log β 11-1 = 30.1. The former value was supported by ITC, with the transfer of Ti(IV) from EDTA to DFOB determined to be both enthalpically and entropically favorable. Computational methods yielded a model of Ti-DFOB. The physiological and environmental implications of this tight interaction and the potential role of DFOB in solubilizing Ti(IV) are discussed.

Determination of binding constants of cyclodextrin inclusion complexes with amino acids and dipeptides by potentiometric titration.

PubMed

Kahle, Claudia; Holzgrabe, Ulrike

2004-10-01

Cyclodextrins are well known for their ability to separate enantiomers of drugs, natural products, and other chiral substances using HPLC, GC, or CE. The resolution of the enantiomers is due to the formation of diastereomeric complexes between the cyclodextrin and the pairs of enantiomers. The aim of this study was to determine the binding constants of the complexes between alpha- and beta-cyclodextrin and the enantiomers of a series of aliphatic and aromatic amino acids, and dipeptides, using a potentiometric titration method. The results of this method are compared to other methods, and correlated to findings in cyclodextrin-modified capillary electrophoresis and possible complex structures. Potentiometric titration was found to be an appropriate tool to determine the binding constants of cyclodextrin inclusion complexes.

A combined spectroscopic, molecular docking and molecular dynamic simulation study on the interaction of quercetin with β-casein nanoparticles.

PubMed

Mehranfar, Fahimeh; Bordbar, Abdol-Khalegh; Parastar, Hadi

2013-10-05

The interaction of quercetin with β-casein nanoparticle micelle was studied at various temperatures in order to do a complete thermodynamic and molecular analysis on the binding process. The results of fluorescence studies showed the possibility of fluorescence energy transfer between excited tryptophan and quercetin. The determined values of critical transfers distance and the mean distance of ligand from Trp-143 residues in β-casein micelle represents a non-radiative energy transfer mechanism for quenching and the existence of a significant interaction between this flavonoid and β-casein nanoparticle. The equilibrium binding of quercetin with β-casein micelle at different temperatures was studied by using UV-Vis absorption spectroscopy. The chemometric analysis (principal component analysis (PCA) and multivariate curve resolution-alternating least squares (MCR-ALS) methods) on spectrophotometric data revealed the existence of two components in solution (quercetin and β-casein-quercetin complex) and resolved their pure concentration and spectral profiles. This information let us to calculate the equilibrium binding constant at various temperatures and the relevant thermodynamic parameters of interaction (enthalpy, entropy and Gibbs free energy) with low uncertainty. The negative values of entropy and enthalpy changes represent the predominate role of hydrogen binding and van der Waals interactions in the binding process. Docking calculations showed the probable binding site of quercetin is located in the hydrophobic core of β-casein where the quercetin molecule is lined by hydrophobic residues and make five hydrogen bonds and several van der Waals contacts with them. Moreover, molecular dynamic (MD) simulation results suggested that this flavonoid can interact with β-casein, without affecting the secondary structure of β-casein. Simulations, molecular docking and experimental data reciprocally supported each other. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of Clostridium botulinum Type B Neurotoxin Associated with Infant Botulism in Japan

PubMed Central

Kozaki, Shunji; Kamata, Yoichi; Nishiki, Tei-ichi; Kakinuma, Hiroaki; Maruyama, Hiromi; Takahashi, Hiroaki; Karasawa, Tadahiro; Yamakawa, Kiyotaka; Nakamura, Shinichi

1998-01-01

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity. PMID:9746583

Chimeric proteins combining phosphatase and cellulose-binding activities: proof-of-concept and application in the hydrolysis of paraoxon.

PubMed

Gonçalves, Larissa M; Chaimovich, Hernan; Cuccovia, Iolanda M; Marana, Sandro R

2014-05-01

Phosphatases for organophosphate degradation and carbohydrate-binding domains (CBMs) have potential biotechnological applications. As a proof-of-concept, a soluble chimeric protein that combines acid phosphatase (AppA) from Escherichia coli and a CBM from Xanthomonas axonopodis pv. citri (AppA-CBM) was produced in E.coli. AppACBM adsorbed in microcrystalline cellulose Avicel PH101 catalyzed the hydrolysis of p-nitrophenyl phosphate (PNPP). The binding to microcrystalline cellulose displayed saturation behavior with an apparent binding constant (Kb) of 22 ± 5 mg and a maximum binding (Bmax) of 1.500 ± 0.001 enzyme units. Binding was highest at pH 2.5 and decreased above pH 6.5, as previously observed for family 2 CBMs. The Km values for PNPP of AppA-CBM and native AppA were identical (2.7 mM). To demonstrate that this strategy for protein engineering has practical applications and is largely functional, even for phosphatases exhibiting diverse folds, a chimeric protein combining human paraoxonase 1 (hPON1) and the CBM was produced. Both PON1-CBM and hPON1 had identical K_m values for paraoxon (1.3 mM). Additionally, hPON1 bound to microcrystalline cellulose with a Kb of 27 ± 3 mg, the same as that observed for AppA-CBM. These data show that the phosphatase domains are as functional in both of the chimeric proteins as they are in the native enzymes and that the CBM domain maintains the same cellulose affinity. Therefore, the engineering of chimeric proteins combining domains of phosphatases and CBMs is fully feasible, resulting in chimeric enzymes that exhibit potential for OP detoxification.

Synthesis, crystal structure and investigation of mononuclear copper(II) and zinc(II) complexes of a new carboxylate rich tripodal ligand and their interaction with carbohydrates in alkaline aqueous solution

PubMed Central

Stewart, Christopher D.; Pedraza, Mayra; Arman, Hadi; Fan, Hua-Jun; Schilling, Eduardo Luiz; Szpoganicz, Bruno; Musie, Ghezai T.

2016-01-01

A new carboxylate rich asymmetric tripodal ligand, N-[2-carboxybenzomethyl]-N-[carboxymethyl]-β-alanine (H3camb), and its di-copper(II), (NH4)2[1]2, and di-zinc(II), ((CH3)4 N)2[2]2, complexes have been synthesized as carbohydrate binding models in aqueous solutions. The ligand and complexes have been fully characterized using several techniques, including single crystal X-ray diffraction. The interactions of (NH4)2[1]2 and ((CH3)4 N)2[2]2 with D-glucose, D-mannose, D-xylose and xylitol in aqueous alkaline media were investigated using UV–Vis and 13C-NMR spectroscopic techniques, respectively. The molar conductance, NMR and ESI–MS studies indicate that the complexes dissociate in solution to produce the respective complex anions, 1− and 2−. Complexes 1− and 2− showed chelating ability towards the naturally abundant and biologically relevant sugars, D-glucose, D-mannose, D-xylose, and xylitol. The complex ions bind to one molar equivalent of the sugars, even in the presence of stoichiometric excess of the substrates, in solution. Experimentally obtained spectroscopic data and computational results suggest that the substrates bind to the metal center in a bidentate fashion. Apparent binding constant values, pKapp, between the complexes and the substrates were determined and a specific mode of substrate binding is proposed. The pKapp and relativistic density functional theory (DFT) calculated Gibbs free energy values indicate that D-mannose displayed the strongest interaction with the complexes. Syntheses, characterizations, detailed substrate binding studies using spectroscopic techniques, single crystal X-ray diffraction and geometry optimizations of the complex-substrates with DFT calculations are also reported. PMID:25969174

Effects of the microbial siderophore DFO-B on Pb and Cd speciation in aqueous solution.

PubMed

Mishra, Bhoopesh; Haack, Elizabeth A; Maurice, Patricia A; Bunker, Bruce A

2009-01-01

This study investigates the complexation environments of aqueous Pb and Cd in the presence of the trihydroxamate microbial siderophore, desferrioxamine-B (DFO-B) as a function of pH. Complexation of aqueous Pb and Cd with DFO-B was predicted using equilibrium speciation calculation. Synchrotron-based X-ray absorption fine structure (XAFS) spectroscopy at Pb L(III) edge and Cd K edge was used to characterize Pb and Cd-DFO-B complexes at pH values predicted to best represent each of the metal-siderophore complexes. Pb was not found to be complexed measurably by DFO-B at pH 3.0, but was complexed by all three hydroxamate groups to form a totally "caged" hexadentate structure at pH 7.5-9.0. At the intermediate pH value (pH 4.8), a mixture of Pb-DFOB complexes involving binding of the metal through one and two hydroxamate groups was observed. Cd, on the other hand, remained as hydrated Cd2+ at pH 5.0, occurred as a mixture of Cd-DFOB and inorganic species at pH 8.0, and was bound by three hydroxamate groups from DFO-B at pH 9.0. Overall, the solution species observed with EXAFS were consistent with those predicted thermodynamically. However, Pb speciation at higher pH values differed from that predicted and suggests that published constants underestimate the binding constant for complexation of Pb with all three hydroxamate groups of the DFO-B ligand. This molecular-level understanding of metal-siderophore solution coordination provides physical evidence for complexes of Pb and Cd with DFO-B, and is an important first step toward understanding processes at the microbial- and/or mineral-water interface in the presence of siderophores.

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

PubMed

Plesniak, Leigh; Horiuchi, Yuki; Sem, Daniel; Meinenger, David; Stiles, Linda; Shaffer, Jennifer; Jennings, Patricia A; Adams, Joseph A

2002-11-26

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.

Mechanism of Sulfide Binding by Ferric Hemeproteins.

PubMed

Boubeta, Fernando M; Bieza, Silvina A; Bringas, Mauro; Estrin, Darío A; Boechi, Leonardo; Bari, Sara E

2018-06-19

The reaction of hydrogen sulfide (H 2 S) with hemeproteins is a key physiological reaction; still, its mechanism and implications are not completely understood. In this work, we propose a combination of experimental and theoretical tools to shed light on the reaction in model system microperoxidase 11 (MP11-Fe III ) and myoglobin (Mb-Fe III ), from the estimation of the intrinsic binding constants of the species H 2 S and hydrosulfide (HS - ), and the computational description of the overall binding process. Our results show that H 2 S and HS - are the main reactive species in Mb-Fe III and MP11-Fe III , respectively, and that the magnitude of their intrinsic binding constants are similar to most of the binding constants reported so far for hemeproteins systems and model compounds. However, while the binding of HS - to Mb-Fe III was negligible, the binding of H 2 S to MP11-Fe III was significant, providing a frame for a discriminated analysis of both species and revealing differential mechanistic aspects. A joint inspection of the kinetic data and the free energy profiles of the binding processes suggests that a dissociative mechanism with the release of a coordinated water molecule as rate limiting step is operative in the binding of H 2 S to Mb-Fe III and that the binding of HS - is prevented in the access to the protein matrix. For the MP11-Fe III case, where no access restrictions for the ligands are present, an associative component in the mechanism seems to be operative. Overall, the results suggest that if accessing the active site then both H 2 S and HS - are capable of binding a ferric heme moiety.

Bringing metabolic networks to life: convenience rate law and thermodynamic constraints

PubMed Central

Liebermeister, Wolfram; Klipp, Edda

2006-01-01

Background Translating a known metabolic network into a dynamic model requires rate laws for all chemical reactions. The mathematical expressions depend on the underlying enzymatic mechanism; they can become quite involved and may contain a large number of parameters. Rate laws and enzyme parameters are still unknown for most enzymes. Results We introduce a simple and general rate law called "convenience kinetics". It can be derived from a simple random-order enzyme mechanism. Thermodynamic laws can impose dependencies on the kinetic parameters. Hence, to facilitate model fitting and parameter optimisation for large networks, we introduce thermodynamically independent system parameters: their values can be varied independently, without violating thermodynamical constraints. We achieve this by expressing the equilibrium constants either by Gibbs free energies of formation or by a set of independent equilibrium constants. The remaining system parameters are mean turnover rates, generalised Michaelis-Menten constants, and constants for inhibition and activation. All parameters correspond to molecular energies, for instance, binding energies between reactants and enzyme. Conclusion Convenience kinetics can be used to translate a biochemical network – manually or automatically - into a dynamical model with plausible biological properties. It implements enzyme saturation and regulation by activators and inhibitors, covers all possible reaction stoichiometries, and can be specified by a small number of parameters. Its mathematical form makes it especially suitable for parameter estimation and optimisation. Parameter estimates can be easily computed from a least-squares fit to Michaelis-Menten values, turnover rates, equilibrium constants, and other quantities that are routinely measured in enzyme assays and stored in kinetic databases. PMID:17173669

Communication: Local energetic analysis of the interfacial and surface energies of graphene from the single layer to graphite

NASA Astrophysics Data System (ADS)

Kocherlakota, Lakshmi S.; Krajina, Brad A.; Overney, René M.

2015-12-01

Recent advances in scanning probe methods that provide direct access to the surface free energy of inorganic layered materials in terms of the Hamaker constant yield energetic values for monolayer graphene that differ substantially, by a factor of around 0.4, from bulk graphite. The onset of bulk deviating energy values was observed at a multilayer slab thickness of ˜3 nm, corresponding to a layer number of 10. The findings, complemented with extractions from water contact angle measurements and calculated interlayer binding energies, find short-range ordinary van der Waals interactions to be most prominently affected by dimensional constraints and many-body interactions.

Communication: Local energetic analysis of the interfacial and surface energies of graphene from the single layer to graphite.

PubMed

Kocherlakota, Lakshmi S; Krajina, Brad A; Overney, René M

2015-12-28

Recent advances in scanning probe methods that provide direct access to the surface free energy of inorganic layered materials in terms of the Hamaker constant yield energetic values for monolayer graphene that differ substantially, by a factor of around 0.4, from bulk graphite. The onset of bulk deviating energy values was observed at a multilayer slab thickness of ∼3 nm, corresponding to a layer number of 10. The findings, complemented with extractions from water contact angle measurements and calculated interlayer binding energies, find short-range ordinary van der Waals interactions to be most prominently affected by dimensional constraints and many-body interactions.

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

PubMed

Van Riesen, Abigail J; Fadock, Kaila L; Deore, Prashant S; Desoky, Ahmed; Manderville, Richard A; Sowlati-Hashjin, Shahin; Wetmore, Stacey D

2018-05-23

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Involvement of histidine residues in the pH-dependent β-galactoside binding activity of human galectin-1.

PubMed

Hiramatsu, Hirotsugu; Takeuchi, Katsuyuki; Takeuchi, Hideo

2013-04-02

The pH dependence of the β-galactoside binding activity of human galectin-1 (hGal-1) was investigated by fluorescence spectroscopy using lactose as a ligand. The obtained binding constant Kb was 2.94 ± 0.10 mM(-1) at pH 7.5. The Kb value decreased at acidic pH with a midpoint of transition at pH 6.0 ± 0.1. To elucidate the molecular mechanism of the pH dependence, we investigated the structures of hGal-1 and its two His mutants (H44Q and H52Q) using fluorescence, circular dichroism, UV absorption, and UV resonance Raman spectroscopy. Analysis of the spectra has shown that the pKa values of His44 and His52 are 5.7 ± 0.2 and 6.3 ± 0.1, respectively. The protonation of His52 below pH 6.3 induces a small change in secondary structure and partly reduces the galactoside binding activity. On the other hand, the protonation of His44 below pH 5.7 exerts a cation-π interaction with Trp68 and largely diminishes the galactoside binding activity. With reference to the literature X-ray structures at pH 7.0 and 5.6, protonated His52 is proposed to move slightly away from the galactoside-binding region with a partial unfolding of the β-strand containing His52. On the other hand, protonated His44 becomes unable to form a hydrogen bond with galactoside and additionally induces a reorientation and/or displacement of Trp68 through cation-π interaction, leading to a loosening of the galactoside-binding pocket. These structural changes associated with His protonation are likely to be the origin of the pH dependence of the galactoside binding activity of hGal-1.

Interaction of Sulforaphane with DNA and RNA

PubMed Central

Abassi Joozdani, Farzaneh; Yari, Faramarz; Abassi Joozdani, Parvaneh; Nafisi, Shohreh

2015-01-01

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO2) interaction. Overall binding constants were estimated to be K(SFN–DNA)=3.01 (± 0.035)×104 M-1 and K(SFN–RNA)= 6.63 (±0.042)×103 M-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure. PMID:26030290

Interaction of Lysozyme with Rhodamine B: A combined analysis of spectroscopic & molecular docking.

PubMed

Millan, Sabera; Satish, Lakkoji; Kesh, Sandeep; Chaudhary, Yatendra S; Sahoo, Harekrushna

2016-09-01

The interaction of Rhodamine B (RB) with Lysozyme (Lys) was investigated by different optical spectroscopic techniques such as absorption, fluorescence, and circular-dichroism (CD), along with molecular docking studies. The fluorescence results (including steady-state and time-resolved mode) revealed that the addition of RB effectively causes strong quenching of intrinsic fluorescence in Lysozyme and mostly, by the static quenching mechanism. Different binding and thermodynamic parameters were calculated at different temperatures and the binding constant value was found to be 2963.54Lmol(-1) at 25°C. The average distance (r0) was found to be 3.31nm according to Förster's theory of non-radiative energy transfer between Lysozyme and RB. The conformational change in Lysozyme during interaction with RB was confirmed from absorbance, synchronous fluorescence, and circular dichroism measurements. Finally, molecular docking studies were done to confirm that the dye binds with Lysozyme. Copyright © 2016 Elsevier B.V. All rights reserved.

Rates and equilibrium constants of the ligand-induced conformational transition of an HCN ion channel protein domain determined by DEER spectroscopy.

PubMed

Collauto, Alberto; DeBerg, Hannah A; Kaufmann, Royi; Zagotta, William N; Stoll, Stefan; Goldfarb, Daniella

2017-06-14

Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (μRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNBD) of the HCN2 ion channel upon binding of the ligand 3',5'-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC + L ⇌ AO + L ⇌ BO ⇌ BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved μRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.

Binding of vitamin A with milk α- and β-caseins.

PubMed

Bourassa, P; N'soukpoé-Kossi, C N; Tajmir-Riahi, H A

2013-05-01

The binding sites of retinol and retinoic acid with milk α- and β-caseins were determined, using constant protein concentration and various retinoid contents. FTIR, UV-visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse retinol and retinoic acid binding sites, the binding constant and the effect of retinoid complexation on the stability and conformation of caseins. Structural analysis showed that retinoids bind caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(retinol-)(α)(-caseins)=1.21 (±0.4)×10(5) M(-1) and K(retinol-)(β)(-caseins)=1.11 (±0.5)×10(5) M(-1) and K(retinoic acid-)(α)(-caseins)=6.2 (±0.6)×10(4) M(-1) and K(retinoic acid-)(β)(-caseins)=6.3 (±0.6)×10(4) M(-1). The number of bound retinol molecules per protein (n) was 1.5 (±0.1) for α-casein and 1.0 (±0.1) for β-casein, while 1 molecule of retinoic acid was bound in the α- and β-casein complexes. Molecular modelling showed different binding sites for retinol and retinoic acid on α- and β-caseins with more stable complexes formed with α-casein. Retinoid-casein complexation induced minor alterations of protein conformation. Caseins might act as carriers for transportation of retinoids to target molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

Modified high-affinity binding of Ni2+, Ca2+ and Zn2+ to natural mutants of human serum albumin and proalbumin.

PubMed

Kragh-Hansen, U; Brennan, S O; Minchiotti, L; Galliano, M

1994-07-01

High-affinity binding of radioactive Ni2+, Ca2+ and Zn2+ to six genetic albumin variants and to normal albumin isolated from the same heterozygote carriers was studied by equilibrium dialysis at pH 7.4. The three cations bind differently to albumin. Ni2+ binds to a site in the N-terminal region of the protein which is partially blocked by the presence of a propeptide as in proalbumin (proAlb) Varese (Arg-2-->His), proAlb Christchurch (Arg-1-->Gln) and proAlb Blenheim (Asp1-->Val) and by the presence of only an extra Arg residue (Arg-1) as in Arg-Alb and albumin (Alb) Redhill. The association constants are decreased by more than one order of magnitude in these cases, suggesting biological consequences for the ligand. The additional structural changes in Alb Redhill have no effect on Ni2+ binding. Finally, the modification of Alb Blenheim (Asp1-->Val) reduces the binding constant to 50%. Ca2+ binding is decreased to about 60-80% by the presence of a propeptide and the mutation Asp1-->Val. Arg-1 alone does not affect binding, whereas Alb Redhill binds Ca2+ more strongly than the normal protein (125%). In contrast with binding of Ni2+ and Ca2+, albumin shows heterogeneity with regard to binding of Zn2+, i.e. the number of high-affinity sites was calculated to be, on average, 0.43. The binding constant for Zn2+ is increased to 125% in the case of proAlb Varese, decreased to 50-60% for proAlb Christchurch and Alb Redhill but is normal for proAlb Blenheim, Alb Blenheim and Arg-Alb. The effects of the mutations on binding of Ca2+ and Zn2+ indicate that primary binding, when operative, is to as yet unidentified sites in domain I of the albumin molecule.

Locating the binding sites of folic acid with milk α- and β-caseins.

PubMed

Bourassa, P; Tajmir-Riahi, H A

2012-01-12

We located the binding sites of folic acid with milk α- and β-caseins at physiological conditions, using constant protein concentration and various folic acid contents. FTIR, UV-visible, and fluorescence spectroscopic methods as well as molecular modeling were used to analyze folic acid binding sites, the binding constant, and the effect of folic acid interaction on the stability and conformation of caseins. Structural analysis showed that folic acid binds caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(folic acid-α-caseins) = 4.8 (±0.6) × 10(4) M(-1) and K(folic acid-β-caseins) = 7.0 (±0.9) × 10(4) M(-1). The number of bound acid molecules per protein was 1.5 (±0.4) for α-casein and 1.4 (±0.3) for β-casein complexes. Molecular modeling showed different binding sites for folic acid on α- and β-caseins. The participation of several amino acids in folic acid-protein complexes was observed, which was stabilized by hydrogen bonding network and the free binding energy of -7.7 kcal/mol (acid-α-casein) and -8.1 kcal/mol (acid-β-casein). Folic acid complexation altered protein secondary structure by the reduction of α-helix from 35% (free α-casein) to 33% (acid-complex) and 32% (free β-casein) to 26% (acid-complex) indicating a partial protein destabilization. Caseins might act as carriers for transportation of folic acid to target molecules.

Interactions and diffusion in fine-stranded β-lactoglobulin gels determined via FRAP and binding.

PubMed

Schuster, Erich; Hermansson, Anne-Marie; Ohgren, Camilla; Rudemo, Mats; Lorén, Niklas

2014-01-07

The effects of electrostatic interactions and obstruction by the microstructure on probe diffusion were determined in positively charged hydrogels. Probe diffusion in fine-stranded gels and solutions of β-lactoglobulin at pH 3.5 was determined using fluorescence recovery after photobleaching (FRAP) and binding, which is widely used in biophysics. The microstructures of the β-lactoglobulin gels were characterized using transmission electron microscopy. The effects of probe size and charge (negatively charged Na2-fluorescein (376Da) and weakly anionic 70kDa FITC-dextran), probe concentration (50 to 200 ppm), and β-lactoglobulin concentration (9% to 12% w/w) on the diffusion properties and the electrostatic interaction between the negatively charged probes and the positively charged gels or solutions were evaluated. The results show that the diffusion of negatively charged Na2-fluorescein is strongly influenced by electrostatic interactions in the positively charged β-lactoglobulin systems. A linear relationship between the pseudo-on binding rate constant and the β-lactoglobulin concentration for three different probe concentrations was found. This validates an important assumption of existing biophysical FRAP and binding models, namely that the pseudo-on binding rate constant equals the product of the molecular binding rate constant and the concentration of the free binding sites. Indicators were established to clarify whether FRAP data should be analyzed using a binding-diffusion model or an obstruction-diffusion model. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Kinetic and thermodynamic study of bovine serum albumin interaction with rifampicin using surface plasmon resonance and molecular docking methods

NASA Astrophysics Data System (ADS)

Sharifi, Maryam; Dolatabadi, Jafar Ezzati Nazhad; Fathi, Farzaneh; Rashidi, Mohammad; Jafari, Behzad; Tajalli, Habib; Rashidi, Mohammad-Reza

2017-03-01

The interaction of bovine serum albumin (BSA) with various drugs, such as antibiotics, due to the importance of BSA in drug delivery has attracted increasing research attention at present. Therefore, the aim of this study was investigation of BSA interaction with rifampicin using surface plasmon resonance (SPR) and molecular docking methods under the imitated physiological conditions (pH=7.4). BSA immobilization on carboxymethyl dextran hydrogel chip has been carried out after activation with N-hydroxysuccinimide/N-ethyl-N-(3-diethylaminopropyl) carbodiimide. The dose-response sensorgrams of BSA upon increasing concentration of refampicin were attained in SPR analysis. The high affinity of rifampicin to BSA was demonstrated by a low equilibrium constants (KD) value (3.46×10-5 at 40°C). The process of kinetic values changing shows that affinity of BSA to rifampicin decreased with rising temperature. The positive value of both enthalpy change (ΔH) and entropy change (ΔS) showed that hydrophobic force plays major role in the BSA interaction with rifampicin. The positive value of ΔG was indicative of nonspontaneous and enthalpy-driven binding process. In addition, according to the molecular docking study, hydrogen binding has some contributions in the interaction of rifampicin with BSA.

Novel tacrine/acridine anticholinesterase inhibitors with piperazine and thiourea linkers.

PubMed

Hamulakova, Slavka; Imrich, Jan; Janovec, Ladislav; Kristian, Pavol; Danihel, Ivan; Holas, Ondrej; Pohanka, Miroslav; Böhm, Stanislav; Kozurkova, Maria; Kuca, Kamil

2014-09-01

A new series of substituted tacrine/acridine and tacrine/tacrine dimers with aliphatic or alkylene-thiourea linkers was synthesized and the potential of these compounds as novel human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE) inhibitors with nanomolar inhibition activity was evaluated. The most potent AChE inhibitor was found to be homodimeric tacrine derivative 14a, which demonstrated an IC50 value of 2 nM; this value indicates an activity rate which is 250-times higher than that of tacrine 1 and 7500-times higher than 7-MEOTA 15, the compounds which were used as standards in the study. IC50 values of derivatives 1, 9, 10, 14b and 15 were compared with the dissociation constants of the enzyme-inhibitor complex, Ki1, and the enzyme-substrate-inhibitor complex, Ki2, for. A dual binding site is presumed for the synthesized compounds which possess two tacrines or tacrine and acridine as terminal moieties show evidence of dual site binding. DFT calculations of theoretical desolvation free energies, ΔΔGtheor, and docking studies elucidate these suggestions in more detail. Copyright © 2014 Elsevier B.V. All rights reserved.

Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

PubMed Central

Zubrienė, Asta; Matulienė, Jurgita; Baranauskienė, Lina; Jachno, Jelena; Torresan, Jolanta; Michailovienė, Vilma; Cimmperman, Piotras; Matulis, Daumantas

2009-01-01

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90αN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90αN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding. PMID:19582223

The effect of glycation on bovine serum albumin conformation and ligand binding properties with regard to gliclazide

NASA Astrophysics Data System (ADS)

Żurawska-Płaksej, Ewa; Rorbach-Dolata, Anna; Wiglusz, Katarzyna; Piwowar, Agnieszka

2018-01-01

Albumin, the major serum protein, plays a variety of functions, including binding and transporting endogenous and exogenous ligands. Its molecular structure is sensitive to different environmental modifiers, among which glucose is one of the most significant. In vivo albumin glycation occurs under physiological conditions, but it is increased in diabetes. Since bovine serum albumin (BSA) may serve as a model protein in in vitro experiments, we aimed to investigate the impact of glucose-mediated BSA glycation on the binding capacity towards gliclazide, as well as the ability of this drug to prevent glycation of the BSA molecule. To reflect normo- and hyperglycemia, the conditions of the glycation process were established. Structural changes of albumin after interaction with gliclazide (0-14 μM) were determined using fluorescence quenching and circular dichroism spectroscopy. Moreover, thermodynamic parameters as well as energy transfer parameters were determined. Calculated Stern-Volmer quenching constants, as well as binding constants for the BSA-gliclazide complex, were lower for the glycated form of albumin than for the unmodified protein. The largest, over 2-fold, decrease in values of binding parameters was observed for the sample with 30 mM of glucose, reflecting the poorly controlled diabetic state, which indicates that the degree of glycation had a critical influence on binding with gliclazide. In contrast to significant changes in the tertiary structure of BSA upon binding with gliclazide, only slight changes in the secondary structure were observed, which was reflected by about a 3% decrease of the α-helix content of glycated BSA (regardless of glucose concentration) in comparison to unmodified BSA. The presence of gliclazide during glycation did not affect its progress. The results of this study indicate that glycation significantly changed the binding ability of BSA towards gliclazide and the scale of these changes depended on glucose concentration. It may have a direct impact on the free drug fraction and its pharmacokinetic behavior, including the risk of hypoglycemic episodes or unexpected interactions with other ligands. The use of BSA in examining binding effects upon glycation seems to be good model for preliminary research and may be used to identify a potential drug response in a diabetic state.

New Parameters for Higher Accuracy in the Computation of Binding Free Energy Differences upon Alanine Scanning Mutagenesis on Protein-Protein Interfaces.

PubMed

Simões, Inês C M; Costa, Inês P D; Coimbra, João T S; Ramos, Maria J; Fernandes, Pedro A

2017-01-23

Knowing how proteins make stable complexes enables the development of inhibitors to preclude protein-protein (P:P) binding. The identification of the specific interfacial residues that mostly contribute to protein binding, denominated as hot spots, is thus critical. Here, we refine an in silico alanine scanning mutagenesis protocol, based on a residue-dependent dielectric constant version of the Molecular Mechanics/Poisson-Boltzmann Surface Area method. We have used a large data set of structurally diverse P:P complexes to redefine the residue-dependent dielectric constants used in the determination of binding free energies. The accuracy of the method was validated through comparison with experimental data, considering the per-residue P:P binding free energy (ΔΔG binding ) differences upon alanine mutation. Different protocols were tested, i.e., a geometry optimization protocol and three molecular dynamics (MD) protocols: (1) one using explicit water molecules, (2) another with an implicit solvation model, and (3) a third where we have carried out an accelerated MD with explicit water molecules. Using a set of protein dielectric constants (within the range from 1 to 20) we showed that the dielectric constants of 7 for nonpolar and polar residues and 11 for charged residues (and histidine) provide optimal ΔΔG binding predictions. An overall mean unsigned error (MUE) of 1.4 kcal mol -1 relative to the experiment was achieved in 210 mutations only with geometry optimization, which was further reduced with MD simulations (MUE of 1.1 kcal mol -1 for the MD employing explicit solvent). This recalibrated method allows for a better computational identification of hot spots, avoiding expensive and time-consuming experiments or thermodynamic integration/ free energy perturbation/ uBAR calculations, and will hopefully help new drug discovery campaigns in their quest of searching spots of interest for binding small drug-like molecules at P:P interfaces.

Simultaneous effects of temperature and pressure on the donor binding energy in a V-groove quantum wire

NASA Astrophysics Data System (ADS)

Khordad, R.

2010-03-01

The influence of temperature and pressure, simultaneously, on the binding energy of a hydrogenic donor impurity in a ridge GaAs/Ga 1- xAl xAs quantum wire is studied using a variational procedure within the effective mass approximation. The subband energy and the binding energy of the donor impurity in its ground state as a function of the wire bend width and impurity location at different temperatures and pressures are calculated. The results show that, when the temperature increases, the donor binding energy decreases for a constant applied pressure for all wire bend widths. Also, the binding energy increases by increasing the pressure for a constant temperature for all wire bend widths. In addition, when the temperature and pressure are applied simultaneously the binding energy decreases as the quantum wire bend width increases. On the whole, it is deduced that the temperature and pressure have important effects on the donor binding energy in a V-groove quantum wire.

Flexible Acyclic Polyol-Chloride Anion Complexes and Their Characterization by Photoelectron Spectroscopy and Variable Temperature Binding Constant Determinations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shokri, Alireza; Wang, Xue B.; Wang, Yangping

2016-03-17

Flexible acyclic alcohols with 1–5 hydroxyl groups were bound to chloride anion and these complexes were interrogated by negative ion photoelectron spectroscopy and companion density functional theory computations. The resulting vertical detachment energies are reproduced on average to 0.10 eV by M06-2X/aug-cc-pVTZ predictions and range from 4.45 – 5.96 eV. These values are 0.84 – 2.35 eV larger than the adiabatic detachment energy of Cl– as a result of the larger hydrogen bond networks in the bigger polyols. Adiabatic detachment energies of the alcohol–Cl– clusters are more difficult to determine both experimentally and computationally. This is due to the largemore » geometry changes that occur upon photodetachment and the large bond dissociation energy of H–Cl which enables the resulting chlorine atom to abstract a hydrogen from any of the methylene (CH2) or methine (CH) positions. Both ionic and non-ionic hydrogen bonds (i.e., OH•••Cl– and OH•••OH•••Cl–) form in the larger polyols complexes, and are found to be energetically comparable. Subtle structural differences, consequently can lead to the formation of different types of hydrogen bonds and maximizing the ionic ones is not always preferred. Solution equilibrium binding constants between the alcohols and tetrrabuylammonium chloride (TBACl) in acetonitrile at -24.2, 22.0, and 53.6 °C were also determined. The free energies of association are nearly identical for all of the substrates (i.e., ΔG° = -2.8 ± 0.7 kcal mol–1). Compensating enthalpy and entropy values reveal, contrary to expectation and the intrinsic gas-phase preferences, that the bigger systems with more hydroxyl groups are entropically favored and enthalpically disfavored relative to the smaller species. This suggests that more solvent molecules are released upon binding TBACl to alcohols with more hydroxyl groups and is consistent with the measured negative heat capacities. These quantities increase with molecular complexity of the substrate, however, contrary to common interpretation of these values.« less

Rational design of biaryl pharmacophore inserted noscapine derivatives as potent tubulin binding anticancer agents.

PubMed

Santoshi, Seneha; Manchukonda, Naresh Kumar; Suri, Charu; Sharma, Manya; Sridhar, Balasubramanian; Joseph, Silja; Lopus, Manu; Kantevari, Srinivas; Baitharu, Iswar; Naik, Pradeep Kumar

2015-03-01

We have strategically designed a series of noscapine derivatives by inserting biaryl pharmacophore (a major structural constituent of many of the microtubule-targeting natural anticancer compounds) onto the scaffold structure of noscapine. Molecular interaction of these derivatives with α,β-tubulin heterodimer was investigated by molecular docking, molecular dynamics simulation, and binding free energy calculation. The predictive binding affinity indicates that the newly designed noscapinoids bind to tubulin with a greater affinity. The predictive binding free energy (ΔG(bind, pred)) of these derivatives (ranging from -5.568 to -5.970 kcal/mol) based on linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model showed improved binding affinity with tubulin compared to the lead compound, natural α-noscapine (-5.505 kcal/mol). Guided by the computational findings, these new biaryl type α-noscapine congeners were synthesized from 9-bromo-α-noscapine using optimized Suzuki reaction conditions for further experimental evaluation. The derivatives showed improved inhibition of the proliferation of human breast cancer cells (MCF-7), human cervical cancer cells (HeLa) and human lung adenocarcinoma cells (A549), compared to natural noscapine. The cell cycle analysis in MCF-7 further revealed that these compounds alter the cell cycle profile and cause mitotic arrest at G2/M phase more strongly than noscapine. Tubulin binding assay revealed higher binding affinity to tubulin, as suggested by dissociation constant (Kd) of 126 ± 5.0 µM for 5a, 107 ± 5.0 µM for 5c, 70 ± 4.0 µM for 5d, and 68 ± 6.0 µM for 5e compared to noscapine (Kd of 152 ± 1.0 µM). In fact, the experimentally determined value of ΔG(bind, expt) (calculated from the Kd value) are consistent with the predicted value of ΔG(bind, pred) calculated based on LIE-SGB. Based on these results, one of the derivative 5e of this series was used for further toxicological evaluation. Treatment of mice with a daily dose of 300 mg/kg and a single dose of 600 mg/kg indicates that the compound does not induce detectable pathological abnormalities in normal tissues. Also there were no significant differences in hematological parameters between the treated and untreated groups. Hence, the newly designed noscapinoid, 5e is an orally bioavailable, safe and effective anticancer agent with a potential for the treatment of cancer and might be a candidate for clinical evaluation.

Preclinical characterization of anticancer gallium(III) complexes: solubility, stability, lipophilicity and binding to serum proteins.

PubMed

Rudnev, Alexander V; Foteeva, Lidia S; Kowol, Christian; Berger, Roland; Jakupec, Michael A; Arion, Vladimir B; Timerbaev, Andrei R; Keppler, Bernhard K

2006-11-01

The discovery and development of gallium(III) complexes capable of inhibiting tumor growth is an emerging area of anticancer drug research. A range of novel gallium coordination compounds with established cytotoxic efficacy have been characterized in terms of desirable chemical and biochemical properties and compared with tris(8-quinolinolato)gallium(III) (KP46), a lead anticancer gallium-based candidate that successfully finished phase I clinical trials (under the name FFC11), showing activity against renal cell cancer. In view of probable oral administration, drug-like parameters, such as solubility in water, saline and 0.5% dimethyl sulfoxide, stability against hydrolysis, measured as the rate constant of hydrolytic degradation in water or physiological buffer using a capillary zone electrophoresis (CZE) assay, and the octanol-water partition coefficient (logP) providing a rational estimate of a drug's lipophilicity, have been evaluated and compared. The differences in bioavailability characteristics between different complexes were discussed within the formalism of structure-activity relationships. The reactivity toward major serum transport proteins, albumin and transferrin, was also assayed in order to elucidate the drug's distribution pathway after intestinal absorption. According to the values of apparent binding rate constants determined by CZE, both KP46 and bis(2-acetylpyridine-4,4-dimethyl-3-thiosemicarbazonato-N,N,S)gallium(III) tetrachlorogallate(III) (KP1089) bind to transferrin faster than to albumin. This implies that transferrin would rather mediate the accumulation of gallium antineoplastic agents in solid tumors. A tendency of being faster converted into the protein-bound form found for KP1089 (due possibly to non-covalent binding) seems complementary to its greater in vitro antiproliferative activity.

Investigations on the Interactions of 5-Fluorouracil with Herring Sperm DNA: Steady State/Time Resolved and Molecular Modeling Studies

NASA Astrophysics Data System (ADS)

Chinnathambi, Shanmugavel; Karthikeyan, Subramani; Velmurugan, Devadasan; Hanagata, Nobutaka; Aruna, Prakasarao; Ganesan, Singaravelu

2015-04-01

In the present study, the interaction of 5-Fluorouracil with herring sperm DNA is reported using spectroscopic and molecular modeling techniques. This binding study of 5-FU with hs-DNA is of paramount importance in understanding chemico-biological interactions for drug design, pharmacy and biochemistry without altering the original structure. The challenge of the study was to find the exact binding mode of the drug 5-Fluorouracil with hs-DNA. From the absorption studies, a hyperchromic effect was observed for the herring sperm DNA in the presence of 5-Fluorouracil and a binding constant of 6.153 × 103 M-1 for 5-Fluorouracil reveals the existence of weak interaction between the 5-Fluorouracil and herring sperm DNA. Ethidium bromide loaded herring sperm DNA showed a quenching in the fluorescence intensity after the addition of 5-Fluorouracil. The binding constants for 5-Fluorouracil stranded DNA and competitive bindings of 5-FU interacting with DNA-EB systems were examined by fluorescence spectra. The Stern-Volmer plots and fluorescence lifetime results confirm the static quenching nature of the drug-DNA complex. The binding constant Kb was 2.5 × 104 L mol-1 and the number of binding sites are 1.17. The 5-FU on DNA system was calculated using double logarithmic plot. From the Forster nonradiative energy transfer study it has been found that the distance of 5-FU from DNA was 4.24 nm. In addition to the spectroscopic results, the molecular modeling studies also revealed the major groove binding as well as the partial intercalation mode of binding between the 5-Fluorouracil and herring sperm DNA. The binding energy and major groove binding as -6.04 kcal mol-1 and -6.31 kcal mol-1 were calculated from the modeling studies. All the testimonies manifested that binding modes between 5-Fluorouracil and DNA were evidenced to be groove binding and in partial intercalative mode.

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

PubMed

Qian, Cheng; Kovalchik, Kevin A; MacLennan, Matthew S; Huang, Xiaohua; Chen, David D Y

2017-06-01

Capillary electrophoresis frontal analysis (CE-FA) can be used to determine binding affinity of molecular interactions. However, its current data processing method mandate specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. This work shows that significant errors are resulted when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real word applications becomes questionable. An electrophoretic mobility-based correction method is developed in this work based on the flux of each species. A simulation program and a pair of model compounds are used to verify the new equations and evaluate the effectiveness of this method. Ibuprofen and hydroxypropyl-β-cyclodextrinare used to demonstrate the differences in the obtained binding constant by CE-FA when different calculation methods are used, and the results are compared with those obtained by affinity capillary electrophoresis (ACE). The results suggest that CE-FA, with the mobility-based correction method, can be a generally applicable method for a much wider range of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of dimethyl sulfoxide, temperature, and sodium chloride on the activity of human matrix metalloproteinase 7 (matrilysin).

PubMed

Oneda, H; Inouye, K

2000-11-01

Effects of dimethyl sulfoxide (DMSO), temperature, and sodium chloride on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2, 4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. DMSO inhibited the matrilysin activity competitively with the inhibitor constant (K(i)) of 0. 59+/-0.04 M, and the binding between them was endothermic and entropy-driven. The binding of matrilysin with MOCAc-PLGL(Dpa)AR was also found to be entropy-driven. The matrilysin activity was increased in a biphasic exponential fashion with increasing concentration of NaCl, and was 5.3 times higher in the presence of 4 M NaCl than that in its absence. The first and second phases were separated at 0.5 M NaCl, and the activation at x M NaCl compared with the activity in the absence of NaCl was expressed as 2.1(x) at [NaCl] < 0.5 M and 1.4(x) at [NaCl] > 0.5 M. The activation was brought about solely through a decrease in the Michaelis constant (K(m)), and the catalytic constant (k(cat)) was not much altered. This suggests that the decrease in the electrostatic interaction and the increase in the hydrophobic interaction between matrilysin and the substrate might enhance the enzyme activity by reducing the K(m) value.

Binding and thermodynamics of REV peptide-ctDNA interaction.

PubMed

Upadhyay, Santosh Kumar

2017-03-01

The thermodynamics of DNA-ligand binding is important as it provides useful information to understand the details of binding processes. HIV-1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV-1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV-1 were investigated using spectroscopic (UV-visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a T m of 75.5 °C, whereas the ctDNA-REV peptide complex exhibited an incremental shift in the T m by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive B→Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr-bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA-REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K-308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low ΔH values indicating that the ctDNA-REV peptide interaction is driven largely by entropy. The heat capacity change (ΔC p ) was insignificant, an unusual finding in the area of DNA-peptide interaction studies. The variation in the values obtained for ΔH, ΔS, and ΔG with temperature further suggests that ctDNA-REV peptide interaction is entropically driven. ITC based analysis of salt dependence of binding constant gave a charge value (Z) = +4.01, as determined for the δlnK/δln[Na + ] parameter, suggesting the participation of only 3-4 Arg out of 11 Arg charge from REV peptide. The stoichiometry observed for the complex was three molar charge of REV peptide binding per molar charge of ctDNA. ITC based analysis further confirmed that the binding between ctDNA and REV peptide is governed by electrostatic interaction. Molecular interactions including H-bonding, van der Waals forces, and solvent molecules rearrangement, underlie the binding of REV peptide to ctDNA. © 2016 Wiley Periodicals, Inc.

Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.

PubMed

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki; Aoki, Kazuhiro

2014-09-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular features of nonionic detergents involved in the binding kinetics and solubilization efficiency, as studied in model (Langmuir films) and biological (Erythrocytes) membranes.

PubMed

Casadei, Bruna Renata; Domingues, Cleyton Crepaldi; Clop, Eduardo M; Couto, Verônica Muniz; Perillo, Maria Angelica; de Paula, Eneida

2018-06-01

The effect of the nonionic detergents Brij-98 and Brij-58 over human erythrocytes was studied through quantitative hemolysis and in Langmuir films. Hemolytic tests revealed that Brijs are stronger membrane solubilizers than Triton X-100 (TX-100), with effective detergent/lipid ratios of 0.18 and 0.37 for Brij-98 and Brij-58, respectively. Experiments with Langmuir films provided significant information on the kinetics and thermodynamics of detergent-membrane interaction. The adsorption (k a ) and desorption (k d ) rate constants of Brijs were lower than those of TX-100. In the case of k a , that is probably due to their larger hydrophilic head (with twice (20) the oxyethylene units of TX-100). As for the thermodynamic binding constant, the linear and longer hydrophobic acyl chains of Brijs favor their stabilization in-between the lipids, through London van der Waals forces. Consequently, K b,m values of Brij-98 (12,500 M -1 ) and Brij-58 (19,300 M -1 ) resulted higher than TX-100 (7500 M -1 ), in agreement with results from the hemolytic tests. Furthermore, Brij-58 binds with higher affinity than Brij-98 to bilayers and monolayers, despite its shorter (palmitic) hydrocarbon chain, showing that unsaturation restrains the detergent insertion into these environments. Our results provide significant information about the mechanism of interaction between Brijs and membranes, supporting their distinct solubilization effect. Copyright © 2018 Elsevier B.V. All rights reserved.

Label-Free Determination of Protein Binding in Aqueous Solution using Overlayer Enhanced Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (OE-ATR-FTIR)

NASA Astrophysics Data System (ADS)

Ruthenburg, Travis; Aweda, Tolulope; Park, Simon; Meares, Claude; Land, Donald

2009-03-01

Protein binding/affinity studies are often performed using Surface Plasmon Resonance techniques that don't produce much spectral information. Measurement of protein binding affinity using FTIR is traditionally performed using high protein concentration or deuterated solvent. By immobilizing a protein near the surface of a gold-coated germanium internal reflection element interactions can be measured between an immobilized protein and free proteins or small molecules in aqueous solution. By monitoring the on and off rates of these interactions, the dissociation constant for the system can be determined. The dissociation constant for the molecule Yttrium-DOTA binding to the antibody 2D12.5 system was determined to be 100nM. Results will also be presented from our measurements of Bovine Serum Albumin (BSA) binding to anti-BSA.

A Receptor-targeted Fluorescent Radiopharmaceutical for Multireporter Sentinel Lymph Node Imaging

PubMed Central

Emerson, Derek K.; Limmer, Karl K.; Hall, David J.; Han, Sung-Ho; Eckelman, William C.; Kane, Christopher J.; Wallace, Anne M.

2012-01-01

Purpose: To determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection. Materials and Methods: The animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m (99mTc) -labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of 99mTc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of 99mTc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test. Results: In vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L ± 0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6 × 103 counts · sec−1 · μW−1 per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P = .005 to P = .042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation. Conclusion: The multireporter probe 99mTc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging. © RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120638/-/DC1 PMID:22753678

Effect of medium acidity on the thermodynamics and kinetics of the reaction of pyridoxal 5'-phosphate with isoniazid in an aqueous solution

NASA Astrophysics Data System (ADS)

Gamov, G. A.; Zavalishin, M. N.; Usacheva, T. R.; Sharnin, V. A.

2017-05-01

Thermodynamic characteristics of the formation of the Schiff base between isoniazid and pyridoxal 5'-phosphate in an aqueous solution at different pH values of a medium are determined by means of spectrophotometry and calorimetric titration. The process kinetics is studied spectrophotometrically, and the reaction rate constants for the formation of the imine at different acidities of a medium are determined. Biochemical aspects of the binding of pyridoxal 5'-phosphate into stable compounds are discussed.

Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

NASA Astrophysics Data System (ADS)

D'Aquino, J. Alejandro; Ringe, Dagmar

2006-08-01

The diphtheria toxin repressor, DtxR, is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear (1 - 3). Calorimetric techniques have demonstrated that while binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 × 10-7, binding site 2 (primary) is a low affinity binding site with a binding constant of 6.3 × 10-4. These two binding sites act independently and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here and the previously reported DtxR(H79A) (4) has allowed us to propose a mechanism of metal ion activation for DtxR.

Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10

PubMed Central

Russ, Ulrich; Metzger, Friedrich; Kickenweiz, Elisabeth; Hambrock, Annette; Krippeit-Drews, Peter; Quast, Ulrich

1997-01-01

The ATP-sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]-P1075, and by electrophysiological techniques. Saturation binding experiments gave a KD value of 9.2±5.2 nM and a binding capacity (BMax) of 140±40 fmol mg−1 protein for [3H]-P1075 binding to A10 cells; from the BMax value a density of binding sites of 5–10 per μm2 plasmalemma was estimated. KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. Resting membrane potential, recorded with microelectrodes, was −51±1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to −25 mV with EC50 values of 170±40 nM and 870±190 nM, respectively. The hyperpolarization induced by levcromakalim (3 μM) was completely reversed by glibenclamide with an IC50 value of 86±17 nM. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 μM) induced a current which reversed around −80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 μM) abolished the effect of levcromakalim. Analysis of the noise of the levcromakalim (10 μM)-induced current at −40 and −20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 μm−2, 8.8 pS and 0.39, respectively. The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells. PMID:9401776

Constrained Surface Complexation Modeling: Rutile in RbCl, NaCl, and NaCF 3SO 3 Media to 250 °C

DOE PAGES

Machesky, Michael L.; Předota, Milan; Ridley, Moira K.; ...

2015-06-01

In this paper, a comprehensive set of molecular-level results, primarily from classical molecular dynamics (CMD) simulations, are used to constrain CD-MUSIC surface complexation model (SCM) parameters describing rutile powder titrations conducted in RbCl, NaCl, and NaTr (Tr = triflate, CF 3SO 3 –) electrolyte media from 25 to 250 °C. Rb + primarily occupies the innermost tetradentate binding site on the rutile (110) surface at all temperatures (25, 150, 250 °C) and negative charge conditions (-0.1 and -0.2 C/m 2) probed via CMD simulations, reflecting the small hydration energy of this large, monovalent cation. Consequently, variable SCM parameters (Stern-layer capacitancemore » values and intrinsic Rb + binding constants) were adjusted relatively easily to satisfactorily match the CMD and titration data. The larger hydration energy of Na + results in a more complex inner-sphere distribution, which shifts from bidentate to tetradentate binding with increasing negative charge and temperature, and this distribution was not matched well for both negative charge conditions, which may reflect limitations in the CMD and/or SCM approaches. Finally, in particular, the CMD axial density profiles for Rb + and Na + reveal that peak binding distances shift toward the surface with increasing negative charge, suggesting that the CD-MUSIC framework may be improved by incorporating CD or Stern-layer capacitance values that vary with charge.« less

Constrained Surface Complexation Modeling: Rutile in RbCl, NaCl, and NaCF 3SO 3 Media to 250 °C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Machesky, Michael L.; Předota, Milan; Ridley, Moira K.

In this paper, a comprehensive set of molecular-level results, primarily from classical molecular dynamics (CMD) simulations, are used to constrain CD-MUSIC surface complexation model (SCM) parameters describing rutile powder titrations conducted in RbCl, NaCl, and NaTr (Tr = triflate, CF 3SO 3 –) electrolyte media from 25 to 250 °C. Rb + primarily occupies the innermost tetradentate binding site on the rutile (110) surface at all temperatures (25, 150, 250 °C) and negative charge conditions (-0.1 and -0.2 C/m 2) probed via CMD simulations, reflecting the small hydration energy of this large, monovalent cation. Consequently, variable SCM parameters (Stern-layer capacitancemore » values and intrinsic Rb + binding constants) were adjusted relatively easily to satisfactorily match the CMD and titration data. The larger hydration energy of Na + results in a more complex inner-sphere distribution, which shifts from bidentate to tetradentate binding with increasing negative charge and temperature, and this distribution was not matched well for both negative charge conditions, which may reflect limitations in the CMD and/or SCM approaches. Finally, in particular, the CMD axial density profiles for Rb + and Na + reveal that peak binding distances shift toward the surface with increasing negative charge, suggesting that the CD-MUSIC framework may be improved by incorporating CD or Stern-layer capacitance values that vary with charge.« less

Bovine serum albumin binding study to erlotinib using surface plasmon resonance and molecular docking methods.

