Generally Applicable NMR Titration Methods for the Determination of Equilibrium Constants for Coordination Complexes: Syntheses and Characterizations of Metallacrown Ethers with α,ω-Bis(phosphite)-polyether Ligands and Determination of Equilibrium Binding Constants to Li+

2011 ◽  
Vol 30 (21) ◽  
pp. 5695-5709 ◽  
Author(s):  
Justin T. Sheff ◽  
Aaron L. Lucius ◽  
Sam B. Owens ◽  
Gary M. Gray
Keyword(s):  
Equilibrium Constants ◽  
Binding Constants ◽  
Coordination Complexes ◽  
Nmr Titration ◽  
Metallacrown Ethers ◽  
Equilibrium Binding

scholarly journals Functional properties of a sarcoplasmic reticulum Ca2+-ATPase with an altered Ca2+-binding mechanism

1995 ◽  
Vol 309 (2) ◽  
pp. 499-505 ◽  
Author(s):  
F Martinez-Azorin ◽  
F Soler ◽  
J C Gomez-Fernandez ◽  
F Fernandez-Belda
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Equilibrium Constants ◽  
Binding Constants ◽  
Binding Mechanism ◽  
Protein Residues ◽  
Equilibrium Binding ◽  
Positive Cooperativity ◽  
Binding Isotherms ◽  
Fitting Procedures ◽  
Equal Amplitude

Treatment of sarcoplasmic reticulum vesicles with diethylpyrocarbonate in the presence of a large excess of reagent, at pH 6.2 and at room temperature, reveals both a fast- and a slow-reacting population of protein residues. The loss of the Ca(2+)-ATPase activity is mainly associated with the fast-reacting population being partially sensitive to hydroxylamine. There is also an effect on the Ca(2+)-binding mechanism. Shorter derivatization times (5 min) produce a loss of the positive cooperativity of Ca2+ binding. When the treatment was prolonged for 30 min there was an additional decrease in the overall Ca2+ affinity. Curve-fitting procedures applied to the non-cooperative binding isotherms provide the equilibrium constants for the two Ca2+ sites, although they cannot discriminate between interacting and independent site mechanisms. Prestationary kinetics assays show 2 Ca2+:1 ATP ratios, at any extent of Ca2+ saturation, indicating that the Ca2+ sites are not independent. The Ca2+ dissociation profile after derivatization shows a decrease in the dissociation constant for the release of the second Ca2+, which is consistent with interacting sites. Isotopic exchange experiments show fast and slow components of equal amplitude even at subsaturating Ca2+ concentrations, which is incompatible with independent binding sites. The experimental data suggest a modification of the equilibrium binding constants making them more similar, but keeping the interacting character. The structural position of the external (cytoplasmic) and the internal (lumenal) Ca2+ sites remains unaltered in the absence of positive cooperativity.

scholarly journals Architecture of Allosteric Structure. Demonstration of the Fundamental Element of Allosteric Structure of Human Hemoglobin. O2-Equilibrium Binding Curve of Purified Human Hemoglobin in an Effector-Free Supporting Electrolyte.

2019 ◽  
Author(s):  
Francis Knowles
Keyword(s):  
Equilibrium Constants ◽  
Supporting Electrolyte ◽  
Binding Constants ◽  
Fold Increase ◽  
Chloride Ions ◽  
Initial Slope ◽  
Human Hemoglobin ◽  
High Concentration ◽  
Equilibrium Binding ◽  
Oxygen Addition

<p>The O<sub>2</sub>-affinity of human hemoglobin, free of effector molecules, was measured in 0.050 <u>M</u> BisTris, pH 7.0 with HCl, 20<sup>o</sup>C. A Hill plot of O<sub>2</sub>-equilibrium binding data reveals an initial slope of 2 and fails to demonstrate an upward inflection. The tetrameric hemoglobin structure in an effector-free supporting electrolyte can be described as two cooperative dimeric subunits. Equilibrium constants of these steps are related to intrinsic O<sub>2</sub>-binding constants for alpha and beta-chains, K(alpha) and K(beta). The equation of state is comprised of four unknown quantities. O<sub>2</sub>-Binding constants are not expected to be identical for each of the dimeric cooperative subunits. The first cooperative subunit binds O<sub>2</sub> while bound to the second O<sub>2</sub>-free cooperative dimer. The second cooperative dimer binds O<sub>2 </sub>while bound to a fully oxygenated cooperative dimer. Effecter-free human hemoglobin is half saturated with1.9 micromolar oxygen. Addition of 0.1 M NaCl to the supporting electrolyte results in an approximately 4-fold increase in the concentration of O<sub>2</sub> required for half saturation of hemoglobin, 8.1 micromolar oxygen. The mechanism of the response to chloride ions is attributed to neutralization of positively charged residues in the central cavity of Hb<sub>4</sub> by the relatively high concentration of chloride ions. </p>

