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Equilibrium Binding Constant (equilibrium + binding_constant)
Selected AbstractsOrtho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: Fluorescence studyBIOPOLYMERS, Issue 5 2002R. F. Turchiello Abstract The peptide hormone bradykinin (BK) (Arg1 -Pro2 -Pro3 -Gly4 -Phe5 -Ser6 -Pro7 -Phe8 -Arg9) and its shorter homolog BK1,5 (Arg1 -Pro2 -Pro3 -Gly4 -Phe5) were labeled with the extrinsic fluorescent probe ortho -aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH2 and Abz-BK1,5 -NH2). The fragment des-Arg9 -BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg9 -BK-DAP(Abz)-NH2. The spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide,lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy,Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK1,5 -NH2 presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 336,346, 2002 [source] Effects of alcohols on micellization and on the reaction methyl 4-nitrobenzenesulfonate + Br, in cetyltrimethylammonium bromide aqueous micellar solutionsINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 12 2004María Muñoz The effects of n -hexanol, n -pentanol, and n -butanol on the critical micelle concentration (cmc), on the micellar ionization degree (,), and on the rate of the reaction methyl 4-nitrobenzenesulfonate + Br, have been investigated in cetyltrimethylammonium bromide (CTAB) aqueous solutions. An increase in the alcohol concentration present in the solution produces a decrease in the cmc and an increase in the micellar ionization degree. Kinetic data show that the observed rate constant decreases as alcohol concentration increases. This result was rationalized by considering variations in the equilibrium binding constant of the methyl 4-nitrobenzenesulfonate molecules to the micelles, variations in the interfacial bromide ion concentration, and variations in the characteristics of the water,alcohol bulk phase provoked by the presence of alcohols. When these operative factors are considered, kinetic data in this and other works show that the second-order rate constants in the micellar pseudophases of water,alcohol micellar solutions are quite similar to those estimated in the absence of alcohols. © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36: 634,641, 2004 [source] Does the ligand-biopolymer equilibrium binding constant depend on the number of bound ligands?,BIOPOLYMERS, Issue 11 2010Daria A. Beshnova Abstract Conventional methods, such as Scatchard or McGhee-von Hippel analyses, used to treat ligand-biopolymer interactions, indirectly make the assumption that the microscopic binding constant is independent of the number of ligands, i, already bound to the biopolymer. Recent results on the aggregation of aromatic molecules (Beshnova et al., J Chem Phys 2009, 130, 165105) indicated that the equilibrium constant of self-association depends intrinsically on the number of molecules in an aggregate due to loss of translational and rotational degrees of freedom on formation of the complex. The influence of these factors on the equilibrium binding constant for ligand-biopolymer complexation was analyzed in this work. It was shown that under the conditions of binding of "small" molecules, these factors can effectively be ignored and, hence, do not provide any hidden systematic error in such widely-used approaches, such as the Scatchard or McGhee-von Hippel methods for analyzing ligand-biopolymer complexation. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 932,935, 2010. [source] Affinity Purification of Lipid VesiclesBIOTECHNOLOGY PROGRESS, Issue 1 2004Boris Peker We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 × 1013 active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 × 108 M,1. We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format. [source] Differing effects of substrate and non-substrate transport inhibitors on glutamate uptake reversalJOURNAL OF NEUROCHEMISTRY, Issue 6 2001Christopher M. Anderson Na+ -dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. lthreo -,-hydroxyaspartate (l -TBHA), dlthreo -,-benzyloxyaspartate (dl -TBOA), ltrans -pyrrolidine-2,4-dicarboxylic acid (ltrans -2,4-PDC) (+/,)- cis -4-methy- trans -pyrrolidine-2,4-dicarboxylic acid (cis -4-methy- trans -2,4-PDC) and lantiendo -3,4-methanopyrrolidine-2,4-dicarboxylic acid (lantiendo -3,4-MPDC) inhibited l -[14C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 µm (dl -TBOA and l -TBHA) , 43 µm (cis -4-methy- trans -2,4-PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While l -TBHA, ltrans -2,4-PDC, cis -4-methy- trans -2,4-PDC and lantiendo -3,4-MPDC displayed significant transporter substrate activities in neurons and astrocytes, dl -TBOA was a substrate only in astrocytes. This effect of dl -TBOA was concentration-dependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal dl -TBOA uptake velocity (, 10 µm), dl -TBOA blocked uptake reversal in ATP-depleted astrocytes; this blockade was negated at concentrations that drove substantial dl -TBOA uptake (> 10 µm). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure conditions. [source] | |||||||||||||