PubMed

Taghipour, Parvin; Zakariazadeh, Mostafa; Sharifi, Maryam; Ezzati Nazhad Dolatabadi, Jafar; Barzegar, Abolfazl

2018-06-01

Bovine serum albumin (BSA) is the most abundant protein in the blood circulation and it is commonly used for drug delivery in blood. Therefore, we aim to study BSA interaction with erlotinib as an anticancer drug using surface plasmon resonance (SPR) and molecular modeling methods under physiological conditions (pH = 7.4). BSA immobilized on carboxymethyl dextran hydrogel Au chip (CMD) after activation with N-hydroxysuccinimide and N-ethyl-N-(3-diethylaminopropyl) carbodiimide and then the erlotinib binding to BSA at different concentrations was evaluated. Increasing of erlotinib concentration led to dose-response sensorgrams of BSA. The amount of equilibrium constant (K D ) at 25 °C (4.25 × 10 -9 ) showed the high affinity of erlotinib to BSA. Thermodynamic parameters were attained at four different temperatures. The positive value of enthalpy and entropy showed that hydrophobic forces play major role in the interaction of erlotinib with BSA. Besides, the positive value of Gibbs free energy demonstrated that the interaction of erlotinib with BSA was nonspontaneous and enthalpy driven and the complexion of drug were dependent on endothermic process. According to the molecular docking study, the most favorable binding sites of erlotinib on the BSA were subdomain IIIA and IB. Moreover, molecular docking study results showed that hydrogen binding has a role in intermolecular force that stabilize erlotinib-BSA complex. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

PubMed

Beeram, Sandya R; Zheng, Xiwei; Suh, Kyungah; Hage, David S

2018-03-03

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha 1 -acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered. Copyright © 2018 Elsevier Inc. All rights reserved.

Metal binding stoichiometry and isotherm choice in biosorption

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schiewer, S.; Wong, M.H.

1999-11-01

Seaweeds that possess a high metal binding capacity may be used as biosorbents for the removal of toxic heavy metals from wastewater. The binding of Cu and Ni by three brown algae (Sargassum, Colpomenia, Petalonia) and one green alga (Ulva) was investigated at pH 4.0 and pH 3.0. The greater binding strength of Cu is reflected in a binding constant that is about 10 times as high as that of Ni. The extent of metal binding followed the order Petalonia {approximately} Sargassum > Colpomenia > Ulva. This was caused by a decreasing number of binding sites and by much lowermore » metal binding constants for Ulva as compared to the brown algae. Three different stoichiometric assumptions are compared for describing the metal binding, which assume either that each metal ion M binds to one binding site B forming a BM complex or that a divalent metal ion M binds to two monovalent sites B forming BM{sub 0.5} or B{sub 2}M complexes, respectively. Stoichiometry plots are proposed as tools to discern the relevant binding stoichiometry. The pH effect in metal binding and the change in proton binding were well predicted for the B{sub 2}M or BM{sub 0.5} stoichiometries with the former being better for Cu and the latter preferable for Ni. Overall, the BM{sub 0.5} model is recommended because it avoids iterations.« less

Spectroscopic studies on the interaction of a water-soluble cationic porphyrin with proteins

NASA Astrophysics Data System (ADS)

Ma, Hong-Min; Chen, Xin; Zhang, Nuo; Han, Yan-Yan; Wu, Dan; Du, Bin; Wei, Qin

2009-04-01

The interaction of a water-soluble cationic porphyrin, meso-tetrakis (4- N, N, N-trimethylanilinium) porphyrin (TMAP), with two proteins, bovine serum albumin (BSA) and human serum albumin (HSA), was studied by UV-vis absorption spectroscopy, fluorescence spectroscopy, fluorescence anisotropy and synchronous fluorescence spectroscopy at neutral aqueous solutions. Free base TMAP bound to proteins as monomers and no aggregation was observed. The binding of TMAP quenched the fluorescence of the protein. On the contrary, the fluorescence of TMAP was enhanced and the fluorescence anisotropy increased due to the binding. The direct static binding mechanism could account for the quenching by TMAP and the binding constants were calculated. TMAP showed a higher quenching efficiency and binding constant of HSA than BSA. The binding of TMAP had no obvious effect on the molecular conformation of the protein. There was only one binding site for TMAP and it was located on the surface of the protein molecule. Electrostatic force played an important role in the binding due to the opposite charges on porphyrin and the proteins.

Spectroscopic studies on the interaction of a water-soluble cationic porphyrin with proteins.

PubMed

Ma, Hong-Min; Chen, Xin; Zhang, Nuo; Han, Yan-Yan; Wu, Dan; Du, Bin; Wei, Qin

2009-04-01

The interaction of a water-soluble cationic porphyrin, meso-tetrakis (4-N,N,N-trimethylanilinium) porphyrin (TMAP), with two proteins, bovine serum albumin (BSA) and human serum albumin (HSA), was studied by UV-vis absorption spectroscopy, fluorescence spectroscopy, fluorescence anisotropy and synchronous fluorescence spectroscopy at neutral aqueous solutions. Free base TMAP bound to proteins as monomers and no aggregation was observed. The binding of TMAP quenched the fluorescence of the protein. On the contrary, the fluorescence of TMAP was enhanced and the fluorescence anisotropy increased due to the binding. The direct static binding mechanism could account for the quenching by TMAP and the binding constants were calculated. TMAP showed a higher quenching efficiency and binding constant of HSA than BSA. The binding of TMAP had no obvious effect on the molecular conformation of the protein. There was only one binding site for TMAP and it was located on the surface of the protein molecule. Electrostatic force played an important role in the binding due to the opposite charges on porphyrin and the proteins.

Synthesis and physicochemical characterization of carbon backbone modified [Gd(TTDA)(H2O)]2- derivatives.

PubMed

Chang, Ya-Hui; Chen, Chiao-Yun; Singh, Gyan; Chen, Hsing-Yin; Liu, Gin-Chung; Goan, Yih-Gang; Aime, Silvio; Wang, Yun-Ming

2011-02-21

The present study was designed to exploit optimum lipophilicity and high water-exchange rate (k(ex)) on low molecular weight Gd(III) complexes to generate high bound relaxivity (r(1)(b)), upon binding to the lipophilic site of human serum albumin (HSA). Two new carbon backbone modified TTDA (3,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic acid) derivatives, CB-TTDA and Bz-CB-TTDA, were synthesized. The complexes [Gd(CB-TTDA)(H(2)O)](2-) and [Gd(Bz-CB-TTDA)(H(2)O)](2-) both display high stability constant (log K(GdL) = 20.28 and 20.09, respectively). Furthermore, CB-TTDA (log K(Gd/Zn) = 4.22) and Bz-CB-TTDA (log K(Gd/Zn) = 4.12) exhibit superior selectivity of Gd(III) against Zn(II) than those of TTDA (log K(Gd/Zn) = 2.93), EPTPA-bz-NO(2) (log K(Gd/Zn) = 3.19), and DTPA (log K(Gd/Zn) = 3.76). However, the stability constant values of [Gd(CB-TTDA)(H(2)O)](2-) and [Gd(Bz-CB-TTDA)(H(2)O)](2-) are lower than that of MS-325. The parameters that affect proton relaxivity have been determined in a combined variable temperature (17)O NMR and NMRD study. The water exchange rates are comparable for the two complexes, 232 × 10(6) s(-1) for [Gd(CB-TTDA)(H(2)O)](2-) and 271 × 10(6) s(-1) for [Gd(Bz-CB-TTDA)(H(2)O)](2-). They are higher than those of [Gd(TTDA)(H(2)O)](2-) (146 × 10(6) s(-1)), [Gd(DTPA)(H(2)O)](2-) (4.1 × 10(6) s(-1)), and MS-325 (6.1 × 10(6) s(-1)). Elevated stability and water exchange rate indicate that the presence of cyclobutyl on the carbon backbone imparts rigidity and steric constraint to [Gd(CB-TTDA)(H(2)O)](2-)and [Gd(Bz-CB-TTDA)(H(2)O)](2-). In addition, the major objective for selecting the cyclobutyl is to tune the lipophilicity of [Gd(Bz-CB-TTDA)(H(2)O)](2-). The binding affinity of [Gd(Bz-CB-TTDA)(H(2)O)](2-) to HSA was evaluated by ultrafiltration study across a membrane with a 30 kDa MW cutoff, and the first three stepwise binding constants were determined by fitting the data to a stoichiometric model. The binding association constants (K(A)) for [Gd(CB-TTDA)(H(2)O)](2-) and [Gd(Bz-CB-TTDA)(H(2)O)](2-) are 1.1 × 10(2) and 1.5 × 10(3), respectively. Although the K(A) value for [Gd(Bz-CB-TTDA)(H(2)O)](2-) is lower than that of MS-325 (K(A) = 3.0 × 10(4)), the r(1)(b) value, r(1)(b) = 66.7 mM(-1) s(-1) for [Gd(Bz-CB-TTDA)(H(2)O)](2-), is significantly higher than that of MS-325 (r(1)(b) = 47.0 mM(-1) s(-1)). As measured by the Zn(II) transmetalation process, the kinetic stabilities of [Gd(CB-TTDA)(H(2)O)](2-), [Gd(Bz-CB-TTDA)(H(2)O)](2-), and [Gd(DTPA)(H(2)O)](2-) are similar and are significantly higher than that of [Gd(DTPA-BMA)(H(2)O)](2-). High thermodynamic and kinetic stability and optimized lipophilicity of [Gd(CB-TTDA)(H(2)O)](2-) make it a favorable blood pool contrast agent for MRI.

Conformation change of tRNA/sub Glu/ in the complex with glutamyl-tRNA synthetase is required for the specific binding of L-glutamate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara-Yokoyama, M.; Yokoyama, S.; Miyazawa, T.

1986-11-04

The binding of Thermus thermophilus glutamyl-tRNA synthetase (GluRS) with T. thermophilus tRNA/sup Glu/, Escherichia coli tRNA/sup Glu/, and amino acids was studied by fluorescence measurements. In the absence of tRNA/sup Glu/, GluRS binds with D-glutamate as well as L-glutamate. However, in the presence of E.coli tRNA/sup Glu/, GluRS binds specifically with L-glutamate. The KCl effects on the Michaelis constants (K/sub m/) for tRNA/sup Glu/, L-glutamate, and ATP were studied for the aminoacylation of the homologous tRNA/sup Glu/ and heterologous tRNA/sup Glu/ species. As the KCl concentration is raised from 0 to 100 mM, the K/sub m/ value for L-glutamate inmore » the heterologous system is remarkably increased whereas the K/sub m/ value for L-glutamate in the homologous system is only slightly increased. The circular dichroism analyses were made mainly of the bands due to the 2-thiouridine derivatives of tRNA/sup Glu/ in the complex. The conformation change of T. thermophilus tRNA/sup Glu/ upon complex formation with GluRS is not affected by addition of KCl. In contrast, the heterologous tRNA/sup Glu/GluRS complex is in equilibrium of two forms that depends on KCl concentration. The predominant form at low KCl concentration is closely related to the small K/sub m/ value for L-glutamate. In this form of the complex, the conformation of tRNA/sup Glu/ is appreciably different from that of free molecule. Accordingly, such a conformation change of tRNA/sup Glu/ in the complex with GluRS is required for the specific binding of L-glutamate as the substrate.« less

Aptamer-Immobilized Surface Plasmon Resonance Biosensor for Rapid and Sensitive Determination of Virulence Determinant.

PubMed

Song, Myeong-Sub; Sekhon, Simranjeet Singh; Shin, Woo-Ri; Rhee, Sung-Keun; Ko, Jung Ho; Kim, Sang Yong; Min, Jiho; Ahn, Ji-Young; Kim, Yang-Hoon

2018-05-01

Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.

Cytotoxic activity of proflavine diureas: synthesis, antitumor, evaluation and DNA binding properties of 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas.

PubMed

Kozurková, Mária; Sabolová, Danica; Janovec, Ladislav; Mikes, Jaromír; Koval', Ján; Ungvarský, Ján; Stefanisinová, Miroslava; Fedorocko, Peter; Kristian, Pavol; Imrich, Ján

2008-04-01

The synthesis of novel 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas was reported. Their biological activity to inhibit cell proliferation was assessed by a MTT assay on two cell lines, HeLa and HCT-116, at micromolar concentration. 1',1''-(Acridin-3,6-diyl)-3',3''-dihexyldiurea hydrochloride was active on a HCT-116 cell line with an IC(50) value of 3.1 microM. The interaction of these compounds with calf thymus DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence and CD spectroscopy. From spectrofluorimetric titrations, binding constants for the DNA-drug complexes were determined (K=0.9-4.2x10(5) M(-1)). Antiproliferative activity of synthesized derivatives might be related to their intercalation into DNA.

Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease.

PubMed

Li, M; Morris, G M; Lee, T; Laco, G S; Wong, C H; Olson, A J; Elder, J H; Wlodawer, A; Gustchina, A

2000-01-01

Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV-1 PR), were cocrystallized with the C2-symmetric inhibitor, TL-3. The mutants of FIV PR were designed to replace residues involved in enzyme-ligand interactions by the corresponding HIV-1 PR residues at the structurally equivalent position. TL-3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3'), small differences are evident in the conformation of the Phe side chains of TL-3 at the P1 and P1' positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL-3 in HIV-1 PR and correlate with a significant improvement in the inhibition constants of TL-3 with the two mutant FIV PRs. Large differences between the HIV-1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL-3 at P4 and P4'. In HIV-1 PR:TL-3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV-1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4' positions. These findings provide a structural explanation of the observed Ki values for TL-3 with the different PRs and will further assist in the development of improved inhibitors.

Binding of polychlorinated biphenyls to aquatic humic substances: The role of substrate and sorbate properties on partitioning

USGS Publications Warehouse

Uhle, M.E.; Chin, Y.-P.; Aiken, G.R.; McKnight, Diane M.

1999-01-01

Two ortho- (2,2',5 and 2,2',5,6') and a non-ortho- (3,3',4,4') substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho- substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1' carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.Two ortho- (2,2???,5 and 2,2???,5,6???) and a non-ortho- (3,3???,4,4???) substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho-substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1??? carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sawada, Y.; Kawai, R.; McManaway, M.

(3H)Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB);more » estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using (18F)CF and positron emission tomography.« less

Interaction of Antiinflammatory Drugs with EPC Liposomes: Calorimetric Study in a Broad Concentration Range

PubMed Central

Matos, Carla; Lima, José L. C.; Reis, Salette; Lopes, António; Bastos, Margarida

2004-01-01

Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient (Kp), and/or the equilibrium constant (Kb). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio >100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, ΔH, and indirectly, ΔG and ΔS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction—possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug. PMID:14747330

Number of independent parameters in the potentiometric titration of humic substances.

PubMed

Lenoir, Thomas; Manceau, Alain

2010-03-16

With the advent of high-precision automatic titrators operating in pH stat mode, measuring the mass balance of protons in solid-solution mixtures against the pH of natural and synthetic polyelectrolytes is now routine. However, titration curves of complex molecules typically lack obvious inflection points, which complicates their analysis despite the high-precision measurements. The calculation of site densities and median proton affinity constants (pK) from such data can lead to considerable covariance between fit parameters. Knowing the number of independent parameters that can be freely varied during the least-squares minimization of a model fit to titration data is necessary to improve the model's applicability. This number was calculated for natural organic matter by applying principal component analysis (PCA) to a reference data set of 47 independent titration curves from fulvic and humic acids measured at I = 0.1 M. The complete data set was reconstructed statistically from pH 3.5 to 9.8 with only six parameters, compared to seven or eight generally adjusted with common semi-empirical speciation models for organic matter, and explains correlations that occur with the higher number of parameters. Existing proton-binding models are not necessarily overparametrized, but instead titration data lack the sensitivity needed to quantify the full set of binding properties of humic materials. Model-independent conditional pK values can be obtained directly from the derivative of titration data, and this approach is the most conservative. The apparent proton-binding constants of the 23 fulvic acids (FA) and 24 humic acids (HA) derived from a high-quality polynomial parametrization of the data set are pK(H,COOH)(FA) = 4.18 +/- 0.21, pK(H,Ph-OH)(FA) = 9.29 +/- 0.33, pK(H,COOH)(HA) = 4.49 +/- 0.18, and pK(H,Ph-OH)(HA) = 9.29 +/- 0.38. Their values at other ionic strengths are more reliably calculated with the empirical Davies equation than any existing model fit.

Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Deepti; Rawat, Surender; Waseem, Mohd

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, K{sub m} values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative propertymore » against BSA and Cu{sup 2+}/H{sub 2}O{sub 2} model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies. - Highlights: • Laccase from Yersinia enterocolitica strain 7 was expressed in Escherichia coli BL21 (DE3). • Recombinant laccase was found to be thermostable and alkali tolerant. • The in silico and experimental studied proves the biotransformation of NSAIDs. • Laccase binds to ligands differentially under different protonation state. • Laccase also possesses free radical scavenging property.« less

Characterization of Cyclodextrin/Volatile Inclusion Complexes: A Review.

PubMed

Kfoury, Miriana; Landy, David; Fourmentin, Sophie

2018-05-17

Cyclodextrins (CDs) are a family of cyclic oligosaccharides that constitute one of the most widely used molecular hosts in supramolecular chemistry. Encapsulation in the hydrophobic cavity of CDs positively affects the physical and chemical characteristics of the guests upon the formation of inclusion complexes. Such a property is interestingly employed to retain volatile guests and reduce their volatility. Within this scope, the starting crucial point for a suitable and careful characterization of an inclusion complex is to assess the value of the formation constant (K f ), also called stability or binding constant. This task requires the application of the appropriate analytical method and technique. Thus, the aim of the present paper is to give a general overview of the main analytical tools used for the determination of K f values for CD/volatile inclusion complexes. This review emphasizes on the advantages, inconvenients and limits of each applied method. A special attention is also dedicated to the improvement of the current methods and to the development of new techniques. Further, the applicability of each technique is illustrated by a summary of data obtained from the literature.

Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: inhibition kinetics and docking studies.

PubMed

de Sousa, Lorena Ramos Freitas; Wu, Hongmei; Nebo, Liliane; Fernandes, João Batista; da Silva, Maria Fátima das Graças Fernandes; Kiefer, Werner; Kanitz, Manuel; Bodem, Jochen; Diederich, Wibke E; Schirmeister, Tanja; Vieira, Paulo Cezar

2015-02-01

NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20 μM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Extracting ligands from receptors by reversed targeted molecular dynamics.

PubMed

Wolf, Romain M

2015-11-01

Short targeted MD trajectories are used to expel ligands from binding sites. The expulsion is governed by a linear increase of the target RMSD value, growing from zero to an arbitrary chosen final RMSD that forces the ligand to a selected distance outside of the receptor. The RMSD lag (i.e., the difference between the imposed and the actual RMSD) can be used to follow barriers encountered by the ligand during its way out of the receptor. The force constant used for the targeted MD can transform the RMSD lag into a strain energy. Integration of the (time-dependent) strain energy over time yields a value with the dimensions of "action" (i.e, energy multiplied by time) and can serve as a measure for the overall effort required to extract the ligand from its binding site. Possibilities to compare (numerically and graphically) the randomly detected exit pathways are discussed. As an example, the method is tested on the exit of bisphenol A from the human estrogen-related receptor [Formula: see text] and of GW0072 from the peroxysome proliferator activated receptor.

Calcium-buffering effects of gluconate and nucleotides, as determined by a novel fluorimetric titration method.

PubMed

Woehler, Andrew; Lin, Kun-Han; Neher, Erwin

2014-11-15

Significantly more Ca(2+) influx is required for eliciting release of neurotransmitter during whole cell patch clamp recording in the Calyx of Held, when gluconate with 3 mm free ATP is used as pipette filling solution, as compared to a methanesulfonate-based solution with excess Mg(2+). This reduction in efficiency of Ca(2+) in eliciting release is due to low-affinity Ca(2+) binding of both gluconate and ATP(2-) anions. To study these effects we developed a simple fluorimeteric titration procedure, which reports the dissociation constant, KD, of a given Ca(2+) indicator dye, multiplied by 1 plus the sum of Ca(2+) binding ratios of any anions, which act as low-affinity Ca(2+) ligands. For solutions without Ca(2+) binding anions we find KD values for Fura2FF ranging from 11.5 ± 1.7 to 15.6 ± 7.47 μm depending on the dominant anion used. For Fura6F and KCl-based solutions we find KD = 17.8 ± 1.3 μm. For solutions with gluconate as the main anion and for solutions that contain nucleotides, such as ATP and GTP, we find much higher values for the product. Assuming that the KD of the indicator dye is equal to that of KCl-based solutions we calculate the summed Ca(2+) binding ratios and find a value of 3.55 for a solution containing 100 mm potassium gluconate and 4 mm ATP. Gluconate contributes a value of 1.75 to this number, while the contribution of ATP depends strongly on the presence of Mg(2+) and varies from 0.8 (with excess Mg(2+)) to 13.8 (in the presence of 3 mm free ATP). Methanesulfonate has negligible Ca(2+) binding capacity. These results explain the reduced efficiency of Ca(2+) influx in the presence of gluconate or nucleotides, as these anions are expected to intercept Ca(2+) ions at short distance. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Probing the interaction of human serum albumin with DPPH in the absence and presence of the eight antioxidants

NASA Astrophysics Data System (ADS)

Li, Xiangrong; Chen, Dejun; Wang, Gongke; Lu, Yan

2015-02-01

Albumin represents a very abundant and important circulating antioxidant in plasma. DPPH radical is also called 2,2-diphenyl-1-picrylhydrazyl. It has been widely used for measuring the efficiency of antioxidants. In this paper, the ability of human serum albumin (HSA) to scavenge DPPH radical was investigated using UV-vis absorption spectra. The interaction between HSA and DPPH was investigated in the absence and presence of eight popular antioxidants using fluorescence spectroscopy. These results indicate the antioxidant activity of HSA against DPPH radical is similar to glutathione and the value of IC50 is 5.200 × 10-5 mol L-1. In addition, the fluorescence experiments indicate the quenching mechanism of HSA, by DPPH, is a static process. The quenching process of DPPH with HSA is easily affected by the eight antioxidants, however, they cannot change the quenching mechanism of DPPH with HSA. The binding of DPPH to HSA primarily takes place in subdomain IIA and exists two classes of binding sites with two different interaction behaviors. The decreased binding constants and the number of binding sites of DPPH with HSA by the introduction of the eight antioxidants may result from the competition of the eight antioxidants and DPPH binding to HSA. The binding of DPPH to HSA may induce the micro-environment of the lone Trp-214 from polar to slightly nonpolar.

Aspartate aminotransferase is potently inhibited by copper complexes: Exploring copper complex-binding proteome.

PubMed

Jia, Yuqi; Lu, Liping; Yuan, Caixia; Feng, Sisi; Zhu, Miaoli

2017-05-01

Recent researches indicated that a copper complex-binding proteome that potently interacted with copper complexes and then influenced cellular metabolism might exist in organism. In order to explore the copper complex-binding proteome, a copper chelating ion-immobilized affinity chromatography (Cu-IMAC) column and mass spectrometry were used to separate and identify putative Cu-binding proteins in primary rat hepatocytes. A total of 97 putative Cu-binding proteins were isolated and identified. Five higher abundance proteins, aspartate aminotransferase (AST), malate dehydrogenase (MDH), catalase (CAT), calreticulin (CRT) and albumin (Alb) were further purified using a SP-, and (or) Q-Sepharose Fast Flow column. The interaction between the purified proteins and selected 11 copper complexes and CuCl 2 was investigated. The enzymes inhibition tests demonstrated that AST was potently inhibited by copper complexes while MDH and CAT were weakly inhibited. Schiff-based copper complexes 6 and 7 potently inhibited AST with the IC 50 value of 3.6 and 7.2μM, respectively and exhibited better selectivity over MDH and CAT. Fluorescence titration results showed the two complexes tightly bound to AST with binding constant of 3.89×10 6 and 3.73×10 6 M -1 , respectively and a stoichiometry ratio of 1:1. Copper complex 6 was able to enter into HepG2 cells and further inhibit intracellular AST activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Adsorption of surfactant ions and binding of their counterions at an air/water interface.

PubMed

Tagashira, Hiroaki; Takata, Youichi; Hyono, Atsushi; Ohshima, Hiroyuki

2009-01-01

An expression for the surface tension of an aqueous mixed solution of surfactants and electrolyte ions in the presence of the common ions was derived from the Helmholtz free energy of an air/water surface. By applying the equation to experimental data for the surface tension, the adsorption constant of surfactant ions onto the air/water interface, the binding constant of counterions on the surfactants, and the surface potential and surface charge density of the interface were estimated. The adsorption constant and binding constant were dependent on the species of surfactant ion and counterion, respectively. Taking account of the dependence of surface potential and surface charge density on the concentration of electrolyte, it was suggested that the addition of electrolyte to the aqueous surfactant solution brings about the decrease in the surface potential, the increase in the surface density of surfactant ions, and consequently, the decrease in the surface tension. Furthermore, it was found that the configurational entropy plays a predominant role for the surface tension, compared to the electrical work.

Integrating a Smartphone and Molecular Modeling for Determining the Binding Constant and Stoichiometry Ratio of the Iron(II)-Phenanthroline Complex: An Activity for Analytical and Physical Chemistry Laboratories

ERIC Educational Resources Information Center

de Morais, Camilo de L. M.; Silva, Se´rgio R. B.; Vieira, Davi S.; Lima, Ka´ssio M. G.

2016-01-01

The binding constant and stoichiometry ratio for the formation of iron(II)-(1,10-phenanthroline) or iron(II)-o-phenanthroline complexes has been determined by a combination of a low-cost analytical method using a smartphone and a molecular modeling method as a laboratory experiment designed for analytical and physical chemistry courses. Intensity…

Multi-spectroscopic and voltammetric evidences for binding, conformational changes of bovine serum albumin with thiamine.