scholarly journals Architecture of Allosteric Structure. Rate Equations, Rate Constants, and Equilibrium Constants for Reaction of: Hb4 with O2 and (HbO2)4 with Dithionate, in the Presence of 2,3-Bisphosphoglycerate

2019 ◽  
Author(s):  
Francis Knowles ◽  
Samantha J. Doyle ◽  
Douglas Magde
Keyword(s):  
Rate Constants ◽  
Binding Constant ◽  
Equilibrium Constants ◽  
Rate Equations ◽  
Rate Law ◽  
First Order ◽  
Equilibrium Binding ◽  
Binding Curve ◽  
O2 Binding

Three unknown quantities are all that is required to describe the O2-equilibrium binding curve for fractional saturation of human hemoglobin in red blood cells, under standard conditions: Kα, the O2-binding constant of equivalent α-chains; KC, the equilibrium constant for the T →R conformation change; Kβ, the O2-binding constant of equivalent β-chains. The model for formulation of the equation of state is a 3-stage ordered sequence of reactions. The values of were established by determination of rate constants for the oxygenation reaction and the dithionite-mediated de oxygenation reaction. The rate law for the forward reaction in the presence of excess O2 yields The same rate law yields for the dithionite-mediated de-oxygenation reaction. The rate constants for binding O2 are pseudo-first-order. The rate constants for release of O2 are first-order. Reactions involving O2, are 2-step ordered sequences of equivalent subunits. Progress curves for a 2-step ordered sequence of equivalent chains collapse to a first order reaction. Progress curves for both oxygenation and dithionite-mediated de-oxygenation reactions return is 0.0580 for the oxygenation reaction and 0.0358 for the dithionite-mediated de-oxygenation reaction. The corresponding values from the O2-equilibrium binding curve are: and = 0.02602. Values of determined from rate constants of progress curves for oxygenation and dithionite-mediated de-oxygenation reactions are close to values of determined by analysis of the O2-equilibrium binding curves for whole blood, by the Perutz/Adair equation.<br>

scholarly journals Architecture of Allosteric Structure. Rate Equations, Rate Constants, and Equilibrium Constants for Reaction of: Hb4 with O2 and (HbO2)4 with Dithionate, in the Presence of 2,3-Bisphosphoglycerate

2019 ◽  
Author(s):  
Francis Knowles ◽  
Samantha J. Doyle ◽  
Douglas Magde
Keyword(s):  
Rate Constants ◽  
Binding Constant ◽  
Equilibrium Constants ◽  
Rate Equations ◽  
Rate Law ◽  
First Order ◽  
Equilibrium Binding ◽  
Binding Curve ◽  
O2 Binding

Three unknown quantities are all that is required to describe the O2-equilibrium binding curve for fractional saturation of human hemoglobin in red blood cells, under standard conditions: Kα, the O2-binding constant of equivalent α-chains; KC, the equilibrium constant for the T →R conformation change; Kβ, the O2-binding constant of equivalent β-chains. The model for formulation of the equation of state is a 3-stage ordered sequence of reactions. The values of were established by determination of rate constants for the oxygenation reaction and the dithionite-mediated de oxygenation reaction. The rate law for the forward reaction in the presence of excess O2 yields The same rate law yields for the dithionite-mediated de-oxygenation reaction. The rate constants for binding O2 are pseudo-first-order. The rate constants for release of O2 are first-order. Reactions involving O2, are 2-step ordered sequences of equivalent subunits. Progress curves for a 2-step ordered sequence of equivalent chains collapse to a first order reaction. Progress curves for both oxygenation and dithionite-mediated de-oxygenation reactions return is 0.0580 for the oxygenation reaction and 0.0358 for the dithionite-mediated de-oxygenation reaction. The corresponding values from the O2-equilibrium binding curve are: and = 0.02602. Values of determined from rate constants of progress curves for oxygenation and dithionite-mediated de-oxygenation reactions are close to values of determined by analysis of the O2-equilibrium binding curves for whole blood, by the Perutz/Adair equation.<br>

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