PubMed

Bagoji, Atmanand M; Gowda, Jayant I; Gokavi, Naveen M; Nandibewoor, Sharanappa T

2017-08-01

The interaction between thiamine hydrochloride (TA) and bovine serum albumin (BSA) was investigated by fluorescence, FTIR, UV-vis spectroscopic and cyclic voltammetric techniques under optimised physiological condition. The fluorescence intensity of BSA is gradually decreased upon addition of TA due to the formation of a BSA-TA complex. The binding parameters were evaluated and their behaviour at different temperatures was analysed. The quenching constants (K sv ) obtained were 2.6 × 10 4 , 2.2 × 10 4 and 2.0 × 10 4  L mol -1 at 288, 298 and 308 K, respectively. The binding mechanism was static-type quenching. The values of ΔH° and ΔS° were found to be 26.87 kJ mol -1 and 21.3 J K -1  mol -1 , and indicated that electrostatic interaction was the principal intermolecular force. The changes in the secondary structure of BSA upon interaction with TA were confirmed by synchronous and 3-D spectral results. Site probe studies reveal that TA is located in site I of BSA. The effects of some common metal ions on binding of BSA-TA complex were also investigated.

Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the alpha-chymotrypsin-proflavin interaction.

PubMed

Bruylants, Gilles; Wintjens, René; Looze, Yvan; Redfield, Christina; Bartik, Kristin

2007-12-01

Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K(obs), DeltaH(obs)(0) ). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between alpha-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between alpha-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria.

Interactions of milk α- and β-casein with malvidin-3-O-glucoside and their effects on the stability of grape skin anthocyanin extracts.

PubMed

He, Zhiyong; Xu, Mingzhu; Zeng, Maomao; Qin, Fang; Chen, Jie

2016-05-15

The interactions of α- and β-casein with malvidin-3-O-glucoside (MG), the major anthocyanin in grape skin anthocyanin extracts (GSAE), were examined at pH 6.3 by fluorescence, fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy. The binding constant (KS), binding force and effects of the interactions on the caseins conformation and GSAE stability were investigated. The results showed that α- and β-casein bound with MG via hydrophilic (van der Waals forces or hydrogen bonding) and hydrophobic interactions, respectively. α-Casein had a slightly stronger binding affinity toward MG than β-casein, with respective KS values of 0.51×10(3)M(-1) and 0.46×10(3)M(-1) at 297K. The secondary structures of α- and β-casein were changed by MG binding, with a decrease in α-helix and an increase in turn for α-casein and no change in α-helix and a decrease in turn for β-casein. The casein-anthocyanin interaction appeared to have a positive effect on the thermal, oxidation and photo stability of GSAE. Copyright © 2015 Elsevier Ltd. All rights reserved.

Competitive counterion complexation allows the true host : guest binding constants from a single titration by ionic receptors.

PubMed

Pessêgo, Márcia; Basílio, Nuno; Muñiz, M Carmen; García-Río, Luis

2016-07-06

Counterion competitive complexation is a background process currently ignored by using ionic hosts. Consequently, guest binding constants are strongly affected by the design of the titration experiments in such a way that the results are dependent on the guest concentration and on the presence of added salts, usually buffers. In the present manuscript we show that these experimental difficulties can be overcome by just considering the counterion competitive complexation. Moreover a single titration allows us to obtain not only the true binding constants but also the stoichiometry of the complex showing the formation of 1 : 1 : 1 (host : guest : counterion) complexes. The detection of high stoichiometry complexes is not restricted to a single titration experiment but also to a displacement assay where both competitive and competitive-cooperative complexation models are taken into consideration.

Spectroscopic investigation on the mechanism of formation of molecular complexes of albendazole and trimethoprim with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone

NASA Astrophysics Data System (ADS)

Ganesh, K.; Balraj, C.; Satheshkumar, A.; Elango, K. P.

2012-06-01

UV-vis, 1H NMR, FT-IR, mass and fluorescence spectral techniques were employed to investigate the mechanism of interaction of albendazole and trimethoprim with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and to characterize the reaction products. The interaction of DDQ with trimethoprim (TMP) and albenadazole (ALB) were found to proceed through the formation of donor-acceptor complex, containing DDQ radical anion and its conversion to the product. Fluorescence quenching studies indicated that the interaction between the donors and the acceptor are spontaneous and the interaction of TMP-DDQ (binding constant = 2.9 × 105) is found to be stronger than that the ALB-DDQ (binding constant = 3 × 103) system. Also, the binding constant increased with an increase in polarity of the medium indicating the involvement of radical anion as intermediate.

Fulvic acid-sulfide ion competition for mercury ion binding in the Florida everglades

USGS Publications Warehouse

Reddy, M.M.; Aiken, G.R.

2001-01-01

Negatively charged functional groups of fulvic acid compete with inorganic sulfide ion for mercury ion binding. This competition is evaluated here by using a discrete site-electrostatic model to calculate mercury solution speciation in the presence of fulvic acid. Model calculated species distributions are used to estimate a mercury-fulvic acid apparent binding constant to quantify fulvic acid and sulfide ion competition for dissolved inorganic mercury (Hg(II)) ion binding. Speciation calculations done with PHREEQC, modified to use the estimated mercury-fulvic acid apparent binding constant, suggest that mercury-fulvic acid and mercury-sulfide complex concentrations are equivalent for very low sulfide ion concentrations (about 10-11 M) in Everglades' surface water. Where measurable total sulfide concentration (about 10-7 M or greater) is present in Everglades' surface water, mercury-sulfide complexes should dominate dissolved inorganic mercury solution speciation. In the absence of sulfide ion (for example, in oxygenated Everglades' surface water), fulvic acid binding should dominate Everglades' dissolved inorganic mercury speciation.

Antioxidative capacity and binding affinity of the complex of green tea catechin and beta-lactoglobulin glycated by the Maillard reaction.

PubMed

Perusko, Marija; Al-Hanish, Ayah; Mihailovic, Jelena; Minic, Simeon; Trifunovic, Sara; Prodic, Ivana; Cirkovic Velickovic, Tanja

2017-10-01

Major green tea catechin, epigallocatechin-3-gallate (EGCG), binds non-covalently to numerous dietary proteins, including beta-lactoglobulin of cow's milk. The effects of glycation of proteins via Maillard reaction on the binding capacity for polyphenols and the antiradical properties of the formed complexes have not been studied previously. Binding constant of BLG glycated by milk sugar lactose to EGCG was measured by the method of fluorophore quenching. Binding of EGCG was confirmed by CD and FTIR. The antioxidative properties of the complexes were examined by measuring ABTS radical scavenging capacity, superoxide anion scavenging capacity and total reducing power assay. Glycation of BLG does not significantly influence the binding constant of EGCG for the protein. Conformational changes were observed for both native and glycated BLG upon complexation with EGCG. Masking effect of polyphenol complexation on the antioxidative potential of the protein was of the similar degree for both glycated BLG and native BLG. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protocols Utilizing Constant pH Molecular Dynamics to Compute pH-Dependent Binding Free Energies

PubMed Central

2015-01-01

In protein–ligand binding, the electrostatic environments of the two binding partners may vary significantly in bound and unbound states, which may lead to protonation changes upon binding. In cases where ligand binding results in a net uptake or release of protons, the free energy of binding is pH-dependent. Nevertheless, conventional free energy calculations and molecular docking protocols typically do not rigorously account for changes in protonation that may occur upon ligand binding. To address these shortcomings, we present a simple methodology based on Wyman’s binding polynomial formalism to account for the pH dependence of binding free energies and demonstrate its use on cucurbit[7]uril (CB[7]) host–guest systems. Using constant pH molecular dynamics and a reference binding free energy that is taken either from experiment or from thermodynamic integration computations, the pH-dependent binding free energy is determined. This computational protocol accurately captures the large pKa shifts observed experimentally upon CB[7]:guest association and reproduces experimental binding free energies at different levels of pH. We show that incorrect assignment of fixed protonation states in free energy computations can give errors of >2 kcal/mol in these host–guest systems. Use of the methods presented here avoids such errors, thus suggesting their utility in computing proton-linked binding free energies for protein–ligand complexes. PMID:25134690

Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.

PubMed

Dogan, Jakob; Jonasson, Josefin; Andersson, Eva; Jemth, Per

2015-08-04

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

Study of fluorescence quenching of Barley α-amylase

NASA Astrophysics Data System (ADS)

Bakkialakshmi, S.; Shanthi, B.; Bhuvanapriya, T.

2012-05-01

The fluorescence quenching of Barley α-amylase by acrylamide and succinimide has been studied in water using steady-state and time-resolved fluorescence techniques. The steady-state fluorescence quenching technique has been performed in three different pHs (i.e., 6, 7 and 8) of water. Ground state and excited state binding constants (Kg &Ke) have been calculated. From the calculated binding constants (Kg &Ke) the free energy changes for the ground (ΔGg) and excited (ΔGe) states have been calculated and are presented in tables. UV and FTIR spectra have also been recorded to prove the binding of Barley α-amylase with acrylamide and succinimide.

Quartz crystal microbalance for the cardiac markers/antibodies binding kinetic measurements in the plasma samples

NASA Astrophysics Data System (ADS)

Agafonova, L. E.; Shumyantseva, V. V.; Archakov, A. I.

2014-06-01

The quartz crystal microbalance (QCM) was exploited for cardiac markers detection and kinetic studies of immunochemical reaction of cardiac troponin I (cTnI) and human heart fatty acid binding protein (H-FABP) with the corresponding monoclonal antibodies in undiluted plasma (serum) and standard solutions. The QCM technique allowed to dynamically monitor the kinetic differences in specific interactions and nonspecific sorption, without multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, all of which were obtained from experimental data.

Solution and fluorescence properties of symmetric dipicolylamine-containing dichlorofluorescein-based Zn2+ sensors.

PubMed

Wong, Brian A; Friedle, Simone; Lippard, Stephen J

2009-05-27

The mechanism by which dipicolylamine (DPA) chelate-appended fluorophores respond to zinc was investigated by the synthesis and study of five new analogues of the 2',7'-dichlorofluorescein-based Zn(2+) sensor Zinpyr-1 (ZP1). With the use of absorption and emission spectroscopy in combination with potentiometric titrations, a detailed molecular picture has emerged of the Zn(2+) and H(+) binding properties of the ZP1 family of sensors. The two separate N(3)O donor atom sets on ZP1 converge to form binding pockets in which all four heteroatoms participate in coordination to either Zn(2+) or protons. The position of the pyridyl group nitrogen atom, 2-pyridyl or 4-pyridyl, has a large impact on the fluorescence response of the dyes to protons despite relatively small changes in pK(a) values. The fluorescence quenching effects of such multifunctional electron-donating units are often taken as a whole. Despite the structural complexity of ZP1, however, we provide evidence that the pyridyl arms of the DPA appendages participate in the quenching process, in addition to the contribution from the tertiary nitrogen amine atom. Potentiometric titrations reveal ZP1 dissociation constants (K(d)) for Zn(2+) of 0.04 pM and 1.2 nM for binding to the first and second binding pockets of the ligand, respectively, the second of which correlates with the value observed by fluorescence titration. This result demonstrates that both binding pockets of this symmetric, ditopic sensor need to be occupied in order for full fluorescence turn-on to be achieved. These results have significant implications for the design and implementation of fluorescent sensors for studies of mobile zinc ions in biology.

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

PubMed

Ketelslegers, J M; Catt, K J

1978-07-03

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.

Saturation behavior: a general relationship described by a simple second-order differential equation.

PubMed

Kepner, Gordon R

2010-04-13

The numerous natural phenomena that exhibit saturation behavior, e.g., ligand binding and enzyme kinetics, have been approached, to date, via empirical and particular analyses. This paper presents a mechanism-free, and assumption-free, second-order differential equation, designed only to describe a typical relationship between the variables governing these phenomena. It develops a mathematical model for this relation, based solely on the analysis of the typical experimental data plot and its saturation characteristics. Its utility complements the traditional empirical approaches. For the general saturation curve, described in terms of its independent (x) and dependent (y) variables, a second-order differential equation is obtained that applies to any saturation phenomena. It shows that the driving factor for the basic saturation behavior is the probability of the interactive site being free, which is described quantitatively. Solving the equation relates the variables in terms of the two empirical constants common to all these phenomena, the initial slope of the data plot and the limiting value at saturation. A first-order differential equation for the slope emerged that led to the concept of the effective binding rate at the active site and its dependence on the calculable probability the interactive site is free. These results are illustrated using specific cases, including ligand binding and enzyme kinetics. This leads to a revised understanding of how to interpret the empirical constants, in terms of the variables pertinent to the phenomenon under study. The second-order differential equation revealed the basic underlying relations that describe these saturation phenomena, and the basic mathematical properties of the standard experimental data plot. It was shown how to integrate this differential equation, and define the common basic properties of these phenomena. The results regarding the importance of the slope and the new perspectives on the empirical constants governing the behavior of these phenomena led to an alternative perspective on saturation behavior kinetics. Their essential commonality was revealed by this analysis, based on the second-order differential equation.

A general and fast scoring function for protein-ligand interactions: a simplified potential approach.

PubMed

Muegge, I; Martin, Y C

1999-03-11

A fast, simplified potential-based approach is presented that estimates the protein-ligand binding affinity based on the given 3D structure of a protein-ligand complex. This general, knowledge-based approach exploits structural information of known protein-ligand complexes extracted from the Brookhaven Protein Data Bank and converts it into distance-dependent Helmholtz free interaction energies of protein-ligand atom pairs (potentials of mean force, PMF). The definition of an appropriate reference state and the introduction of a correction term accounting for the volume taken by the ligand were found to be crucial for deriving the relevant interaction potentials that treat solvation and entropic contributions implicitly. A significant correlation between experimental binding affinities and computed score was found for sets of diverse protein-ligand complexes and for sets of different ligands bound to the same target. For 77 protein-ligand complexes taken from the Brookhaven Protein Data Bank, the calculated score showed a standard deviation from observed binding affinities of 1.8 log Ki units and an R2 value of 0.61. The best results were obtained for the subset of 16 serine protease complexes with a standard deviation of 1.0 log Ki unit and an R2 value of 0.86. A set of 33 inhibitors modeled into a crystal structure of HIV-1 protease yielded a standard deviation of 0.8 log Ki units from measured inhibition constants and an R2 value of 0.74. In contrast to empirical scoring functions that show similar or sometimes better correlation with observed binding affinities, our method does not involve deriving specific parameters that fit the observed binding affinities of protein-ligand complexes of a given training set. We compared the performance of the PMF score, Böhm's score (LUDI), and the SMOG score for eight different test sets of protein-ligand complexes. It was found that for the majority of test sets the PMF score performs best. The strength of the new approach presented here lies in its generality as no knowledge about measured binding affinities is needed to derive atomic interaction potentials. The use of the new scoring function in docking studies is outlined.

Modeling the Concentrations and Efficiencies for the Interacting Species of Pyropheophorbide Methyl Ester-Copper Association

NASA Astrophysics Data System (ADS)

Al-Omari, S.

2013-07-01

The interaction between pyropheophorbide methyl ester (PPME) and Cu2+ was investigated using UV-vis and fluorescence spectrscopy. Study of the binding interaction between PPME and Cu2+ could contribute to understanding of its pharmacokinetics and pharmacodynamics. Parameters of the static and dynamic fluorescence quenching of PPME-Cu2+ association were calculated at different temperatures. For binding site of 1:1 at 299 K, the static binding constant (kS), the static isosbestic concentration (CS{ iso}), the dynamic binding constant (kD), and the dynamic isosbestic concentration (CD{ iso }) are, respectively, 61 M-1, 0.0164 M, 75 M-1, and 0.0133 M. The concentrations and efficiencies of the intermediates species were modeled. Satisfactory correspondence between the experimental and calculated results was found.

Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

PubMed

Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

2016-12-15

In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

A hierarchical approach to cooperativity in macromolecular and self-assembling binding systems.

PubMed

Garcés, Josep Lluís; Acerenza, Luis; Mizraji, Eduardo; Mas, Francesc

2008-04-01

The study of complex macromolecular binding systems reveals that a high number of states and processes are involved in their mechanism of action, as has become more apparent with the sophistication of the experimental techniques used. The resulting information is often difficult to interpret because of the complexity of the scheme (large size and profuse interactions, including cooperative and self-assembling interactions) and the lack of transparency that this complexity introduces into the interpretation of the indexes traditionally used to describe the binding properties. In particular, cooperative behaviour can be attributed to very different causes, such as direct chemical modification of the binding sites, conformational changes in the whole structure of the macromolecule, aggregation processes between different subunits, etc. In this paper, we propose a novel approach for the analysis of the binding properties of complex macromolecular and self-assembling systems. To quantify the binding behaviour, we use the global association quotient defined as K(c) = [occupied sites]/([free sites] L), L being the free ligand concentration. K(c) can be easily related to other measures of cooperativity (such as the Hill number or the Scatchard plot) and to the free energies involved in the binding processes at each ligand concentration. In a previous work, it was shown that K(c) could be decomposed as an average of equilibrium constants in two ways: intrinsic constants for Adair binding systems and elementary constants for the general case. In this study, we show that these two decompositions are particular cases of a more general expression, where the average is over partial association quotients, associated with subsystems from which the system is composed. We also show that if the system is split into different subsystems according to a binding hierarchy that starts from the lower, microscopic level and ends at the higher, aggregation level, the global association quotient can be decomposed following the hierarchical levels of macromolecular organisation. In this process, the partial association quotients of one level are expressed, in a recursive way, as a function of the partial quotients of the level that is immediately below, until the microscopic level is reached. As a result, the binding properties of very complex macromolecular systems can be analysed in detail, making the mechanistic explanation of their behaviour transparent. In addition, our approach provides a model-independent interpretation of the intrinsic equilibrium constants in terms of the elementary ones.

[Binding interaction of harpagoside and bovine serum albumin: spectroscopic methodologies and molecular docking].

PubMed

Cao, Tuan-Wu; Huang, Wen-Bing; Shi, Jian-Wei; He, Wei

2018-03-01

Scrophularia ningpoensis has exhibited a variety of biological activities and been used as a pharmaceutical product for the treatment of inflammatory ailment, rheumatoid arthritis, osteoarthritis and so on. Harpagoside (HAR) is considerer as a main bioactive compound in this plant. Serum albumin has important physiological roles in transportation, distribution and metabolism of many endogenous and exogenous substances in body. It is of great significance to study the interaction mechanism between HAR and bovine serum albumin (BSA). The mechanism of interaction between HAR and BSA was investigated using 2D and 3D fluorescence, synchronous florescence, ultraviolet spectroscopy and molecular docking. According to the analysis of fluorescence spectra, HAR could strongly quench the fluorescence of BSA, and the static quenching process indicated that the decrease in the quenching constant was observed with the increase in temperature. The magnitude of binding constants (KA) was more than 1×10⁵ L·mol⁻¹, and the number of binding sites(n) was approximate to 1. The thermodynamic parameters were calculated through analysis of fluorescence data with Stern-Volmer and Van't Hoff equation. The calculated enthalpy change (ΔH) and entropy change (ΔS) implied that the main interaction forces of HAR with BSA were the bonding interaction between van der Waals forces and hydrogen. The negative values of energy (ΔG) demonstrated that the binding of HAR with BSA was a spontaneous and exothermic process. The binding distance(r) between HAR and BSA was calculated to be about 2.80 nm based on the theory of Frster's non-radiation energy transfer, which indicated that energy is likely to be transfer from BSA to HAR. Both synchronous and 3D florescence spectroscopy clearly revealed that the microenvironment and conformation of BSA changed during the binding interaction between HAR and BSA. The molecular docking analysis revealed HAR is more inclined to BSA and human serum albumin (HSA) in subdomain ⅡA (Sudlow's site I). This study will provide valuable information for understanding the action mechanism of HAR. Copyright© by the Chinese Pharmaceutical Association.

Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase.

PubMed

Volkán-Kacsó, Sándor; Marcus, Rudolph A

2016-10-25

A recently proposed chemomechanical group transfer theory of rotary biomolecular motors is applied to treat single-molecule controlled rotation experiments. In these experiments, single-molecule fluorescence is used to measure the binding and release rate constants of nucleotides by monitoring the occupancy of binding sites. It is shown how missed events of nucleotide binding and release in these experiments can be corrected using theory, with F 1 -ATP synthase as an example. The missed events are significant when the reverse rate is very fast. Using the theory the actual rate constants in the controlled rotation experiments and the corrections are predicted from independent data, including other single-molecule rotation and ensemble biochemical experiments. The effective torsional elastic constant is found to depend on the binding/releasing nucleotide, and it is smaller for ADP than for ATP. There is a good agreement, with no adjustable parameters, between the theoretical and experimental results of controlled rotation experiments and stalling experiments, for the range of angles where the data overlap. This agreement is perhaps all the more surprising because it occurs even though the binding and release of fluorescent nucleotides is monitored at single-site occupancy concentrations, whereas the stalling and free rotation experiments have multiple-site occupancy.

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein. Characterization and regulation by uridine and guanosine nucleotides

PubMed Central

Jørgensen, Casper Møller; Fields, Christopher J.; Chander, Preethi; Watt, Desmond; Burgner, John W.; Smith, Janet L.; Switzer, Robert L.

2011-01-01

Summary The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5’ leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. Apparent dissociation constants at 0° C ranged from 0.13 to 0.87 nM in the absence of effectors; dissociation constants were decreased by 3 to 12 fold by uridine nucleotides and increased by 40 to 200 fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied. PMID:18190533

Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Aquino,J.; Tetenbaum-Novatt, J.; White, A.

2005-01-01

The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10{sup -7}, binding site 2 (primary) is a low-affinity binding site with amore » binding constant of 6.3 x 10{sup -4}. These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A, C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.« less

[Derivative spectrophotometric and NMR spectroscopic study in pharmaceutical science].

PubMed

Kitamura, Keisuke

2007-10-01

This review starts with an introduction of derivative spectrophotometry followed by a description on the construction of a personal computer-assisted derivative spectrophotometric (DS) system. An acquisition system for inputting digitalized absorption spectra into personal computers and a BASIC program for calculating derivative spectra were developed. Then, applications of the system to drug analyses that are difficult with traditional absorption methods are described. Following this, studies on the interactions of drugs with biological macromolecules by the DS and NMR methods were discussed. An (1)H NMR study elucidated that the small unilamellar vesicle (SUV) has a single membrane made of a phosphatidylcholine bilayer, and that chlorpromazine interacts with both the outer and inner layers. (13)C NMR revealed a reduction of the dissociation constants of phenothiazine drugs due to their interaction with SUV. The partition coefficients of phenothiazine, benzodiazepine and steroid drugs in an SUV-water system and the effects of cholesterol or amino lipids content on these partition coefficients were examined by the DS method. The binding constants of phenothiazine drugs to bovine serum albumin (BSA) and the influence of Na(+), K(+), Cl(-), Br(-), and I(-) on these binding constants were determined by DS. It was found that I(-), Br(-), Cl(-) reduce the binding constants in this order, and that Na(+) and K(+) have no effect. A (19)F NMR study revealed that triflupromazine binds to BSA and human serum albumin in two regions including Site II with different populations, and that a nonsteroidal anti-inflammatory drug, niflumic acid, binds Sites Ia and Ib.

Application of pulsed field gradient NMR techniques for investigating binding of flavor compounds to macromolecules.

PubMed

Jung, Da-Mi; De Ropp, Jeffrey S; Ebeler, Susan E

2002-07-17

Two diffusion-based NMR techniques are presented and used to investigate the binding of selected flavor compounds to macromolecules. A pulsed field gradient NMR (PFG-NMR) method was applied to measure the apparent diffusion coefficients of four alkanone compounds as they associated with bovine serum albumin (BSA). The change in the apparent diffusion coefficient as a function of the BSA/alkanone ratio was fitted to yield binding constants (K(a)()) and binding stoichiometry (n) for each alkanone. The results showed that the apparent diffusion coefficients of alkanones increased with a decrease in the BSA/alkanone ratios, and the measured values of K(a)() and n were comparable with those obtained with other methods and depended on the alkanone structure. A diffusion-based nuclear Overhauser effect (called diffusion NOE pumping) method was also applied to screen mixtures of flavor compounds and identify those that have a binding affinity to complex macromolecules. Using this technique benzaldehyde and vanillin were observed to bind with bovine serum albumin, whereas 2-phenylethanol was identified as a nonbinding or weakly binding ligand with BSA. The diffusion NOE pumping method was also applied to a hydro alcoholic solution of cacao bean tannin extracts to which a mixture of ethylbenzoate, benzaldehyde, and 2-phenylethanol was added. The diffusion NOE pumping technique clearly indicated that ethylbenzoate had a stronger binding affinity to the polymeric (-)-epicatechin units of the cacao bean tannin extracts than the other two flavor compounds. The results successfully demonstrate the potential applications of diffusion-based NMR techniques for studying flavors and nonvolatile food matrix interactions.

Effects of organic ligands on fractionation of rare earth elements (REEs) in hydroponic plants: an application to the determination of binding capacities by humic acid for modeling.

PubMed

Ding, ShiMing; Liang, Tao; Zhang, ChaoSheng; Yan, JunCai; Zhang, ZiLi

2006-12-01

Previous studies have revealed the fractionation processes of rare earth elements (REEs) in hydroponic plants, with a heavy REE (HREE, the elements from Gd to Lu) enrichment in leaves. In this study, effects on the HREE enrichment in soybean leaves with additions of carboxylic acids (acetate, malate, citrate, NTA, EDTA and DTPA) and two soil humic acids (HAs) were investigated. REE speciation in carboxylic acid and HA solutions was simulated using Visual MINTEQ and Model V, respectively. The results showed that the effects caused by carboxylic acids were strongly dependent on the differences between their binding strengths for light REEs (LREEs, the elements from La to Eu) and those for HREEs. A good correlation existed between these effects and the changes of free REE ions in solutions. This relationship was also observed for the HA treatments, provided that the intrinsic equilibrium constants of REEs for cation-proton exchange with HA (i.e., pK(MHA)) in Model V were estimated using a free-energy relationship with the stability constants for REE complexation with lactic acid. It is suggested that this set of pK(MHA) values is more suitable for use in Model V for the simulation of REE complexation with HA.

Soluble 1:1 complexes and insoluble 3:2 complexes - Understanding the phase-solubility diagram of hydrocortisone and γ-cyclodextrin.

PubMed

Schönbeck, Christian; Madsen, Tobias L; Peters, Günther H; Holm, René; Loftsson, Thorsteinn

2017-10-15

The molecular mechanisms underlying the drug-solubilizing properties of γ-cyclodextrin were explored using hydrocortisone as a model drug. The B S -type phase-solubility diagram of hydrocortisone with γ-cyclodextrin was thoroughly characterized by measuring the concentrations of hydrocortisone and γ-cyclodextrin in solution and the solid phase. The drug-solubilizer interaction was also studied by isothermal titration calorimetry from which a precise value of the 1:1 binding constant (K 11 =4.01mM -1 at 20°C) was obtained. The formation of water-soluble 1:1 complexes is responsible for the initial increase in hydrocortisone solubility while the precipitation of entities with a 3:2 ratio of γ-cyclodextrin:hydrocortisone is responsible for the plateau and the ensuing strong decrease in solubility once all solid hydrocortisone is used up. The complete phase-solubility diagram is well accounted for by a model employing the 1:1 binding constant and the solubility product of the precipitating 3:2 entity (K 32 S =5.51 mM 5 ). For such systems, a small surplus of γ-cyclodextrin above the optimum concentration may result in a significant decrease in drug solubility, and the implications for drug formulations are briefly discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Assessment of amsacrine binding with DNA using UV-visible, circular dichroism and Raman spectroscopic techniques.

PubMed

Jangir, Deepak Kumar; Dey, Sanjay Kumar; Kundu, Suman; Mehrotra, Ranjana

2012-09-03

Proper understanding of the mechanism of binding of drugs to their targets in cell is a fundamental requirement to develop new drug therapy regimen. Amsacrine is a rationally designed anticancer drug, used to treat leukemia and lymphoma. Binding with cellular DNA is a crucial step in its mechanism of cytotoxicity. Despite numerous studies, DNA binding properties of amsacrine are poorly understood. Its reversible binding with DNA does not permit X-ray crystallography or NMR spectroscopic evaluation of amsacrine-DNA complexes. In the present work, interaction of amsacrine with calf thymus DNA is investigated at physiological conditions. UV-visible, FT-Raman and circular dichroism spectroscopic techniques were employed to determine the binding mode, binding constant, sequence specificity and conformational effects of amsacrine binding to native calf thymus DNA. Our results illustrate that amsacrine interacts with DNA by and large through intercalation between base pairs. Binding constant of the amsacrine-DNA complex was found to be K=1.2±0.1×10(4) M(-1) which is indicative of moderate type of binding of amsacrine to DNA. Raman spectroscopic results suggest that amsacrine has a binding preference of intercalation between AT base pairs of DNA. Minor groove binding is also observed in amsacrine-DNA complexes. These results are in good agreement with in silico investigation of amsacrine binding to DNA and thus provide detailed insight into DNA binding properties of amsacrine, which could ultimately, renders its cytotoxic efficacy. Copyright © 2012 Elsevier B.V. All rights reserved.

Mercury(II) sorption to two Florida Everglades peat--Evidence for strong and weak binding and competition by dissolved organic matter released from the peat

USGS Publications Warehouse

Drexel, R. Todd; Haitzer, Markus; Ryan, Joseph N.; Aiken, George R.; Nagy, Kathryn L.

2002-01-01

The binding of mercury(II) to two peats from Florida Everglades sites with different rates of mercury methylation was measured at pH 6.0 and 0.01 M ionic strength. The mercury(II) sorption isotherms, measured over a total mercury(II) range of 10-7.4 to 10-3.7 M, showed the competition for mercury(II) between the peat and dissolved organic matter released from the peat and the existence of strong and weak binding sites for mercury(II). Binding was portrayed by a model accounting for strong and weak sites on both the peat and the released DOM. The conditional binding constants (for which the ligand concentration was set as the concentration of reduced sulfur in the organic matter as measured by X-ray absorption near-edge structure spectroscopy) determined for the strong sites on the two peats were similar (Kpeat,s = 1021.8±0.1and 1022.0±0.1 M-1), but less than those determined for the DOM strong sites (Kdom,s = 1022.8±0.1and 1023.2±0.1 M-1), resulting in mercury(II) binding by the DOM at low mercury(II) concentrations. The magnitude of the strong site binding constant is indicative of mercury(II) interaction with organic thiol functional groups. The conditional binding constants determined for the weak peat sites (Kpeat,w = 1011.5±0.1 and 1011.8±0.1 M-1) and weak DOM sites (Kdom,w = 108.7±3.0 and 107.3±4.5 M-1) were indicative of mercury(II) interaction with carboxyl and phenol functional groups.

Prediction of binding constants of protein ligands: A fast method for the prioritization of hits obtained from de novo design or 3D database search programs

NASA Astrophysics Data System (ADS)

Böhm, Hans-Joachim

1998-07-01

A dataset of 82 protein-ligand complexes of known 3D structure and binding constant Ki was analysed to elucidate the important factors that determine the strength of protein-ligand interactions. The following parameters were investigated: the number and geometry of hydrogen bonds and ionic interactions between the protein and the ligand, the size of the lipophilic contact surface, the flexibility of the ligand, the electrostatic potential in the binding site, water molecules in the binding site, cavities along the protein-ligand interface and specific interactions between aromatic rings. Based on these parameters, a new empirical scoring function is presented that estimates the free energy of binding for a protein-ligand complex of known 3D structure. The function distinguishes between buried and solvent accessible hydrogen bonds. It tolerates deviations in the hydrogen bond geometry of up to 0.25 Å in the length and up to 30 °Cs in the hydrogen bond angle without penalizing the score. The new energy function reproduces the binding constants (ranging from 3.7 × 10-2 M to 1 × 10-14 M, corresponding to binding energies between -8 and -80 kJ/mol) of the dataset with a standard deviation of 7.3 kJ/mol corresponding to 1.3 orders of magnitude in binding affinity. The function can be evaluated very fast and is therefore also suitable for the application in a 3D database search or de novo ligand design program such as LUDI. The physical significance of the individual contributions is discussed.

Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals.

PubMed

Escher, Beate I; Cowan-Ellsberry, Christina E; Dyer, Scott; Embry, Michelle R; Erhardt, Susan; Halder, Marlies; Kwon, Jung-Hwan; Johanning, Karla; Oosterwijk, Mattheus T T; Rutishauser, Sibylle; Segner, Helmut; Nichols, John

2011-07-18

Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values used in previous modeling efforts. Bioconcentration factors (BCF) predicted using the experimental binding data were substantially higher than the predicted values obtained in earlier studies and correlated poorly with measured BCF values in fish. One possible explanation for this finding is that chemicals bound to proteins can desorb rapidly and thus contribute to metabolic turnover of the chemicals. This hypothesis remains to be investigated in future studies, ideally with chemicals of higher hydrophobicity. © 2011 American Chemical Society

Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity.

PubMed

Valko, Klara; Nunhuck, Shenaz; Bevan, Chris; Abraham, Michael H; Reynolds, Derek P

2003-11-01

A fast gradient HPLC method (cycle time 15 min) has been developed to determine Human Serum Albumin (HSA) binding of discovery compounds using chemically bonded protein stationary phases. The HSA binding values were derived from the gradient retention times that were converted to the logarithm of the equilibrium constants (logK HSA) using data from a calibration set of molecules. The method has been validated using literature plasma protein binding data of 68 known drug molecules. The method is fully automated, and has been used for lead optimization in more than 20 company projects. The HSA binding data obtained for more than 4000 compounds were suitable to set up global and project specific quantitative structure binding relationships that helped compound design in early drug discovery. The obtained HSA binding of known drug molecules were compared to the Immobilized Artificial Membrane binding data (CHI IAM) obtained by our previously described HPLC-based method. The solvation equation approach has been used to characterize the normal binding ability of HSA, and this relationship shows that compound lipophilicity is a significant factor. It was found that the selectivity of the "baseline" lipophilicity governing HSA binding, membrane interaction, and octanol/water partition are very similar. However, the effect of the presence of positive or negative charges have very different effects. It was found that negatively charged compounds bind more strongly to HSA than it would be expected from the lipophilicity of the ionized species at pH 7.4. Several compounds showed stronger HSA binding than can be expected from their lipophilicity alone, and comparison between predicted and experimental binding affinity allows the identification of compounds that have good complementarities with any of the known binding sites. Copyright 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2236-2248, 2003

Molecular Interaction Between Salivary Proteins and Food Tannins.

PubMed

Silva, Mafalda Santos; García-Estévez, Ignacio; Brandão, Elsa; Mateus, Nuno; de Freitas, Victor; Soares, Susana

2017-08-09

Polyphenols interaction with salivary proteins (SP) has been related with organoleptic features such as astringency. The aim of this work was to study the interaction between some human SP and tannins through two spectroscopic techniques, fluorescence quenching, and saturation transfer difference-nuclear magnetic resonance (STD-NMR). Generally, the results showed a significant interaction between SP and both condensed tannins and ellagitannins. Herein, STD-NMR proved to be a useful tool to map tannins' epitopes of binding, while fluorescence quenching allowed one to discriminate binding affinities. Ellagitannins showed the greatest binding constants values (K SV from 20.1 to 94.1 mM -1 ; K A from 0.7 to 8.3 mM -1 ) in comparison with procyanidins (K SV from 5.4 to 40.0 mM -1 ; K A from 1.1 to 2.7 mM -1 ). In fact, punicalagin was the tannin that demonstrated the highest affinity for all three SP. Regarding SP, P-B peptide was the one with higher affinity for ellagitannins. On the other hand, cystatins showed in general the lower K SV and K A values. In the case of condensed tannins, statherin was the SP with the highest affinity, contrasting with the other two SP. Altogether, these results are evidence that the distinct SP present in the oral cavity have different abilities to interact with food tannins class.

A Model for Amplification of Hair-Bundle Motion by Cyclical Binding of Ca2+ to Mechanoelectrical-Transduction Channels

NASA Astrophysics Data System (ADS)

Choe, Yong; Magnasco, Marcelo O.; Hudspeth, A. J.

1998-12-01

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of Ca2+ to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system's behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for Ca2+ binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken's cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.

Millimeter Wave Spectrum of the Weakly Bound Complex CH2═CHCN·H2O: Structure, Dynamics, and Implications for Astronomical Search.

PubMed

Calabrese, Camilla; Vigorito, Annalisa; Maris, Assimo; Mariotti, Sergio; Fathi, Pantea; Geppert, Wolf D; Melandri, Sonia

2015-12-03

The weakly bound 1:1 complex between acrylonitrile (CH2═CHCN) and water has been characterized spectroscopically in the millimeter wave range (59.6-74.4 GHz) using a Free Jet Absorption Millimeter Wave spectrometer. Precise values of the rotational and quartic centrifugal distortion constants have been obtained from the measured frequencies of the normal and isotopically substituted water moiety (DOH, DOD, H(18)OH). Structural parameters have been estimated from the rotational constants and their differences among isotopologues: the complex has a planar structure with the two subunits held together by a O-H···N (2.331(3) Å) and a C-H···O (2.508(4) Å) interaction. The ab initio intermolecular binding energy, obtained at the counterpoise corrected MP2/aug-cc-pVTZ level of calculation, is De = 24.4 kJ mol(-1).

Motion of kinesin in a viscoelastic medium

NASA Astrophysics Data System (ADS)

Knoops, Gert; Vanderzande, Carlo

2018-05-01

Kinesin is a molecular motor that transports cargo along microtubules. The results of many in vitro experiments on kinesin-1 are described by kinetic models in which one transition corresponds to the forward motion and subsequent binding of the tethered motor head. We argue that in a viscoelastic medium like the cytosol of a cell this step is not Markov and has to be described by a nonexponential waiting time distribution. We introduce a semi-Markov kinetic model for kinesin that takes this effect into account. We calculate, for arbitrary waiting time distributions, the moment generating function of the number of steps made, and determine from this the average velocity and the diffusion constant of the motor. We illustrate our results for the case of a waiting time distribution that is Weibull. We find that for realistic parameter values, viscoelasticity decreases the velocity and the diffusion constant, but increases the randomness (or Fano factor).

Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

PubMed Central

Alfonso, Amparo; Pazos, María-José; Fernández-Araujo, Andrea; Tobio, Araceli; Alfonso, Carmen; Vieytes, Mercedes R.; Botana, Luis M.

2013-01-01

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs). From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods. PMID:24379088

A reductionist biomimetic model system that demonstrates highly effective Zn(II)-catalyzed cleavage of an RNA model.

PubMed

Liu, C Tony; Neverov, Alexei A; Brown, R Stan

2007-03-05

The cyclization of the RNA model 2-hydroxypropyl p-nitrophenyl phosphate (HPNPP, 1) promoted by Zn2+ alone and the 1,5,9-triazacyclododecane complex of Zn2+ (Zn2+:[12]aneN3) is studied in ethanol in the presence of 0.5 equiv of -OEt/Zn2+ to investigate the effect of a low polarity/dielectric medium on a metal-catalyzed reaction of biological relevance. Ethanol exerts a medium effect that promotes strong binding of HPNPP to Zn2+, followed by a dimerization to form a catalytically active complex (HPNPP:Zn2+)2 in which the phosphate undergoes cyclization with a rate constant of kcat = 2.9 s(-1) at s(s)pH 7.1. In the presence of the triaza ligand:Zn2+ complex, the change from water to methanol and then to ethanol brings about a mechanism where two molecules of the complex, suggested as EtOH:Zn2+:[12]aneN3 and its basic form, EtO-:Zn2+:[12]aneN3, bind to HPNPP and catalyze its decomposition with a rate constant of kcat of 0.13 s(-1) at s(s)pH 7.1. Overall, the acceleration exhibited in these two situations is 4 x 10(14)-fold and 1.7 x 10(12)-fold relative to the background ethoxide-promoted reactions at the respective s(s)pH values. The implications of these findings are discussed within the context of the idea that enzymatic catalysis is enhanced by a reduced effective dielectric constant within the active site.

Cloning and characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa.

PubMed

Palmer, Stephanie O; Rangel, Edna Y; Montalvo, Alberto E; Tran, Alexis T; Ferguson, Kate C; Bullard, James M

2013-01-01

We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be K M = 33 μM, k cat (obs) = 0.003 s(-1), and the specificity constant k cat (obs)/K M was 0.1 × 10(-3) s(-1) μM(-1). In the presence of EF-Ts, these values were shifted to K M = 2 μM, k cat (obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K M was 2.5 × 10(-3) s(-1) μM(-1). The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K GDP) were 30-75 nM and to GTP (K GTP) were 125-200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNA(Phe) at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

PubMed Central

Jankowsky, E; Schwenzer, B

1996-01-01

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

Surface plasmon resonance biosensor method for palytoxin detection based on Na+,K+-ATPase affinity.

PubMed

Alfonso, Amparo; Pazos, María-José; Fernández-Araujo, Andrea; Tobio, Araceli; Alfonso, Carmen; Vieytes, Mercedes R; Botana, Luis M

2013-12-27

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (Kobs). From the representation of Kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (K(D)) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is K(D) = 6.38 × 10-7 ± 6.67 × 10-8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.

Studies of interaction of emodin and DNA in the presence of ethidium bromide by spectroscopic method

NASA Astrophysics Data System (ADS)

Bi, Shuyun; Zhang, Hanqi; Qiao, Chunyu; Sun, Ying; Liu, Chunming

2008-01-01

Emodin interacting with deoxyribonucleic acid (DNA) has been studied by different spectroscopic techniques, such as fluorescence, ultraviolet and visible (UV-vis), and fourier transform infared (FT-IR) spectroscopies, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA-EB system on addition of emodin shows that the fluorescence quenching of DNA-EB complex by emodin occurs. The binding constants of emodin with DNA in the presence of EB are 6.02 × 10 4, 9.20 × 10 4 and 1.17 × 10 5 L mol -1 at 20, 35 and 50 °C, respectively. FT-IR spectrum further suggests that both the phosphate groups and the bases of DNA react with emodin. The reaction of DNA with emodin in the presence of EB is affected by ionic strength and temperature. The values of melting temperature ( Tm) of DNA-EB complex and emodin-DNA-EB complexes were determined, respectively. From the experiment evidences, the major binding mode of emodin with DNA should be the groove binding.

Fluorescent bovine serum albumin interacting with the antitussive quencher dextromethorphan: a spectroscopic insight.

PubMed

Durgannavar, Amar K; Patgar, Manjanath B; Nandibewoor, Sharanappa T; Chimatadar, Shivamurti A

2016-05-01

The interaction of dextromethorphan hydrobromide (DXM) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV-vis absorption, synchronous fluorescence spectra (SFS), 3D fluorescence spectra, Fourier transform infrared (FTIR) spectroscopy and circular dichroism under simulated physiological conditions. DXM effectively quenched the intrinsic fluorescence of BSA. Values of the binding constant, K(A), are 7.159 × 10(3), 9.398 × 10(3) and 16.101 × 10(3)  L/mol; the number of binding sites, n, and the corresponding thermodynamic parameters ΔG°, ΔH° and ΔS° between DXM and BSA were calculated at different temperatures. The interaction between DXM and BSA occurs through dynamic quenching and the effect of DXM on the conformation of BSA was analyzed using SFS. The average binding distance, r, between the donor (BSA) and acceptor (DXM) was determined based on Förster's theory. The results of fluorescence spectra, UV-vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of DXM. Copyright © 2015 John Wiley & Sons, Ltd.

Determination of berberine and the study of fluorescence quenching mechanism between berberine and enzyme-catalyzed product

NASA Astrophysics Data System (ADS)

Wang, Huaiyou; Zhang, Miao; Lv, Qingluan; Yue, Ningning; Gong, Bin

2009-08-01

A new method for determining berberine has been established based on the principle of fluorescence quenching. The calibration curve was found to be linear between F0/ F and the concentration of berberine with the range of 3.00-20.0 μg mL -1. The detection limit was 0.51 μg mL -1 and the relative standard derivative was 0.18%. Effects of pH, foreign ions and the optimization of variables on the determination of berberine have been examined. The mechanism of the fluorescence quenching has been discussed. The binding constant and the number of binding sites were 1.70 × 10 6 L mol -1 and 1.14, respectively. The data, Δ H = 42.71 kJ mol -1, Δ S = 264.3 J K -1 mol -1 and the mean value Δ G = -39.65 kJ mol -1 were estimated which showed that the reaction was spontaneous and endothermic. The main binding force was hydrophobic force because both Δ H and Δ S were positive.

Synthesis, characterization, and sol-gel entrapment of a crown ether-styryl fluoroionophore

PubMed Central

Sui, Zhijie; Hanan, Nathan J.; Phimphivong, Sam; Wysocki, Ronald J.; Saavedra, S. Scott

2011-01-01

The synthesis and initial evaluation of a new dye-functionalized crown-ether, 2-[2-(2,3,5,6,8,9,11,12,14,15-decahydro-1,4,7,10.13.16-benzohexaoxacyclooctadecin)ethenyl]-3-methyl benzothiazolium iodide (denoted BSD), is reported. This molecule contains a benzyl 18-crown-6 moiety as the ionophore and a benzothiazolium to spectrally transduce ion binding. Binding of K+ to BSD in methanol causes shifts in the both absorbance and fluorescence emission maxima, as well as changes in the molar absorptivity and the emission intensity. Apparent dissociation constants (Kd) in the range of 30 – 65 μM were measured. In water and neutral buffer, Kd values were approximately 1 mM. BSD was entrapped in sol-gel films composed of methyltriethoxysilane (MTES) and tetraethylorthosilicate (TEOS) with retention of its spectral properties and minimal leaching. K+ binding to BSD in sol-gels films immersed in pH 7.4 buffer causes significant fluorescence quenching, with an apparent response time of approximately 2 min and an apparent Kd of 1.5 mM. PMID:19253273

Chemometrics-assisted investigation of interactions of Tasmar with human serum albumin at a glassy carbon disk: Application to electrochemical biosensing of electro-inactive serum albumin.

PubMed

Mohammadi, Ghobad; Faramarzi, Elahe; Mahmoudi, Majid; Ghobadi, Sirous; Ghiasvand, Ali Reza; Goicoechea, Hector C; Jalalvand, Ali R

2018-07-15

In this work, voltammetric data recorded by a glassy carbon electrode (GCE) was used to investigate the interactions of tolcapone (Tasmar, TAS) with human serum albumin (HSA) at the electrode surface. The recorded voltammetric data was also combined with spectroscopic data to construct an augmented data matrix which was analysed by multivariate curve resolution-alternating least squares (MCR-ALS) as an efficient chemometric tool to obtain more information about TAS-HSA interactions. The results of MCR-ALS confirmed formation of one complex species (HSA-TAS 2 ) and application of MCR-BANDS to the results of MCR-ALS confirmed the absence of rotational ambiguities and existing unambiguous and reliable results. Binding of TAS to HSA was also modeled by molecular docking and the results showed that the TAS was bound to sub-domain IIA of HSA which were compatible with the ones obtained by recording experimental data. Hard-modeling of combined voltammetric and spectroscopic data by EQUISPEC helped us to compute binding constant of HSA-TAS 2 complex species which was compatible with the binding constant value obtained by direct analysis of experimental data. Finally, a new electroanalytical method was developed based on TAS-HSA interactions for determination of HSA in two ranges of 0-541 nM and 541-1200 nM with a limit of detection of 0.04 nM and a sensitivity of 0.02 μA nM -1 . Copyright © 2018 Elsevier B.V. All rights reserved.

Gigantol from Dendrobium chrysotoxum Lindl. binds and inhibits aldose reductase gene to exert its anti-cataract activity: An in vitro mechanistic study.

PubMed

Wu, Jie; Li, Xue; Wan, Wencheng; Yang, Qiaohong; Ma, Weifeng; Chen, Dan; Hu, Jiangmiao; Chen, C-Y Oliver; Wei, Xiaoyong

2017-02-23

Dendrobium. chrysotoxum Lindl is a commonly used species of medicinal Dendrobium which belongs to the family of Orchidaceae, locally known as "Shihu" or "Huangcao". D. chrysotoxum Lindl is widely known for medicinal values in traditional Chinese medicine as it possesses anti-inflammatory, anti-hyperglycemic induction, antitumor and antioxidant properties. To characterize the interaction between gigantol extracted from D. chrysotoxum Lindl and the AR gene, and determine gigantol's efficacy against cataractogenesis. Human lens epithelial cells (HLECs) were induced by glucose as the model group. Reverse transcription polymerase chain reaction (RT-PCR) was used to assess AR gene expression. Then, the mode of interaction of gigantol with the AR gene was evaluated by UV-visible spectroscopy, atomic force microscope (AFM) and surface-enhanced Raman spectroscopy (SERS). The binding constant was determined by UV-visible. Gigantol depressed AR gene expression in HLECs. UV-visible spectra preliminarily indicated that interaction between the AR gene and gigantol may follow the groove mode, with a binding constant of 1.85×10 3 L/mol. Atomic force microscope (AFM) data indicated that gigantol possibly bound to insert AR gene base pairs of the double helix. Surface-enhanced Raman spectroscopy (SERS) studies further supported these observations. Gigantol extracted from D. chrysotoxum Lindl not only has inhibitory effects on aldose reductase, but also inhibits AR gene expression. These findings provide a more comprehensive theoretical basis for the use of Dendrobium for the treatment of diabetic cataract. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Hydrogen molecules and chains in a superstrong magnetic field

NASA Technical Reports Server (NTRS)

Lai, Dong; Salpeter, Edwin E.; Shapiro, Stuart L.

1992-01-01

The electronic structures of hydrogen polymolecules H(n) (n = 2,3,4,...) is studied in a superstrong magnetic field (B greater than about 10 exp 12 G) typically found on the surface of a neutron star. Simple analytical scaling relations for several limiting cases (e.g., large n, high B field) are derived. The binding energies of H(n) molecules are numerically calculated for various magnetic-field strengths. For a given magnetic-field strength, the binding energy per atom in the H(n) molecules is found to approach a constant value as n increases. For typical field strengths of interest, energy saturation is essentially achieved once n exceeds 3 to 4. Also considered is the structure of negative H ions in a high magnetic field. For B about 10 exp 12 G, the dissociation energy of an atom in a hydrogen chain and the ionization potential of H(-) are smaller than the ionization potential of neutral atomic hydrogen.

Naringenin and quercetin--potential anti-HCV agents for NS2 protease targets.

PubMed

Lulu, S Sajitha; Thabitha, A; Vino, S; Priya, A Mohana; Rout, Madhusmita

2016-01-01

Nonstructural proteins of hepatitis C virus had drawn much attention for the scientific fraternity in drug discovery due to its important role in the disease. 3D structure of the protein was predicted using molecular modelling protocol. Docking studies of 10 medicinal plant compounds and three drugs available in the market (control) with NS2 protease were employed by using rigid docking approach of AutoDock 4.2. Among the molecules tested for docking study, naringenin and quercetin revealed minimum binding energy of - 7.97 and - 7.95 kcal/mol with NS2 protease. All the ligands were docked deeply within the binding pocket region of the protein. The docking study results showed that these compounds are potential inhibitors of the target; and also all these docked compounds have good inhibition constant, vdW+Hbond+desolv energy with best RMSD value.

Quasiclassical analysis of Bloch oscillations in non-Hermitian tight-binding lattices

NASA Astrophysics Data System (ADS)

Graefe, E. M.; Korsch, H. J.; Rush, A.

2016-07-01

Many features of Bloch oscillations in one-dimensional quantum lattices with a static force can be described by quasiclassical considerations for example by means of the acceleration theorem, at least for Hermitian systems. Here the quasiclassical approach is extended to non-Hermitian lattices, which are of increasing interest. The analysis is based on a generalised non-Hermitian phase space dynamics developed recently. Applications to a single-band tight-binding system demonstrate that many features of the quantum dynamics can be understood from this classical description qualitatively and even quantitatively. Two non-Hermitian and PT-symmetric examples are studied, a Hatano-Nelson lattice with real coupling constants and a system with purely imaginary couplings, both for initially localised states in space or in momentum. It is shown that the time-evolution of the norm of the wave packet and the expectation values of position and momentum can be described in a classical picture.

Molecular Containers Bind Drugs of Abuse in Vitro and Reverse the Hyperlocomotive Effect of Methamphetamine in Rats.

PubMed

Ganapati, Shweta; Grabitz, Stephanie D; Murkli, Steven; Scheffenbichler, Flora; Rudolph, Maíra I; Zavalij, Peter Y; Eikermann, Matthias; Isaacs, Lyle

2017-08-17

We measured the affinity of five molecular container compounds (calabadions 1 and 2, CB[7], sulfocalix[4]arene, and HP-β-CD) toward seven drugs of abuse in homogenous aqueous solution at physiological pH by various methods ( 1 H NMR, UV/Vis, isothermal titration calorimetry [ITC]) and found binding constants (K a values) spanning from <10 2 to >10 8  m -1 . We also report X-ray crystal structures of CB[7]⋅methamphetamine and 1⋅methamphetamine. We found that 2, but not CB[7], was able to ameliorate the hyperlocomotive activity of rats treated with methamphetamine. The bioavailability of the calabadions and their convergent building block synthesis suggest potential for further structural optimization as reversal agents for intoxication with nonopioid drugs of abuse for which no treatments are currently available. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effective solubilization of chalcones in micellar phase: Conductivity and voltammetric study

NASA Astrophysics Data System (ADS)

Ahmed, Safeer; Khan, Gul Tiaz; Shah, Syed Sakhawat

2013-12-01

The solubilization of four chalcones, between aqueous and micellar phases of ionic surfactants (SDS and CTAB), was investigated by conductivity and cyclic voltammetry (CV) techniques. From conductivity data, a decrease in the critical micellar concentration (CMC) of the surfactants, in presence of the chalcones was ascribed to the decreased charge density over the surfactants. The results were seconded by thermodynamic parameters including degree of ionization (α), counter ion binding (β), and standard Gibbs free energy of micellization (Δ G {m/○}). The added surfactant decreased the peak current of the oxidized chalcone and shifted the peak potential either positively (in presence of SDS) or negatively (in presence of CTAB). The effect is rationalized as chalcone-surfactant interaction and quantitated as binding constant ( K b) assorting values from 8.78 to 552.97 M-1. The preferred solubilization of the chalcones in the micellar phase has been inferred.

The death of the Job plot, transparency, open science and online tools, uncertainty estimation methods and other developments in supramolecular chemistry data analysis.

PubMed

Brynn Hibbert, D; Thordarson, Pall

2016-10-25

Data analysis is central to understanding phenomena in host-guest chemistry. We describe here recent developments in this field starting with the revelation that the popular Job plot method is inappropriate for most problems in host-guest chemistry and that the focus should instead be on systematically fitting data and testing all reasonable binding models. We then discuss approaches for estimating uncertainties in binding studies using case studies and simulations to highlight key issues. Related to this is the need for ready access to data and transparency in the methodology or software used, and we demonstrate an example a webportal () that aims to address this issue. We conclude with a list of best-practice protocols for data analysis in supramolecular chemistry that could easily be translated to other related problems in chemistry including measuring rate constants or drug IC 50 values.

Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

ERIC Educational Resources Information Center

Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

2010-01-01

The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

Photoaffinity labeling and partial purification of the beta cell sulfonylurea receptor using a novel, biologically active glyburide analog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aguilar-Bryan, L.; Nelson, D.A.; Vu, Q.A.

1990-05-15

An iodinated analog of the sulfonylurea, glyburide, has been synthesized which can be labeled to high specific activity and used to photolabel the sulfonylurea receptor. 5-Iodo-2-hydroxy-glyburide, has an iodo group replacing the chlorine at position 5 and a methoxy residue replacing the hydroxy group at position 2 on the benzamido ring. This analog retains biologic activity stimulating insulin secretion from a hamster beta cell line (HIT cells) at the same ED50 (0.4 nM) as glyburide. Scatchard analysis demonstrated high and low affinity binding sites on HIT cell membranes (Kd values of 0.36 nM and 277 nM and Bmax values ofmore » 1.6 and 100 pmol/mg of membrane protein, respectively). Competitive binding assays with unlabeled glyburide or 5-iodo-2-hydroxyglyburide yield Ki values of 0.5 and 1.0 nM, respectively. The analog can be covalently linked by ultraviolet irradiation to a membrane protein of Mr = 140,000. The photolabeling is completely blocked by unlabeled glyburide or the analog. Two other species of Mr = 65,000 and 43,000 are also photolabeled; these may be the low affinity sites. After photolabeling, the receptor has been purified partially by chromatographic procedures and is suitable for obtaining peptide sequence. The 140,000 molecular weight protein is identified as the sulfonylurea receptor since its binding constant, 0.36 nM, is closely correlated with its ability to stimulate insulin secretion (ED50 congruent to 0.4 nM).« less

An x-ray absorption spectroscopy study of Cd binding onto a halophilic archaeon

NASA Astrophysics Data System (ADS)

Showalter, Allison R.; Szymanowski, Jennifer E. S.; Fein, Jeremy B.; Bunker, Bruce A.

2016-05-01

X-ray absorption spectroscopy (XAS) and cadmium (Cd) isotherm experiments determine how Cd adsorbs to the surface of halophilic archaeon Halobacterium noricense. This archaeon, isolated from the Waste Isolation Pilot Plant (WIPP) near Carlsbad, New Mexico could be involved with the transport of toxic metals stored in the transuranic waste in the salt mine. The isotherm experiments show that adsorption is relatively constant across the tolerable pH range for H. noricense. The XAS results indicate that Cd adsorption occurs predominately via a sulfur site, most likely sulfhydryl, with the same site dominating all measured pH values.

Anion binding by bambus[6]uril probed in the gas phase and in solution.

PubMed

Révész, Agnes; Schröder, Detlef; Svec, Jan; Wimmerová, Michaela; Sindelar, Vladimir

2011-10-20

Electrospray ionization mass spectrometry (ESI-MS) is used to probe the binding of small anions to the macrocycle of bambus[6]uril. For the halide ions, the experimental patterns suggest F(-) < Cl(-) < Br(-) < I(-), which is consistent with the order of anion binding found in the condensed phase. Parallel equilibrium studies in the condensed phase establish the association constants of halide anions and bambus[6]uril in mixed solvents. A detailed analysis of the mass spectrometric data is used to shed light on the correlations between the binding constants in the condensed phase and the ion abundances observed using ESI-MS. From the analysis it becomes apparent that ESI-MS can indeed represent the situation in solution to some extent, but the sampling in the gas-phase experiment is not 1:1 compared to that in solution.

Interaction of milk whey protein with common phenolic acids

NASA Astrophysics Data System (ADS)

Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

2014-01-01

Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

Method bacterial endospore quantification using lanthanide dipicolinate luminescence

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri J. (Inventor); Kirby, James Patrick (Inventor); Ponce, Adrian (Inventor)

2007-01-01

A lanthanide is combined with a medium to be tested for endospores. The dipicolinic acid released from the endospores binds the lanthanides, which have distinctive emission (i.e., luminescence) spectra, and are detected using photoluminescence. The concentration of spores is determined by preparing a calibration curve generated from photoluminescence spectra of lanthanide complex mixed with spores of a known concentration. A lanthanide complex is used as the analysis reagent, and is comprised of lanthanide ions bound to multidentate ligands that increase the dipicolinic acid binding constant through a cooperative binding effect with respect to lanthanide chloride. The resulting combined effect of increasing the binding constant and eliminating coordinated water and multiple equilibria increase the sensitivity of the endospore assay by an estimated three to four orders of magnitude over prior art of endospore detection based on lanthanide luminescence.

Interactions of poly(amidoamine) dendrimers with human serum albumin: binding constants and mechanisms.

PubMed

Giri, Jyotsnendu; Diallo, Mamadou S; Simpson, André J; Liu, Yi; Goddard, William A; Kumar, Rajeev; Woods, Gwen C

2011-05-24

The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K(b)) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To gain insight into the mechanisms of HSA binding to PAMAM dendrimers, we combined (1)H NMR, saturation transfer difference (STD) NMR, and NMR diffusion ordered spectroscopy (DOSY) of dendrimer-HSA complexes with atomistic molecular dynamics (MD) simulations of dendrimer conformation in aqueous solutions. The binding measurements show that the HSA binding constants (K(b)) of PAMAM dendrimers depend on dendrimer size and terminal group chemistry. The NMR (1)H and DOSY experiments indicate that the interactions between HSA and PAMAM dendrimers are relatively weak. The (1)H NMR STD experiments and MD simulations suggest that the inner shell protons of the dendrimers groups interact more strongly with HSA proteins. These interactions, which are consistently observed for different dendrimer generations (G0-NH(2)vs G4-NH(2)) and terminal groups (G4-NH(2)vs G4-OH with amidoethanol groups), suggest that PAMAM dendrimers adopt backfolded configurations as they form weak complexes with HSA proteins in aqueous solutions at physiological pH (7.4).

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

PubMed

Das, Pratyusa; Chaudhari, Sunil Kumar; Das, Asmita; Kundu, Somashree; Saha, Chabita

2018-04-24

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 10 7  M -1 and 4.2 × 10 6  M -1 , respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

Calorimetric study of mutant human lysozymes with partially introduced Ca2+ binding sites and its efficient refolding system from inclusion bodies.

PubMed

Koshiba, T; Tsumoto, K; Masaki, K; Kawano, K; Nitta, K; Kumagai, I

1998-08-01

During the process of evolution, ancestral lysozymes evolved into calcium-binding lysozymes by acquiring three critical aspartate residues at positions 86, 91 and 92. To investigate the process of the acquisition of calcium-binding ability, two of the aspartates were partially introduced into human lysozyme at positions 86, 91 and 92. These mutants (HLQ86D, HLA92D and HLQ86D/D91Q/A92D), having two critical aspartates in calcium-binding sites, were expressed in Escherichia coli as non-active inclusion bodies. For the preparation of lysozyme samples, a refolding system using thioredoxin was established. This system allowed for effective refolding of wild-type and mutant lysozymes, and 100% of activity was recovered within 4 days. The calcium ion dependence of the melting temperature (Tm) of wild-type and mutant lysozymes was investigated by differential scanning calorimetry at pH 4.5. The Tm values of wild-type, HLQ86D and HLA92D mutants were not dependent on calcium ion concentration. However, the Tm of HLQ86D/D91Q/A92D was 4 degrees higher in the presence of 50 mM CaCl2 than in its absence, and the calcium-binding constant of this mutant was estimated to be 2.25(+/-0.25)x10(2) M(-1) at pH 4.5. Moreover, the calcium-binding ability of this mutant was confirmed by the result using Sephadex G-25 gel chromatography. These results indicate that it is indispensable to have at least two aspartates at positions 86 and 92 for acquisition of calcium-binding ability. The process of the acquisition of calcium-binding site during evolution of calcium-binding lysozyme is discussed.

Utilizing time-lapse micro-CT-correlated bisphosphonate binding kinetics and soft tissue-derived input functions to differentiate site-specific changes in bone metabolism in vivo.

PubMed

Tower, R J; Campbell, G M; Müller, M; Glüer, C C; Tiwari, S

2015-05-01

The turnover of bone is a tightly regulated process between bone formation and resorption to ensure skeletal homeostasis. This process differs between bone types, with trabecular bone often associated with higher turnover than cortical bone. Analyses of bone by micro-computed tomography (micro-CT) reveal changes in structure and mineral content, but are limited in the study of metabolic activity at a single time point, while analyses of serum markers can reveal changes in bone metabolism, but cannot delineate the origin of any aberrant findings. To obtain a site-specific assessment of bone metabolic status, bisphosphonate binding kinetics were utilized. Using a fluorescently-labeled bisphosphonate, we show that early binding kinetics monitored in vivo using fluorescent molecular tomography (FMT) can monitor changes in bone metabolism in response to bone loss, stimulated by ovariectomy (OVX), or bone gain, resulting from treatment with the anabolic bone agent parathyroid hormone (PTH), and is capable of distinguishing different, metabolically distinct skeletal sites. Using time-lapse micro-CT, longitudinal bone turnover was quantified. The spine showed a significantly greater percent resorbing volume and surface in response to OVX, while mice treated with PTH showed significantly greater resorbing volume per bone surface in the spine and significantly greater forming surfaces in the knee. Correlation studies between binding kinetics and micro-CT suggest that forming surfaces, as assessed by time-lapse micro-CT, are preferentially reflected in the rate constant values while forming and resorbing bone volumes primarily affect plateau values. Additionally, we developed a blood pool correction method which now allows for quantitative multi-compartment analyses to be conducted using FMT. These results further expand our understanding of bisphosphonate binding and the use of bisphosphonate binding kinetics as a tool to monitor site-specific changes in bone metabolism in vivo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Cyanide binding to human plasma heme-hemopexin: A comparative study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it; Istituto Nazionale di Biostrutture e Biosistemi, Roma; Leboffe, Loris

Highlights: Black-Right-Pointing-Pointer Cyanide binding to ferric HHPX-heme-Fe. Black-Right-Pointing-Pointer Cyanide binding to ferrous HHPX-heme-Fe. Black-Right-Pointing-Pointer Dithionite-mediated reduction of ferric HHPX-heme-Fe-cyanide. Black-Right-Pointing-Pointer Cyanide binding to HHPX-heme-Fe is limited by ligand deprotonation. Black-Right-Pointing-Pointer Cyanide dissociation from HHPX-heme-Fe-cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0 Degree-Sign C,more » are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 {+-} 0.4) Multiplication-Sign 10{sup -6} M, k{sub on} = (6.9 {+-} 0.5) Multiplication-Sign 10{sup 1} M{sup -1} s{sup -1}, and k{sub off} = 2.8 Multiplication-Sign 10{sup -4} s{sup -1}; and H = (6 {+-} 1) Multiplication-Sign 10{sup -1} M, h{sub on} = 1.2 Multiplication-Sign 10{sup -1} M{sup -1} s{sup -1}, and h{sub off} = (7.1 {+-} 0.8) Multiplication-Sign 10{sup -2} s{sup -1}, respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 {+-} 0.8 M{sup -1/2} s{sup -1}. HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.« less

Particle dynamics and particle-cell interaction in microfluidic systems

NASA Astrophysics Data System (ADS)

Stamm, Matthew T.

Particle-laden flow in a microchannel resulting in aggregation of microparticles was investigated to determine the dependence of the cluster growth rate on the following parameters: suspension void fraction, shear strain rate, and channel-height to particle-diameter ratio. The growth rate of an average cluster was found to increase linearly with suspension void fraction, and to obey a power-law relationships with shear strain rate as S 0.9 and channel-height to particle-diameter ratio as (h/d )--3.5. Ceramic liposomal nanoparticles and silica microparticles were functionalized with antibodies that act as targeting ligands. The bio-functionality and physical integrity of the cerasomes were characterized. Surface functionalization allows cerasomes to deliver drugs with selectivity and specificity that is not possible using standard liposomes. The functionalized particle-target cell binding process was characterized using BT-20 breast cancer cells. Two microfluidic systems were used; one with both species in suspension, the other with cells immobilized inside a microchannel and particle suspension as the mobile phase. Effects of incubation time, particle concentration, and shear strain rate on particle-cell binding were investigated. With both species in suspension, the particle-cell binding process was found to be reasonably well-described by a first-order model. Particle desorption and cellular loss of binding affinity in time were found to be negligible; cell-particle-cell interaction was identified as the limiting mechanism in particle-cell binding. Findings suggest that separation of a bound particle from a cell may be detrimental to cellular binding affinity. Cell-particle-cell interactions were prevented by immobilizing cells inside a microchannel. The initial stage of particle-cell binding was investigated and was again found to be reasonably well-described by a first-order model. For both systems, the time constant was found to be inversely proportional to particle concentration. The second system revealed the time constant to obey a power-law relationship with shear strain rate as tau∝ S-.37+/-.06. Under appropriate scaling, the behavior displayed in both systems is well-described by the same exponential curve. Identification of the appropriate scaling parameters allows for extrapolation and requires only two empirical values. This could provide a major head-start in any dosage optimization studies.

Theory for rates, equilibrium constants, and Brønsted slopes in F1-ATPase single molecule imaging experiments

PubMed Central

Volkán-Kacsó, Sándor; Marcus, Rudolph A.

2015-01-01

A theoretical model of elastically coupled reactions is proposed for single molecule imaging and rotor manipulation experiments on F1-ATPase. Stalling experiments are considered in which rates of individual ligand binding, ligand release, and chemical reaction steps have an exponential dependence on rotor angle. These data are treated in terms of the effect of thermodynamic driving forces on reaction rates, and lead to equations relating rate constants and free energies to the stalling angle. These relations, in turn, are modeled using a formalism originally developed to treat electron and other transfer reactions. During stalling the free energy profile of the enzymatic steps is altered by a work term due to elastic structural twisting. Using biochemical and single molecule data, the dependence of the rate constant and equilibrium constant on the stall angle, as well as the Børnsted slope are predicted and compared with experiment. Reasonable agreement is found with stalling experiments for ATP and GTP binding. The model can be applied to other torque-generating steps of reversible ligand binding, such as ADP and Pi release, when sufficient data become available. PMID:26483483

Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies.

PubMed

Patra, Malay; Mitra, Madhurima; Chakrabarti, Abhijit; Mukhopadhyay, Chaitali

2014-01-01

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (Kd) of .50 and .17 μM, for prodan, and .04 and .02 μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain > SH3 domain > Self-association domain > N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.

Binding of caffeine with caffeic acid and chlorogenic acid using fluorescence quenching, UV/vis and FTIR spectroscopic techniques.

PubMed

Belay, Abebe; Kim, Hyung Kook; Hwang, Yoon-Hwae

2016-03-01

The interactions of caffeine (CF) with chlorogenic acid (CGA) and caffeic acid (CFA) were investigated by fluorescence quenching, UV/vis and Fourier transform infrared (FTIR) spectroscopic techniques. The results of the study indicated that the fluorescence quenching between caffeine and hydroxycinnamic acids could be rationalized in terms of static quenching or the formation of non-fluorescent CF-CFA and CF-CGA complexes. From fluorescence quenching spectral analysis, the quenching constant (KSV), quenching rate constant (kq), number of binding sites (n), thermodynamic properties and conformational changes of the interaction were determined. The quenching constants (KSV) between CF and CGA, CFA are 1.84 × 10(4) and 1.04 × 10(4) L/mol at 298 K and their binding site n is ~ 1. Thermodynamic parameters determined using the Van't Hoff equation indicated that hydrogen bonds and van der Waal's forces have a major role in the reaction of caffeine with caffeic acid and chlorogenic acid. The 3D fluorescence, UV/vis and FTIR spectra also showed that the binding of CF with CFA and CGA induces conformational changes in CFA and CGA. Copyright © 2015 John Wiley & Sons, Ltd.

Critical evaluation of methods to incorporate entropy loss upon binding in high-throughput docking.

PubMed

Salaniwal, Sumeet; Manas, Eric S; Alvarez, Juan C; Unwalla, Rayomand J

2007-02-01

Proper accounting of the positional/orientational/conformational entropy loss associated with protein-ligand binding is important to obtain reliable predictions of binding affinity. Herein, we critically examine two simplified statistical mechanics-based approaches, namely a constant penalty per rotor method, and a more rigorous method, referred to here as the partition function-based scoring (PFS) method, to account for such entropy losses in high-throughput docking calculations. Our results on the estrogen receptor beta and dihydrofolate reductase proteins demonstrate that, while the constant penalty method over-penalizes molecules for their conformational flexibility, the PFS method behaves in a more "DeltaG-like" manner by penalizing different rotors differently depending on their residual entropy in the bound state. Furthermore, in contrast to no entropic penalty or the constant penalty approximation, the PFS method does not exhibit any bias towards either rigid or flexible molecules in the hit list. Preliminary enrichment studies using a lead-like random molecular database suggest that an accurate representation of the "true" energy landscape of the protein-ligand complex is critical for reliable predictions of relative binding affinities by the PFS method. Copyright 2006 Wiley-Liss, Inc.

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

NASA Astrophysics Data System (ADS)

Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary; Benkovic, Stephen J.; Hammes, Gordon G.

2002-10-01

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

PubMed Central

Wharton, Christopher W.; Cornish-Bowden, Athel; Brocklehurst, Keith; Crook, Eric M.

1974-01-01

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S0]/vi (initial substrate concn./initial velocity) versus [S0] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S0]/vi versus [S0] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S0]/vi versus [S0] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25°C is characterized by Km1 (the dissociation constant of ES)=1.22±0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57×10−2±0.32×10−2s−1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38±0.06m, and k′ (the rate constant for the breakdown of SES to E+P+S)=0.45±0.04s−1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of α-N-benzoyl-l-arginine ethyl ester and of α-N-benzoyl-l-arginine amide; Km1 and k for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine amide hydrolysis and Kas and k′ for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kcat. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km=52±4mm, kcat.=2.80±0.1s−1 at pH7.0, I=0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain. PMID:4455211

Investigation into the interaction of losartan with human serum albumin and glycated human serum albumin by spectroscopic and molecular dynamics simulation techniques: A comparison study.

PubMed

Moeinpour, Farid; Mohseni-Shahri, Fatemeh S; Malaekeh-Nikouei, Bizhan; Nassirli, Hooriyeh

2016-09-25

The interaction between losartan and human serum albumin (HSA), as well as its glycated form (gHSA) was studied by multiple spectroscopic techniques and molecular dynamics simulation under physiological conditions. The binding information, including the binding constants, effective quenching constant and number of binding sites showed that the binding partiality of losartan to HSA was higher than to gHSA. The findings of three-dimensional fluorescence spectra demonstrated that the binding of losartan to HSA and gHSA would alter the protein conformation. The distances between Trp residue and the binding sites of the drug were evaluated on the basis of the Förster theory, and it was indicated that non-radiative energy transfer from HSA and gHSA to the losartan happened with a high possibility. According to molecular dynamics simulation, the protein secondary and tertiary structure changes were compared in HSA and gHSA for clarifying the obtained results. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Mussel-inspired histidine-based transient network metal coordination hydrogels

PubMed Central

Fullenkamp, Dominic E.; He, Lihong; Barrett, Devin G.; Burghardt, Wesley R.; Messersmith, Phillip B.

2013-01-01

Transient network hydrogels cross-linked through histidine-divalent cation coordination bonds were studied by conventional rheologic methods using histidine-modified star poly(ethylene glycol) (PEG) polymers. These materials were inspired by the mussel, which is thought to use histidine-metal coordination bonds to impart self-healing properties in the mussel byssal thread. Hydrogel viscoelastic mechanical properties were studied as a function of metal, pH, concentration, and ionic strength. The equilibrium metal-binding constants were determined by dilute solution potentiometric titration of monofunctional histidine-modified methoxy-PEG and were found to be consistent with binding constants of small molecule analogs previously studied. pH-dependent speciation curves were then calculated using the equilibrium constants determined by potentiometric titration, providing insight into the pH dependence of histidine-metal ion coordination and guiding the design of metal coordination hydrogels. Gel relaxation dynamics were found to be uncorrelated with the equilibrium constants measured, but were correlated to the expected coordination bond dissociation rate constants. PMID:23441102

Constants for mercury binding by organic matter isolates from the Florida Everglades

USGS Publications Warehouse

Benoit, J.M.; Mason, R.P.; Gilmour, C.C.; Aiken, G.R.

2001-01-01

Dissolved organic matter (DOM) has been implicated as an important complexing agent for Hg that can affect its mobility and bioavailability in aquatic ecosystems. However, binding constants for natural Hg-DOM complexes are not well known. We employed a competitive ligand approach to estimate conditional stability constants for Hg complexes with DOM isolates collected from Florida Everglades surface waters. The isolates examined were the hydrophobic fraction of DOM from a eutrophic, sulfidic site (F1-HPoA) and the hydrophilic fraction from an oligotrophic, low-sulfide site (2BS-HPiA). Our experimental determinations utilized overall octanol-water partitioning coefficients (Dow) for 203Hg at 0.01 M chloride and across pH and DOM concentration gradients. Use of this radioisotope allowed rapid determinations of Hg concentrations in both water and octanol phases without problems of matrix interference. Conditional stability constants (1 = 0.06, 23??C) were log K??? = 11.8 for F1-HPoA and log K' = 10.6 for 2BS-HPiA. These are similar to previously published stability constants for Hg binding to low-molecular-weight thiols. Further, F1-HPoA showed a pH-dependent decline in Dow that was consistent with models of Hg complexation with thiol groups as the dominant Hg binding sites in DOM. These experiments demonstrate that the DOM isolates are stronger ligands for Hg than chloride ion or ethylenediamine-tetraacetic acid. Speciation calculations indicate that at the DOM concentrations frequently measured in Everglades, 20 to 40 ??M, significant complexation of Hg by DOM would be expected in aerobic (sulfide-free) surface waters. Copyright ?? 2001 Elsevier Science Ltd.

Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8.

PubMed

Eron, Scott J; MacPherson, Derek J; Dagbay, Kevin B; Hardy, Jeanne A

2018-05-18

Zinc is emerging as a widely used and important biological regulatory signal. Cellular zinc levels are tightly regulated by a complex array of zinc importers and exporters to control processes such as apoptotic cell death. While caspase inhibition by zinc has been reported previously, the reported inhibition constants were too weak to suggest a critical biological role for zinc-mediated inhibition. In this work, we have adopted a method of assessing available zinc. This allowed assessment of accurate inhibition constants for apoptotic caspases, caspase-3, -6, -7, and -8. Each of these caspases are inhibited by zinc at intracellular levels but with widely differing inhibition constants and different zinc binding stoichiometries. Caspase-3, -6, and -8 appear to be constitutively inhibited by typical zinc levels, and this inhibition must be lifted to allow activation. The inhibition constant for caspase-7 (76 nM) is much weaker than for the other apoptotic caspases (2.6-6.9 nM) suggesting that caspase-7 is not inactivated by normal zinc concentrations but can be inhibited under conditions of zinc stress. Caspase-3, -7, and -8 were found to bind three, one, and two zincs, respectively. In each of these caspases, zinc was present in the active site, in contrast to caspase-6, which binds one zinc allosterically. The most notable new mechanism to emerge from this work is for zinc-mediated inhibition of caspase-8. Zinc binds caspase-8 directly at the active site and at a second site. Zinc binding inhibits formation of the caspase-8 dimer, the activated form of the enzyme. Together these findings suggest that zinc plays a critical role in regulation of apoptosis by direct inactivation of caspases, in a manner that is unique for each caspase.

Mechanism of host-guest complexation by cucurbituril.

PubMed

Márquez, César; Hudgins, Robert R; Nau, Werner M

2004-05-12

The factors affecting host-guest complexation between the molecular container compound cucurbit[6]uril (CB6) and various guests in aqueous solution are studied, and a detailed complexation mechanism in the presence of cations is derived. The formation of the supramolecular complex is studied in detail for cyclohexylmethylammonium ion as guest. The kinetics and thermodynamics of complexation is monitored by NMR as a function of temperature, salt concentration, and cation size. The binding constants and the ingression rate constants decrease with increasing salt concentration and cation-binding constant, in agreement with a competitive binding of the ammonium site of the guest and the metal cation with the ureido carbonyl portals of CB6. Studies as a function of guest size indicate that the effective container volume of the CB6 cavity is approximately 105 A(3). It is suggested that larger guests are excluded for two reasons: a high activation barrier for ingression imposed by the tight CB6 portals and a destabilization of the complex due to steric repulsion inside. For example, in the case of the nearly spherical azoalkane homologues 2,3-diazabicyclo[2.2.1]hept-2-ene (DBH, volume ca. 96 A(3)) and 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO, volume ca. 110 A(3)), the former forms the CB6 complex promptly with a sizable binding constant (1300 M(-1)), while the latter does not form a complex even after several months at optimized complexation conditions. Molecular mechanics calculations are performed for several CB6/guest complexes. A qualitative agreement is found between experimental and calculated activation energies for ingression as a function of both guest size and state of protonation. The potential role of constrictive binding by CB6 is discussed.

Biophysical insights into the interaction of hen egg white lysozyme with therapeutic dye clofazimine: modulation of activity and SDS induced aggregation of model protein.

PubMed

Ajmal, Mohammad Rehan; Chaturvedi, Sumit Kumar; Zaidi, Nida; Alam, Parvez; Zaman, Masihuz; Siddiqi, Mohammad Khursheed; Nusrat, Saima; Jamal, Mohammad Sarwar; Mahmoud, Mohamed H; Badr, Gamal; Khan, Rizwan Hasan

2017-08-01

The present study details the binding process of clofazimine to hen egg white lysozyme (HEWL) using spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), and molecular docking techniques. Clofazimine binds to the protein with binding constant (K b ) in the order of 1.57 × 10 4 at 298 K. Binding process is spontaneous and exothermic. Molecular docking results suggested the involvement of hydrogen bonding and hydrophobic interactions in the binding process. Bacterial cell lytic activity in the presence of clofazimine increased to more than 40% of the value obtained with HEWL only. Interaction of the drug with HEWL induced ordered secondary structure in the protein and molecular compaction. Clofazimine also effectively inhibited the sodium dodecyl sulfate (SDS) induced amyloid formation in HEWL and caused disaggregation of preformed fibrils, reinforcing the notion that there is involvement of hydrophobic interactions and hydrogen bonding in the binding process of clofazimine with HEWL and clofazimine destabilizes the mature fibrils. Further, TEM images confirmed that fibrillar species were absent in the samples where amyloid induction was performed in the presence of clofazimine. As clofazimine is a drug less explored for the inhibition of fibril formation of the proteins, this study reports the inhibition of SDS-induced amyloid formation of HEWL by clofazimine, which will help in the development of clofazimine-related molecules for the treatment of amyloidosis.

Monitoring binding affinity between drug and α1-acid glycoprotein in real time by Venturi easy ambient sonic-spray ionization mass spectrometry.

PubMed

Liu, Ning; Lu, Xin; Yang, YuHan; Yao, Chen Xi; Ning, BaoMing; He, Dacheng; He, Lan; Ouyang, Jin

2015-10-01

A new approach for monitoring the binding affinity between drugs and alpha 1-acid glycoprotein in real time was developed based on a combination of drug-protein reaction followed by Venturi easy ambient sonic-spray ionization mass spectrometry determination of the free drug concentrations. A known basic drug, propranolol was used to validate the new built method. Binding constant values calculated by venturi easy ambient sonic-spray ionization mass spectrometry was in good accordance with a traditional ultrafiltration combined with high performance liquid chromatography method. Then six types of basic drugs were used as the samples to conduct the real time analysis. Upon injection of alpha 1-acid glycoprotein to the drug mixture, the ion chromatograms were extracted to show the changes in the free drug concentrations in real time. By observing the drop-out of six types of drugs during the whole binding reaction, the binding affinities of different drugs were distinguished. A volume shift validating experiment and an injection delay correcting experiment were also performed to eliminate extraneous factors and verify the reliability of our experiment. Therefore, the features of Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS) and the experimental results indicate that our technique is likely to become a powerful tool for monitoring drug-AGP binding affinity in real time. Copyright © 2015 Elsevier B.V. All rights reserved.

Probing the interaction of human serum albumin with DPPH in the absence and presence of the eight antioxidants.

PubMed

Li, Xiangrong; Chen, Dejun; Wang, Gongke; Lu, Yan

2015-02-25

Albumin represents a very abundant and important circulating antioxidant in plasma. DPPH radical is also called 2,2-diphenyl-1-picrylhydrazyl. It has been widely used for measuring the efficiency of antioxidants. In this paper, the ability of human serum albumin (HSA) to scavenge DPPH radical was investigated using UV-vis absorption spectra. The interaction between HSA and DPPH was investigated in the absence and presence of eight popular antioxidants using fluorescence spectroscopy. These results indicate the antioxidant activity of HSA against DPPH radical is similar to glutathione and the value of IC50 is 5.200×10(-5) mol L(-1). In addition, the fluorescence experiments indicate the quenching mechanism of HSA, by DPPH, is a static process. The quenching process of DPPH with HSA is easily affected by the eight antioxidants, however, they cannot change the quenching mechanism of DPPH with HSA. The binding of DPPH to HSA primarily takes place in subdomain IIA and exists two classes of binding sites with two different interaction behaviors. The decreased binding constants and the number of binding sites of DPPH with HSA by the introduction of the eight antioxidants may result from the competition of the eight antioxidants and DPPH binding to HSA. The binding of DPPH to HSA may induce the micro-environment of the lone Trp-214 from polar to slightly nonpolar. Copyright © 2014 Elsevier B.V. All rights reserved.

A spectroscopic and molecular docking approach on the binding of tinzaparin sodium with human serum albumin

NASA Astrophysics Data System (ADS)

Abdullah, Saleh M. S.; Fatma, Sana; Rabbani, Gulam; Ashraf, Jalaluddin M.

2017-01-01

Protein bound toxins are poorly removed by conventional extracorporeal therapies. Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients with cancer. The interaction between tinzaparin, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by tinzaparin (TP). The binding constants and binding stoichiometry can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG° reveals that the binding process is a spontaneous process. Thermodynamic analysis shows that the HSA-TP complex formation occurs via hydrogen bonds, hydrophobic interactions and undergoes slight structural changes as evident by far-UV CD. It indicated that the hydrophobic interactions play a main role in the binding of TP to human serum albumin. In addition, the distance between TP (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 2.21 nm according to the Förster's resonance energy transfer theory. For the deeper understanding of the interaction, thermodynamic, and molecular docking studies were performed as well. Our docking results suggest that TP forms stable complex with HSA (Kb ∼ 104) and its primary binding site is located in subdomain IIA (Sudlow Site I). The results obtained herein will be of biological significance in pharmacology and clinical medicine.

Combined spectroscopies and molecular docking approach to characterizing the binding interaction of enalapril with bovine serum albumin.

PubMed

Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi; Shi, Jie-Hua

2017-06-01

The binding interaction between bovine serum albumin (BSA) and enalapril (ENPL) at the imitated physiological conditions (pH = 7.4) was investigated using UV-vis absorption spectroscopy (UV-vis), fluorescence emission spectroscopy (FES), synchronous fluorescence spectroscopy (SFS), Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) and molecular docking methods. It can be deduced from the experimental results from the steady-state fluorescence spectroscopic titration that the intrinsic BSA fluorescence quenching mechanism induced by ENPL is static quenching, based on the decrease in the BSA quenching constants in the presence of ENPL with increase in temperature and BSA quenching rates >10 10  L mol -1  sec -1 . This result indicates that the ENPL-BSA complex is formed through an intermolecular interaction of ENPL with BSA. The main bonding forces for interaction of BSA and ENPL are van der Waal's forces and hydrogen bonding interaction based on negative values of Gibbs free energy change (ΔG 0 ), enthalpic change (ΔH 0 ) and entropic change (ΔS 0 ). The binding of ENPL with BSA is an enthalpy-driven process due to |ΔH°| > |TΔS°| in the binding process. The results of competitive binding experiments and molecular docking confirm that ENPL binds in BSA sub-domain IIA (site I) and results in a slight change in BSA conformation, but BSA still retains its α-helical secondary structure. Copyright © 2016 John Wiley & Sons, Ltd.

The Influence of Spatial Variation in Chromatin Density Determined by X-Ray Tomograms on the Time to Find DNA Binding Sites

PubMed Central

Larabell, Carolyn A.; Le Gros, Mark A.; McQueen, David M.; Peskin, Charles S.

2014-01-01

In this work, we examine how volume exclusion caused by regions of high chromatin density might influence the time required for proteins to find specific DNA binding sites. The spatial variation of chromatin density within mouse olfactory sensory neurons is determined from soft X-ray tomography reconstructions of five nuclei. We show that there is a division of the nuclear space into regions of low-density euchromatin and high-density heterochromatin. Volume exclusion experienced by a diffusing protein caused by this varying density of chromatin is modeled by a repulsive potential. The value of the potential at a given point in space is chosen to be proportional to the density of chromatin at that location. The constant of proportionality, called the volume exclusivity, provides a model parameter that determines the strength of volume exclusion. Numerical simulations demonstrate that the mean time for a protein to locate a binding site localized in euchromatin is minimized for a finite, nonzero volume exclusivity. For binding sites in heterochromatin, the mean time is minimized when the volume exclusivity is zero (the protein experiences no volume exclusion). An analytical theory is developed to explain these results. The theory suggests that for binding sites in euchromatin there is an optimal level of volume exclusivity that balances a reduction in the volume searched in finding the binding site, with the height of effective potential barriers the protein must cross during the search process. PMID:23955281

Elucidation of intermolecular interaction of bovine serum albumin with Fenhexamid: A biophysical prospect.

PubMed

Shi, Jie-Hua; Lou, Yan-Yue; Zhou, Kai-Li; Pan, Dong-Qi

2018-03-01

Fenhexamid, as a hydroxyanilide, is widely applied to control Botrytis cinerea for protecting crops and fruits. But it could adversely affect human and animals health due to accumulation of residues in food production. Here, the affinity characteristics of fenhexamid on bovine serum albumin (BSA) was studied via a series of spectroscopic methods such as steady-state fluorescence spectroscopy, ultraviolet spectroscopy (UV), synchronous fluorescence spectroscopy (SFS), 3D fluorescence spectroscopy, and fourier transform infrared spectroscopy (FT-IR). The experimental results illustrated that the fluorescence quenching mechanism of BSA induced by fenhexamid was a static quenching. The binding constant (K b ) of fenhexamid with BSA was 2.399 × 10 4  M -1 at 298 K and the combination ratio was about 1:1. The competitive experiment demonstrated that fenhexamid was binding on the BSA at site II (subdomain IIIA), which was confirmed by the molecular docking studies. The negative values of thermodynamic parameter (ΔH 0 , ΔS 0 and ΔG 0 ) revealed that the reaction of fenhexamid with BSA could proceed spontaneously, the van der Waals force and hydrogen bonding interaction conducted the main effect, and the binding process was enthalpy-driven. What's more, the 8-Anilino-1-naphthalenesulfonate (ANS) and sucrose binding studies were also performed and further verified the binding force between BSA and fenhexamid. Copyright © 2018 Elsevier B.V. All rights reserved.

Insights from spectroscopic and in-silico techniques for the exploitation of biomolecular interactions between Human serum albumin and Paromomycin.

PubMed

Raza, Muslim; Jiang, Yang; Wei, Yun; Ahmad, Aftab; Khan, Ajmal; Qipeng, Yuan

2017-09-01

The study of molecular interactions of drug-protein are extremely important from the biological aspect in all living organisms, and therefore such type of investigation hold a tremendous significance in rational drug design and discovery. In the present study, the molecular interactions between paromomycin (PAR) and human serum albumin (HSA) have been studied by different biophysical techniques and validated by in-silico approaches. The results obtained from Ultraviolet-visible spectroscopy (UV) and Fourier transform infrared spectroscopy (FT-IR) demonstrated a remarkable change upon the complexation of PAR with HSA. Circular Dichroism (CD), Dynamic Light Scattering (DLS) and Resonance Rayleigh scattering (RRS) results revealed a significant secondary structure alteration and reduction of hydrodynamic radii upon the conjugation of PAR with HSA. The fluorescence spectroscopy results also apparently revealed the static quenching mechanism. The number of binding sites, binding constants, and Gibbs free energy values were calculated to illustrate the nature of intermolecular interactions. Similarly, the in-silico docking and molecular dynamics simulation clearly explain the theoretical basis of the binding mechanism of PAR with HSA. The experimental and docking approaches suggested that PAR binds to the hydrophobic cavity site I of HSA. The finding of present investigation will provide binding insight of PAR and associated alterations in the stability and conformation of HSA. Copyright © 2017 Elsevier B.V. All rights reserved.

Investigation of Non-Covalent Interactions of Aflatoxins (B1, B2, G1, G2, and M1) with Serum Albumin

PubMed Central

Poór, Miklós; Bálint, Mónika; Hetényi, Csaba; Gődér, Beatrix; Kunsági-Máté, Sándor; Lemli, Beáta

2017-01-01

Aflatoxins are widely spread mycotoxins produced mainly by Aspergillus species. Consumption of aflatoxin-contaminated foods and drinks causes serious health risks for people worldwide. It is well-known that the reactive epoxide metabolite of aflatoxin B1 (AFB1) forms covalent adducts with serum albumin. However, non-covalent interactions of aflatoxins with human serum albumin (HSA) are poorly characterized. Thus, in this study the complex formation of aflatoxins was examined with HSA applying spectroscopic and molecular modelling studies. Our results demonstrate that aflatoxins form stable complexes with HSA as reflected by binding constants between 2.1 × 104 and 4.5 × 104 dm3/mol. A binding free energy value of −26.90 kJ mol−1 suggests a spontaneous binding process between AFB1 and HSA at room-temperature, while the positive entropy change of 55.1 JK−1 mol−1 indicates a partial decomposition of the solvation shells of the interacting molecules. Modeling studies and investigations with site markers suggest that Sudlow’s Site I of subdomain IIA is the high affinity binding site of aflatoxins on HSA. Interaction of AFB1 with bovine, porcine, and rat serum albumins was also investigated. Similar stabilities of the examined AFB1-albumin complexes were observed suggesting the low species differences of the albumin-binding of aflatoxins. PMID:29068381

Rapid characterization of a novel taspine derivative-HMQ1611 binding to EGFR by a cell membrane chromatography method.

PubMed

Du, Hui; Lv, Nan; Wang, Sicen; He, Langchong

2013-05-01

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor's concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (K(D)) was (9.11 ± 1.89) × 10(-6) M; HMQ1611 had two major binding regions on the EGFR, and the K(D) values obtained from the model were (2.39 ± 0.33) × 10(-7) and (3.87 ± 0.93) × 10(-5) M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

Revealing multi-binding sites for taspine to VEGFR-2 by cell membrane chromatography zonal elution.

PubMed

Du, Hui; Wang, Sicen; Ren, Jing; Lv, Nan; He, Langchong

2012-03-01

A new high-expression vascular endothelial growth factor receptor-2 (VEGFR-2) cell membrane chromatography (CMC) method was developed to investigate the affinity of ligands for VEGFR-2. An HEK293 VEGFR-2/CMC system was applied to specifically recognize ligands acting on VEGFR-2. Sorafenib was used as a mobile phase additive to evaluate the effect of the marker's concentration on the retention of sorafenib and taspine, respectively. The relationship among the retention, the types of binding sites and the affinity of taspine binding to VEGFR-2 has also been concerned. The retention behavior indicated that sorafenib had two major binding regions on VEGFR-2, and that taspine might act as a multi-target VEGFR-2 inhibitor with similar biological activity to sorafenib. The equilibrium dissociation constants (K(D)) obtained from the model are (5.25 ± 0.31) × 10⁻⁷ and (9.88 ± 0.54) × 10⁻⁵ mol L⁻¹ for sorafenib at the high- and low-affinity sites, respectively, and the corresponding values for taspine are (3.88 ± 0.31) × 10⁻⁶ and (7.04 ± 0.49)×10⁻⁵ mol L⁻¹. The two types of binding sites contributed about a 1:2 ratio on the retention of taspine. Copyright © 2012 Elsevier B.V. All rights reserved.

A simple model for solvation in mixed solvents. Applications to the stabilization and destabilization of macromolecular structures.

PubMed

Schellman, J A

1990-08-31

The properties of a simple model for solvation in mixed solvents are explored in this paper. The model is based on the supposition that solvent replacement is a simple one-for-one substitution reaction at macromolecular sites which are independent of one another. This leads to a new form for the binding polynomial in which all terms are associated with ligand interchange rather than ligand addition. The principal solvent acts as one of the ligands. Thermodynamic analysis then shows that thermodynamic binding (i.e., selective interaction) depends on the properties of K'-1, whereas stoichiometric binding (site occupation) depends on K'. K' is a 'practical' interchange equilibrium constant given by (f3/f1)K, where K is the true equilibrium constant for the interchange of components 3 and 1 on the site and f3 and f4 denote their respective activity coefficients on the mole fraction scale. Values of K' less than unity lead to negative selective interaction. It is selective interaction and not occupation number which determines the thermodynamic effects of solvation. When K' greater than 100 on the mole fraction scale or K' greater than 2 on the molality scale (in water), the differences between stoichiometric binding and selective interaction become less than 1%. The theory of this paper is therefore necessary only for very weak binding constants. When K'-1 is small, large concentrations of the added solvent component are required to produce a thermodynamic effect. Under these circumstances the isotherms for the selective interaction and for the excess (or transfer) free energy are strongly dependent on the behavior of the activity coefficients of both solvent components. Two classes of behavior are described depending on whether the components display positive or negative deviations from Raoult's law. Examples which are discussed are aqueous solutions of urea and guanidinium chloride for positive deviations and of sucrose and glucose for negative deviations. Examination of the few studies which have been reported in the literature shows that most of the qualitative features of the stabilization of proteins by sugars and their destabilization by urea and guanidinium chloride are faithfully represented with the model. This includes maxima in the free energy of stabilization and destabilization, decreased and zero selective interaction at high concentrations, etc. These phenomena had no prior explanation. Deficiencies in the model as a representation of solvation in aqueous solution are discussed in the appendix.

Photochemical and DFT studies on DNA-binding ability and antibacterial activity of lanthanum(III)-phenanthroline complex

NASA Astrophysics Data System (ADS)

Niroomand, Sona; Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Jahani, Shohreh; Moodi, Asieh

2017-02-01

The binding of the lanthanum(III) complex containing 1,10-phenanthroline (phen), [La(phen)3Cl3·OH2], to DNA is investigated by absorption and emission methods. This complex shows absorption decreasing in a charge transfer band, and fluorescence decrement when it binds to DNA. Electronic absorption spectroscopy (UV-Vis), fluorescence spectra, iodide quenching experiments, salt effect and viscosity measurements, ethidium bromide (EB) competition test, circular dichroism (CD) spectra as well as variable temperature experiments indicate that the La(III) complex binds to fish salmon (FS) DNA, presumably via groove binding mode. The binding constants (Kb) of the La(III) complex with DNA is (2.55 ± 0.02) × 106 M-1. Furthermore, the binding site size, n, the Stern-Volmer constant KSV and thermodynamic parameters; enthalpy change (ΔH0) and entropy change (ΔS0) and Gibb's free energy (ΔG0), are calculated according to relevant fluorescent data and the Van't Hoff equation. The La(III) complex has been screened for its antibacterial activities by the disc diffusion method. Also, in order to supplement the experimental findings, DFT computation and NBO analysis are carried out.

Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin.

PubMed

Xu, Yujing; Hong, Tingting; Chen, Xueping; Ji, Yibing

2017-05-01

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S-omeprazole, S-OME) and its R-enantiomer (R-omeprazole, R-OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10 3 M -1 and 5.36 × 10 3 M -1 , respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Applications of isothermal titration calorimetry - the research and technical developments from 2011 to 2015.

PubMed

Falconer, Robert J

2016-10-01

Isothermal titration calorimetry is a widely used biophysical technique for studying the formation or dissociation of molecular complexes. Over the last 5 years, much work has been published on the interpretation of isothermal titration calorimetry (ITC) data for single binding and multiple binding sites. As over 80% of ITC papers are on macromolecules of biological origin, this interpretation is challenging. Some researchers have attempted to link the thermodynamics constants to events at the molecular level. This review highlights work carried out using binding sites characterized using x-ray crystallography techniques that allow speculation about individual bond formation and the displacement of individual water molecules during ligand binding and link these events to the thermodynamic constants for binding. The review also considers research conducted with synthetic binding partners where specific binding events like anion-π and π-π interactions were studied. The revival of assays that enable both thermodynamic and kinetic information to be collected from ITC data is highlighted. Lastly, published criticism of ITC research from a physical chemistry perspective is appraised and practical advice provided for researchers unfamiliar with thermodynamics and its interpretation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins).

PubMed

Athwal, Navjot Singh; Alagurajan, Jagannathan; Andreotti, Amy H; Hargrove, Mark S

2016-10-18

Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.

New dinuclear palladium(II) complexes: Studies of the nucleophilic substitution reactions, DNA/BSA interactions and cytotoxic activity.

PubMed

Ćoćić, Dušan; Jovanović, Snežana; Nišavić, Marija; Baskić, Dejan; Todorović, Danijela; Popović, Suzana; Bugarčić, Živadin D; Petrović, Biljana

2017-10-01

Six new dinuclear Pd(II) complexes, [{Pd(2,2'-bipy)Cl} 2 (μ-pz)](ClO 4 ) 2 (Pd1), [{Pd(dach)Cl} 2 (μ-pz)](ClO 4 ) 2 (Pd2), [{Pd(en)Cl} 2 (μ-pz)](ClO 4 ) 2 (Pd3), [{Pd(2,2'-bipy)Cl} 2 (μ-4,4'-bipy)](ClO 4 ) 2 (Pd4), [{Pd(dach)Cl} 2 (μ-4,4'-bipy)](ClO 4 ) 2 (Pd5) and [{Pd(en)Cl} 2 (μ-4,4'-bipy)](ClO 4 ) 2 (Pd6) (where 2,2'-bipy=2,2'-bipyridyl, pz=pyrazine, dach=trans-(±)-1,2-diaminocyclohexane, en=ethylenediamine, 4,4'-bipy=4,4'-bipyridyl) have been synthesized and characterized by elemental microanalysis, IR, 1 H NMR and MALDI-TOF mass spectrometry. The pK a values of corresponding diaqua complexes were determined by spectrophotometric pH titration. Substitution reactions with thiourea (Tu), l-methionine (l-Met), l-cysteine (l-Cys), l-histidine (l-His) and guanosine-5'-monophosphate (5'-GMP) were studied under the pseudo-first order conditions at pH7.2. Reactions of Pd1 with Tu, l-Met and l-Cys were followed by decomposition of complexes, while structures of dinuclear complexes were preserved during the substitution with nitrogen donors. Interactions with calf-thymus DNA (CT-DNA) were followed by absorption spectroscopy and fluorescence quenching measurements. All complexes can bind to CT-DNA exhibiting high intrinsic binding constants (K b =10 4 -10 5 M -1 ). Competitive studies with ethidium bromide (EB) have shown that complexes can displace DNA-bound EB. High values of binding constants towards bovine serum albumin protein (BSA) indicate good binding affinity. Finally, all complexes showed moderate to high cytotoxic activity against HeLa (human cervical epithelial carcinoma cell lines) and MDA-MB-231 (human breast epithelial carcinoma cell lines) tumor cell lines inducing apoptotic type cell death, whereas normal fibroblasts were significantly less sensitive. The impact on cell cycle of these cells was distinctive, where Pd4, Pd5 and Pd6 showed the most prominent effect arresting MDA-MB-231 (human lung fibroblast cell lines) cell in G1/S phase of cell cycle. Copyright © 2017 Elsevier Inc. All rights reserved.

The structural and electronical factors that contribute affinity for the time-dependent inhibition of PGHS-1 by indomethacin, diclofenac and fenamates

NASA Astrophysics Data System (ADS)

Pouplana, R.; Pérez, C.; Sánchez, J.; Lozano, J. J.; Puig-Parellada, P.

1999-05-01

PGHS-1 and PGHS-2 are the targets of nonsteroidal anti- inflammatory drugs (NSAIDs). It appears that the high degree of selectivity for inhibition of PGHS-2 shown by certain compounds is the result of two mechanisms (time-dependent and time-independent inhibition), by which they interact with each isoform. The fenamic acids can be divided into competitive inhibitors of substrate binding and competitive inhibitors that cause time-dependent losses of cyclooxygenase activity. The cyclooxygenase activity was measured by oxygen consumption following preincubation of the enzyme and the inhibitor for increasing periods of time. The rate constants associated with binding inhibition kinetics and structure-activity relationships were calculated for a large number of fenamates, diclofenac and indomethacin. The KI* values are similar but the individual rate constants are markedly different: KI is two-fold lower, and k2 is six-fold slower for diclofenac than for indomethacin. All the active time-dependent compounds show MEPs with a negative conical surface, with their vertex on the minimum of the carboxyl group, which extends around the first aromatic ring to the central region. The conical surface keeps an open angle of 61° or larger, and a close contact surface with the residues Ala527, Ileu523, Val349, and Ser530, in the zones surrounding the bridging amino group and the chlorine atoms for meclofenamate and diclofenac, or in the region around the carbonyl group for indomethacin. The KI* and IC50 values indicate that the interactions that promote the slow binding kinetics must be examined in relation to the reaction energies of formation (ΔHr) of an ionic bond between the deprotonated carboxylic acid group of acid NSAIDs with the monocationic guanidinum group of Arg120, the free energies of solvation in aqueous solution, and the molecular volumes measured. Presumably indomethacin, diclofenac and meclofenamate cause the enzyme to undergo a subtle conformational change to a form that binds compounds even more tightly, with some slight structural changes confined to reorientations of the Arg277 and Gln358 side chains. These results show that the model has reliably chosen regions of biological significance consistent with both the X-ray crystallographic and kinetic results.

Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin

PubMed Central

Weisemann, Jasmin; Stern, Daniel; Mahrhold, Stefan; Dorner, Brigitte G.; Rummel, Andreas

2016-01-01

Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A‐C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain HC of BoNT/A. Here, we deciphered the BoNT/A‐SV2 interactions in more detail. In pull down assays, the binding of HCA to SV2-LD4 isoforms decreases from SV2C >> SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for HCA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of HCA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A‐SV2C interaction is of different nature compared to BoNT/B‐Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone–backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A‐SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic. PMID:27196927

Determination of thermodynamic parameters for complexation of calcium and magnesium with chondroitin sulfate isomers using isothermal titration calorimetry: Implications for calcium kidney-stone research

NASA Astrophysics Data System (ADS)

Rodgers, Allen L.; Jackson, Graham E.

2017-04-01

Chondroitin sulfate (CS) occurs in human urine. It has several potential binding sites for calcium and as such may play an inhibitory role in calcium oxalate and calcium phosphate (kidney stone disease by reducing the supersaturation (SS) and crystallization of these salts. Urinary magnesium is also a role player in determining speciation in stone forming processes. This study was undertaken to determine the thermodynamic parameters for binding of the disaccharide unit of two different CS isomers with calcium and magnesium. These included the binding constant K. Experiments were performed using an isothermal titration calorimeter (ITC) at 3 different pH levels in the physiological range in human urine. Data showed that interactions between the CS isomers and calcium and magnesium occur via one binding site, thought to be sulfate, and that log K values are 1.17-1.93 and 1.77-1.80 for these two metals respectively. Binding was significantly stronger in Mg-CS than in Ca-CS complexes and was found to be dependent on pH in the latter but not in the former. Furthermore, binding in Ca-CS complexes was dependent on the location of the sulfate binding site. This was not the case in the Mg-CS complexes. Interactions were shown to be entropy driven and enthalpy unfavourable. These findings can be used in computational modeling studies to predict the effects of the calcium and magnesium CS complexes on the speciation of calcium and the SS of calcium salts in real urine samples.

Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase

PubMed Central

Yuan, Ji; Chow, Dar-Chone; Huang, Wanzhi; Palzkill, Timothy

2011-01-01

The β-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits treatment strategies for Staphylococcus infections. The goal of this study was to determine the binding affinity of BLIP for S. aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (Ki) for the PC1 β-lactamase was measured at 350 nM and isothermal titration calorimetry (ITC) experiments indicated a binding constant (Kd) of 380 nM. A total of 23 residue positions in BLIP that contact β-lactamase were randomized and phage display was used to sort the libraries for tight binders to immobilized PC1 β-lactamase. The BLIP K74G mutant was the dominant clone selected and it was found to inhibit the PC1 β-lactamase with a Ki of 42 nM while calorimetry indicated a Kd of 26 nM. Molecular modeling studies suggested BLIP binds weakly to the PC1 β-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme where it forms a salt bridge with BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the A104E PC1 enzyme binds BLIP with 15-fold greater affinity than wild type PC1 β-lactamase. Kinetic measurements indicated similar association rates for all complexes with the variation in affinity due to altered dissociation rate constants suggesting changes in short-range interactions are responsible for the altered binding properties of the mutants. PMID:21238457

Actinomycin D binding mode reveals the basis for its potent HIV-1 and cancer activity

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan; Vladescu, Ioana D.; McCauley, Micah J.; Rouzina, Ioulia; Williams, Mark C.

2011-03-01

Actinomycin D (ActD) is one of the most studied antibiotics, which has been used as an anti-cancer agent and also shown to inhibit HIV reverse transcription. Initial studies with ActD established that it intercalates double stranded DNA (dsDNA). However, recent studies have shown that ActD binds with even higher affinity to single stranded DNA (ssDNA). In our studies we use optical tweezers to stretch and hold single dsDNA molecule at constant force in the presence of varying ActD concentrations until the binding reaches equilibrium. The change in dsDNA length upon ActD binding measured as a function of time yields the rate of binding in addition to the equilibrium lengthening of DNA. The results suggest extremely slow kinetics, on the order of several minutes and 0.52 +/- 0.06 μ M binding affinity. Holding DNA at constant force while stretching and relaxing suggests that ActD binds to two single strands that are close to each other rather than to pure dsDNA or ssDNA. This suggests that biological activity of ActD that contributes towards the inhibition of cellular replication is due to its ability to bind at DNA bubbles during RNA transcription, thereby stalling the transcription process.

In vitro DNA binding studies of Aspartame, an artificial sweetener.

PubMed

Kashanian, Soheila; Khodaei, Mohammad Mehdi; Kheirdoosh, Fahimeh

2013-03-05

A number of small molecules bind directly and selectively to DNA, by inhibiting replication, transcription or topoisomerase activity. In this work the interaction of native calf thymus DNA (CT-DNA) with Aspartame (APM), an artificial sweeteners was studied at physiological pH. DNA binding study of APM is useful to understand APM-DNA interaction mechanism and to provide guidance for the application and design of new and safer artificial sweeteners. The interaction was investigated using spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD). Hypochromism and red shift are shown in UV absorption band of APM. A strong fluorescence quenching reaction of DNA to APM was observed and the binding constants (Kf) of DNA with APM and corresponding number of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be +181kJmol(-1) and +681Jmol(-1)K(-1) according to Van't Hoff equation, which indicated that reaction is predominantly entropically driven. Moreover, spectrofluorometric competition experiment and circular dichroism (CD) results are indicative of non-intercalative DNA binding nature of APM. We suggest that APM interacts with calf thymus DNA via groove binding mode with an intrinsic binding constant of 5×10(+4)M(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca2+-ATPase: kinetic pathway and effects of methionine oxidation

PubMed Central

Slaughter, Brian D.; Bieber Urbauer, Ramona J.; Urbauer, Jeffrey L.; Johnson, Carey K.

2008-01-01

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma-membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide (C28W(1b)) corresponding to the CaM binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or on the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism where an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the lower effectiveness of oxidized CaM in activating the Ca2+ pump. PMID:17343368

Interactions of tea tannins and condensed tannins with proteins.

PubMed

Frazier, Richard A; Deaville, Eddie R; Green, Rebecca J; Stringano, Elisabetta; Willoughby, Ian; Plant, John; Mueller-Harvey, Irene

2010-01-20

Binding parameters for the interactions of four types of tannins: tea catechins, grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins, and sorghum procyanidins (mDP=17), with gelatin and bovine serum albumin (BSA) have been determined from isothermal titration calorimetry data. Equilibrium binding constants determined for the interaction with gelatin were in the range 10(4) to 10(6) M(-1) and in the order: sorghum procyanidins > grape seed proanthocyanidins > mimosa 5-deoxy proanthocyanidins > tea catechins. Interaction with BSA was generally weaker, with equilibrium binding constants of < or =10(3)M(-1) for grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins and tea catechins, and 10(4)M(-1) for the sorghum procyanidins. In all cases the interactions with proteins were exothermic and involved multiple binding sites on the protein. The data are discussed in relation to the structures and the known nutritional effects of the condensed tannins.

Photocaged Competitor Guests: A General Approach Toward Light-Activated Cargo Release From Cucurbiturils.

PubMed

Romero, Miguel A; Basílio, Nuno; Moro, Artur J; Domingues, Mara; González-Delgado, José A; Arteaga, Jesús F; Pischel, Uwe

2017-09-21

A general approach toward the light-induced guest release from cucurbit[7]uril by means of a photoactivatable competitor was devised. An o-nitrobenzyl-caged competitor is photolyzed to generate a competitive guest that can displace cargo from the host macrocycle solely based on considerations of chemical equilibrium. With this method the release of terpene guests from inclusion complexes with cucurbit[7]uril was demonstrated. The binding of the herein investigated terpenes, all being lead fragrant components in essential oils, has been characterized for the first time. They feature binding constants of up to 10 8  L mol -1 and a high differential binding selectivity (spanning four orders of magnitude for the binding constants for the particular set of terpenes). By fine-tuning the photoactivatable competitor guest, selective and also sequential release of the terpenes was achieved. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computational Approaches to the Chemical Equilibrium Constant in Protein-ligand Binding.

PubMed

Montalvo-Acosta, Joel José; Cecchini, Marco

2016-12-01

The physiological role played by protein-ligand recognition has motivated the development of several computational approaches to the ligand binding affinity. Some of them, termed rigorous, have a strong theoretical foundation but involve too much computation to be generally useful. Some others alleviate the computational burden by introducing strong approximations and/or empirical calibrations, which also limit their general use. Most importantly, there is no straightforward correlation between the predictive power and the level of approximation introduced. Here, we present a general framework for the quantitative interpretation of protein-ligand binding based on statistical mechanics. Within this framework, we re-derive self-consistently the fundamental equations of some popular approaches to the binding constant and pinpoint the inherent approximations. Our analysis represents a first step towards the development of variants with optimum accuracy/efficiency ratio for each stage of the drug discovery pipeline. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rational design of biaryl pharmacophore inserted noscapine derivatives as potent tubulin binding anticancer agents

NASA Astrophysics Data System (ADS)

Santoshi, Seneha; Manchukonda, Naresh Kumar; Suri, Charu; Sharma, Manya; Sridhar, Balasubramanian; Joseph, Silja; Lopus, Manu; Kantevari, Srinivas; Baitharu, Iswar; Naik, Pradeep Kumar

2015-03-01

We have strategically designed a series of noscapine derivatives by inserting biaryl pharmacophore (a major structural constituent of many of the microtubule-targeting natural anticancer compounds) onto the scaffold structure of noscapine. Molecular interaction of these derivatives with α,β-tubulin heterodimer was investigated by molecular docking, molecular dynamics simulation, and binding free energy calculation. The predictive binding affinity indicates that the newly designed noscapinoids bind to tubulin with a greater affinity. The predictive binding free energy (ΔGbind, pred) of these derivatives (ranging from -5.568 to -5.970 kcal/mol) based on linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model showed improved binding affinity with tubulin compared to the lead compound, natural α-noscapine (-5.505 kcal/mol). Guided by the computational findings, these new biaryl type α-noscapine congeners were synthesized from 9-bromo-α-noscapine using optimized Suzuki reaction conditions for further experimental evaluation. The derivatives showed improved inhibition of the proliferation of human breast cancer cells (MCF-7), human cervical cancer cells (HeLa) and human lung adenocarcinoma cells (A549), compared to natural noscapine. The cell cycle analysis in MCF-7 further revealed that these compounds alter the cell cycle profile and cause mitotic arrest at G2/M phase more strongly than noscapine. Tubulin binding assay revealed higher binding affinity to tubulin, as suggested by dissociation constant (Kd) of 126 ± 5.0 µM for 5a, 107 ± 5.0 µM for 5c, 70 ± 4.0 µM for 5d, and 68 ± 6.0 µM for 5e compared to noscapine (Kd of 152 ± 1.0 µM). In fact, the experimentally determined value of ΔGbind, expt (calculated from the Kd value) are consistent with the predicted value of ΔGbind, pred calculated based on LIE-SGB. Based on these results, one of the derivative 5e of this series was used for further toxicological evaluation. Treatment of mice with a daily dose of 300 mg/kg and a single dose of 600 mg/kg indicates that the compound does not induce detectable pathological abnormalities in normal tissues. Also there were no significant differences in hematological parameters between the treated and untreated groups. Hence, the newly designed noscapinoid, 5e is an orally bioavailable, safe and effective anticancer agent with a potential for the treatment of cancer and might be a candidate for clinical evaluation.

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

PubMed

Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

2003-05-13

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.

Study on the interaction of the toxic food additive carmoisine with serum albumins: a microcalorimetric investigation.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2014-05-30

The interaction of the synthetic azo dye and food colorant carmoisine with human and bovine serum albumins was studied by microcalorimetric techniques. A complete thermodynamic profile of the interaction was obtained from isothermal titration calorimetry studies. The equilibrium constant of the complexation process was of the order of 10(6)M(-1) and the binding stoichiometry was found to be 1:1 with both the serum albumins. The binding was driven by negative standard molar enthalpy and positive standard molar entropy contributions. The binding affinity was lower at higher salt concentrations in both cases but the same was dominated by mostly non-electrostatic forces at all salt concentrations. The polyelectrolytic forces contributed only 5-8% of the total standard molar Gibbs energy change. The standard molar enthalpy change enhanced whereas the standard molar entropic contribution decreased with rise in temperature but they compensated each other to keep the standard molar Gibbs energy change almost invariant. The negative standard molar heat capacity values suggested the involvement of a significant hydrophobic contribution in the complexation process. Besides, enthalpy-entropy compensation phenomenon was also observed in both the systems. The thermal stability of the serum proteins was found to be remarkably enhanced on binding to carmoisine. Copyright © 2014 Elsevier B.V. All rights reserved.

The Effect of Salts in Promoting Specific and Competitive Interactions between Zinc Finger Proteins and Metals

NASA Astrophysics Data System (ADS)

Li, Gongyu; Yuan, Siming; Zheng, Shihui; Chen, Yuting; Zheng, Zhen; Liu, Yangzhong; Huang, Guangming

2017-12-01

Specific protein-metal interactions (PMIs) fulfill essential functions in cells and organic bodies, and activation of these functions in vivo are mostly modulated by the complex environmental factors, including pH value, small biomolecules, and salts. Specifically, the role of salts in promoting specific PMIs and their competition among various metals has remained untapped mainly due to the difficulty to distinguish nonspecific PMIs from specific PMIs by classic spectroscopic techniques. Herein, we report Hofmeister salts differentially promote the specific PMIs by combining nanoelectrospray ionization mass spectrometry and spectroscopic techniques (fluorescence measurement and circular dichroism). Furthermore, to explore the influence of salts in competitive binding between metalloproteins and various metals, we designed a series of competitive experiments and applied to a well-defined model system, the competitive binding of zinc (II) and arsenic (III) to holo-promyelocytic leukemia protein (PML). These experiments not only provided new insights at the molecular scale as complementary to previous NMR and spectroscopic results, but also deduced the relative binding ability between zinc finger proteins and metals at the molecular scale, which avoids the mass spectrometric titration-based determination of binding constants that is frequently affected and often degraded by variable solution conditions including salt contents. [Figure not available: see fulltext.

Kinetics of protein–ligand unbinding: Predicting pathways, rates, and rate-limiting steps

PubMed Central

Tiwary, Pratyush; Limongelli, Vittorio; Salvalaglio, Matteo; Parrinello, Michele

2015-01-01

The ability to predict the mechanisms and the associated rate constants of protein–ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin–benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate koff is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate kon. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein–ligand systems and a valid support for drug design. PMID:25605901

Efficient interrupting skills of amino acid metallointercalators with DNA at physiological pH: Evaluation of biological assays

NASA Astrophysics Data System (ADS)

Raman, Natarajan; Selvaganapathy, Muthusamy; Radhakrishnan, Srinivasan

2014-06-01

The 4-aminoantipyrine derivatives (sbnd NO2, sbnd OCH3) and their mixed-ligand complexes with amino acids have been synthesized and investigated for their binding with CT DNA using UV-visible spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH (stomach 4.7; blood 7.4). The results from all techniques i.e. binding constant (Kb), and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds 1 and 4 showed comparatively greater binding at pH 7.4 as evident from its greater Kb values. All the complexes exhibit oxidative cleavage of supercoiled (SC) pBR322 plasmid DNA in the presence of H2O2 as an activator. It is remarkable that at 25 μM concentration 1 and 4 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. Among the new complexes, complexes 1 and 4 have highest potential against all the microorganisms tested. The results of the above biological experiments also reveal that the choice of different metal ions has little influence on the DNA binding, DNA cleavage and antimicrobial assay.

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

PubMed Central

Ballermann, B J; Hoover, R L; Karnovsky, M J; Brenner, B M

1985-01-01

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus. Images PMID:3001139

Highly Preorganized Ligand 1,10-Phenanthroline-2,9-dicarboxylic Acid for the Selective Recovery of Uranium from Seawater in the Presence of Competing Vanadium Species

DOE PAGES

Lashley, Mark A.; Ivanov, Alexander S.; Bryantsev, Vyacheslav S.; ...

2016-09-30

Studies of the complexation of new promising ligands with uranyl (UO 2 2+) and other seawater cations can aid the development of more efficient, selective, and robust sorbents for the recovery of uranium from seawater. Here, we propose that the ligand design principles based on structural preorganization can be successfully applied to obtain a dramatic enhancement in UO 2 2+ ion binding affinity and selectivity. This concept is exemplified through the investigation of the com-plexes of UO 2 2+, VO 2+, and VO 2+ with the highly preorganized ligand PDA (1,10-phenanthroline-2,9-dicarboxylic acid) using a combination of fluores-cence and absorbance techniques,more » along with den-sity functional theory (DFT) calculations. Moreover, the measured stability constant value, log K1, of 16.5 for the UO 2 2+/PDA complex is very high compared to uranyl complexes with other dicarboxylic ligands. Moreover, PDA exhibits strong selectivity for uranyl over vanadium ions, since the determined sta-bility constant values of the PDA complexes of the vanadium ions are quite low (V(IV) log K1 = 7.4, V(V) = 7.3). Finally, the structures of the corresponding UO 2 2+, VO 2+, and VO 2+ complexes with PDA were identified by systematic DFT calculations, and helped to interpret the stronger binding affinity for uranium over the vanadium ions. Due to its high chemical stability, selectivity, and structural preor-ganization for UO 2 2+ complexation, PDA is a very promising candidate that can be potentially used in the development of novel adsorbent materials for the selective extraction of uranium from sea-water.« less

Highly Preorganized Ligand 1,10-Phenanthroline-2,9-dicarboxylic Acid for the Selective Recovery of Uranium from Seawater in the Presence of Competing Vanadium Species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lashley, Mark A.; Ivanov, Alexander S.; Bryantsev, Vyacheslav S.

Studies of the complexation of new promising ligands with uranyl (UO 2 2+) and other seawater cations can aid the development of more efficient, selective, and robust sorbents for the recovery of uranium from seawater. Here, we propose that the ligand design principles based on structural preorganization can be successfully applied to obtain a dramatic enhancement in UO 2 2+ ion binding affinity and selectivity. This concept is exemplified through the investigation of the com-plexes of UO 2 2+, VO 2+, and VO 2+ with the highly preorganized ligand PDA (1,10-phenanthroline-2,9-dicarboxylic acid) using a combination of fluores-cence and absorbance techniques,more » along with den-sity functional theory (DFT) calculations. Moreover, the measured stability constant value, log K1, of 16.5 for the UO 2 2+/PDA complex is very high compared to uranyl complexes with other dicarboxylic ligands. Moreover, PDA exhibits strong selectivity for uranyl over vanadium ions, since the determined sta-bility constant values of the PDA complexes of the vanadium ions are quite low (V(IV) log K1 = 7.4, V(V) = 7.3). Finally, the structures of the corresponding UO 2 2+, VO 2+, and VO 2+ complexes with PDA were identified by systematic DFT calculations, and helped to interpret the stronger binding affinity for uranium over the vanadium ions. Due to its high chemical stability, selectivity, and structural preor-ganization for UO 2 2+ complexation, PDA is a very promising candidate that can be potentially used in the development of novel adsorbent materials for the selective extraction of uranium from sea-water.« less

Lactose-installed poly(ethylene glycol)-poly(d,l-lactide) block copolymer micelles exhibit fast-rate binding and high affinity toward a protein bed simulating a cell surface. A surface plasmon resonance study.

PubMed

Jule, Eduardo; Nagasaki, Yukio; Kataoka, Kazunori

2003-01-01

Lactose molecules were installed on the surface of poly(ethylene glycol)-poly(d,l-lactide) (PEG-PLA) block copolymer micelles in the scope of seeking specific recognition by cell surface receptors at hepatic sites. This, in turn, is expected to result in the formation of a complex displaying prolonged retention times and thus enhanced cellular internalization by receptor-mediated endocytosis. The so-obtained particles based on a block copolymer of molecular weight 9400 g/mol (4900/4500 g/mol for the PEG and PLA blocks, respectively) were found to have an average hydrodynamic diameter of 31.8 nm, as measured by dynamic light scattering. Further, the particle size distribution (micro(2)/Gamma(2)) was found to be lower than 0.08. Lactose-PEG-PLA micelles (Lac-micelles) were then injected over a gold surface containing Ricinus communis agglutinin lectins simulating the aforementioned glycoreceptors, and their interaction was studied by surface plasmon resonance. Then, a kinetic evaluation was carried out, by fitting the observed data mathematically. It appears that Lac-micelles bind in a multivalent manner to the lectin protein bed, which logically results in low dissociation constants. Micelles bearing a ligand density of 80% (Lac-micelles 80%: 80 lactose molecules per 100 copolymer chains) exhibit fast association phases (k(a1) = 3.2 x 10(4) M(-)(1) s(-)(1)), but also extremely slow dissociation phases (k(d1) = 1.3 x 10(-)(4) s(-)(1)). Recorded sensorgrams were fitted with a trivalent model, conveying a calculated equilibrium dissociation constant (K(D1) = k(d1)/k(a1)) of about 4 nM. The importance of cooperative binding was also assessed, by preparing Lac-micelles bearing different ligand densities, and by discussing the influence of the latter on kinetic constants. Interestingly enough, whereas Lac-micelles 80% bind in a trivalent manner to the protein bed, Lac-micelles 20% are still capable of forming bivalent complexes with the same protein bed (K(D1) = 1360 nM). Therefore, despite enhanced kinetic values brought about by a supplementary bond, lower ligand densities appear to be more effective on a molecular basis.

Molecularly imprinted polymers for RGD selective recognition and separation.

PubMed

Papaioannou, Emmanuel; Koutsas, Christos; Liakopoulou-Kyriakides, Maria

2009-03-01

Molecularly imprinted polymers that could recognize the tripeptide Arg-Gly-Asp have been produced with the use of two functional monomers and three different cross-linkers, respectively. Methacrylic acid and acrylamide were used as functional monomers and the role of the ethylene glycol dimethacrylate, trimethylpropane trimethacrylate and N,N'-methylene-bisacrylamide as crosslinking monomers, was investigated on their recognition capability. The % net rebinding and the imprinting factor values were obtained, giving for the methacrylic acid-trimethylpropane trimethacrylate polymer the highest values 12.3% and 2.44, respectively. In addition, this polymer presented lower dissociation constant (K(D)) value and the higher B (max)% of theoretical total binding sites than all the other polymers. Rebinding experiments with Lys-Gly-Asp, an analogue of Arg-Gly-Asp, and other different peptides, such as cholecystokinin C-terminal tri- and pentapeptide and gramicidin, further indicated the selectivity of methacrylic acid-trimethylpropane trimethacrylate copolymer for Arg-Gly-Asp giving specific selectivity factor values 1.27, 1.98, 1.31 and 1.67, respectively.

Study of Interaction between Cadmium and Bovine Serum Albumin with UV-Vis Spectrocopy Approach

NASA Astrophysics Data System (ADS)

Suhartono, E.; Thalib, I.; Aflanie, I.; Noor, Z.; Idroes, R.

2018-05-01

This study aims to explain the interaction of cadmium (Cd) with serum albumin through visible light (UV-Vis) spectroscopy approach. This study is an in vitro experimental study using Cd with several concentrations and Bovine Serum Albumin (BSA). Each solution was then incubated for 10 min at 37°C, and measured the absorbance at 220-300 nm. The absorbance data is then presented in graphical form. From the graph, a linear equation will appear to calculate the value of metal binding constants (K) to proteins. Also, in this present study we analsyed the ratio between A220 and A220 to identify changes in the protein region especially tyrosine and peptide bonds. The results show that the addition of Cd in different concentrations could increase the absorbance with a constant value (K) = 1.634. Based on the result, it seems the addition of Cd in different concentrations will lead the reaction to form BSA-Cd. Also, the result shows that the ration of A220/A280 were decreased with the increasing of Cd concentration. In conclusion, the addition of Cd could interact and changes the protein structure in BSA.

Study on the interaction of antiviral drug 'Tenofovir' with human serum albumin by spectral and molecular modeling methods

NASA Astrophysics Data System (ADS)

Shahabadi, Nahid; Hadidi, Saba; Feizi, Foroozan

2015-03-01

This study was designed to examine the interaction of Tenofovir (Ten) with human serum albumin (HSA) under physiological conditions. The binding of drugs with human serum albumin is a crucial factor influencing the distribution and bioactivity of drugs in the body. To understand the action mechanisms between Ten and HSA, the binding of Ten with HSA was investigated by a combined experimental and computational approach. UV-vis results confirmed that Ten interacted with HSA to form a ground-state complex and values of the Stern-Volmer quenching constant indicate the presence of a static component in the quenching mechanism. As indicated by the thermodynamic parameters (positive ΔH and ΔS values), hydrophobic interaction plays a major role in the Ten-HSA complex. Through the site marker competitive experiment, Ten was confirmed to be located in site I of HSA. Furthermore, UV-vis absorption spectra, synchronous fluorescence spectrum and CD data were used to investigate the structural change of HSA molecules with addition of Ten, the results indicate that the secondary structure of HSA molecules was changed in the presence of Ten. The experimental results were in agreement with the results obtained via molecular docking study.