Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

2019 ◽  
Vol 131 (20) ◽  
pp. 6707-6711
Author(s):  
Nicolas Sisavath ◽  
Jean‐Luc Rukundo ◽  
J. C. Yves Le Blanc ◽  
Victor A. Galievsky ◽  
Jiayin Bao ◽  
...  
Keyword(s):  
Dissociation Constant ◽  
Small Molecule ◽  
Equilibrium Dissociation Constant ◽  
Equilibrium Dissociation

scholarly journals Accurate Kd via Transient Incomplete Separation

2019 ◽  
Author(s):  
Nicolas Sisavath ◽  
Jean Luc Rukundo ◽  
J.C. Yves Le Blanc ◽  
Victor A. Galievsky ◽  
Jiayin Bao ◽  
...  
Keyword(s):  
Computer Simulation ◽  
Dissociation Constant ◽  
Small Molecule ◽  
Equilibrium Dissociation Constant ◽  
Practical Application ◽  
Constant Concentration ◽  
Binding Isotherm ◽  
Equilibrium Dissociation

<div>Current methods for finding the equilibrium dissociation constant, <i>K</i><sub>d</sub>, of protein-small molecule complexes have inherent sources of inaccuracy.</div><div><br></div><div>We introduce “Accurate <i>K</i><sub>d</sub> via Transient Incomplete Separation” (AKTIS), an approach that is free of known sources of inaccuracy. Conceptually, in AKTIS, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit as a function of time, for different concentrations of the protein and a constant concentration of the small molecule. Finally, a classical binding isotherm is built and used to find accurate <i>K</i><sub>d</sub> value. <br></div><div><br></div><div>Here we prove AKTIS validity theoretically and by computer simulation, present a fluidic system satisfying AKTIS requirements, and demonstrate practical application of AKTIS to finding <i>K</i><sub>d</sub> of protein-small molecule complexes.</div>

Accurate Kd via Transient Incomplete Separation

2019 ◽  
Author(s):  
Nicolas Sisavath ◽  
Jean Luc Rukundo ◽  
J.C. Yves Le Blanc ◽  
Victor A. Galievsky ◽  
Jiayin Bao ◽  
...  
Keyword(s):  
Computer Simulation ◽  
Dissociation Constant ◽  
Small Molecule ◽  
Equilibrium Dissociation Constant ◽  
Practical Application ◽  
Constant Concentration ◽  
Binding Isotherm ◽  
Equilibrium Dissociation

<div>Current methods for finding the equilibrium dissociation constant, <i>K</i><sub>d</sub>, of protein-small molecule complexes have inherent sources of inaccuracy.</div><div><br></div><div>We introduce “Accurate <i>K</i><sub>d</sub> via Transient Incomplete Separation” (AKTIS), an approach that is free of known sources of inaccuracy. Conceptually, in AKTIS, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit as a function of time, for different concentrations of the protein and a constant concentration of the small molecule. Finally, a classical binding isotherm is built and used to find accurate <i>K</i><sub>d</sub> value. <br></div><div><br></div><div>Here we prove AKTIS validity theoretically and by computer simulation, present a fluidic system satisfying AKTIS requirements, and demonstrate practical application of AKTIS to finding <i>K</i><sub>d</sub> of protein-small molecule complexes.</div>

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

2019 ◽  
Vol 58 (20) ◽  
pp. 6635-6639 ◽  
Author(s):  
Nicolas Sisavath ◽  
Jean‐Luc Rukundo ◽  
J. C. Yves Le Blanc ◽  
Victor A. Galievsky ◽  
Jiayin Bao ◽  
...  
Keyword(s):  
Dissociation Constant ◽  
Small Molecule ◽  
Equilibrium Dissociation Constant ◽  
Equilibrium Dissociation

scholarly journals Accurate Kd via Transient Incomplete Separation

2019 ◽  
Author(s):  
Nicolas Sisavath ◽  
Jean Luc Rukundo ◽  
J.C. Yves Le Blanc ◽  
Victor A. Galievsky ◽  
Jiayin Bao ◽  
...  
Keyword(s):  
Computer Simulation ◽  
Dissociation Constant ◽  
Small Molecule ◽  
Equilibrium Dissociation Constant ◽  
Practical Application ◽  
Constant Concentration ◽  
Binding Isotherm ◽  
Equilibrium Dissociation

<div>Current methods for finding the equilibrium dissociation constant, <i>K</i><sub>d</sub>, of protein-small molecule complexes have inherent sources of inaccuracy.</div><div><br></div><div>We introduce “Accurate <i>K</i><sub>d</sub> via Transient Incomplete Separation” (AKTIS), an approach that is free of known sources of inaccuracy. Conceptually, in AKTIS, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit as a function of time, for different concentrations of the protein and a constant concentration of the small molecule. Finally, a classical binding isotherm is built and used to find accurate <i>K</i><sub>d</sub> value. <br></div><div><br></div><div>Here we prove AKTIS validity theoretically and by computer simulation, present a fluidic system satisfying AKTIS requirements, and demonstrate practical application of AKTIS to finding <i>K</i><sub>d</sub> of protein-small molecule complexes.</div>

Ontogeny of gastric mucosal muscarinic receptor and sensitivity to carbachol

1984 ◽  
Vol 246 (5) ◽  
pp. G550-G555
Author(s):  
E. R. Seidel ◽  
L. R. Johnson
Keyword(s):  
Dissociation Constant ◽  
Cholinergic Receptor ◽  
Equilibrium Dissociation Constant ◽  
Muscarinic Cholinergic Receptor ◽  
Fetal Rat ◽  
Gastric Lumen ◽  
Basal Acid ◽  
Muscarinic Cholinergic ◽  
Equilibrium Dissociation ◽  
Apparent Equilibrium

Development of the muscarinic cholinergic receptor and sensitivity of oxyntic gland mucosa to a muscarinic agonist were studied in rats of various ages. The gastric lumen of the fetal rat at the 20th day of gestation contained a statistically significant amount of basal pepsin, which increased log linearly over the first 30 days of life. Carbachol was effective in stimulating the secretion of pepsin as early as 12 h after birth. Basal acid could be measured in the gastric lumen 12 h after birth. The secretion of basal acid increased log linearly over the first 30 days of life. Carbachol was an effective secretagogue even in the fetal rat. The density of the muscarinic cholinergic receptor of the adult rat oxyntic gland mucosa was 99.3 fmol/mg protein with an apparent equilibrium dissociation constant for quinuclidinyl benzilate of 0.40 nM. The receptor was well developed even in the fetal rat, which bound 79.6 fmol/mg protein with an apparent equilibrium dissociation constant of 0.26 nM. Except for immediately after birth, receptor density was maintained between 70 and 90% of the adult level over the first 30 days of life. These results suggest that cholinergic regulation of gastric acid and pepsin secretion is probably functional by either late gestation or at least immediately after birth.

SARS CoV Main Proteinase:  The Monomer−Dimer Equilibrium Dissociation Constant

Biochemistry ◽  
2006 ◽  
Vol 45 (49) ◽  
pp. 14632-14641 ◽  
Author(s):  
Vito Graziano ◽  
William J. McGrath ◽  
Lin Yang ◽  
Walter F. Mangel
Keyword(s):  
Dissociation Constant ◽  
Equilibrium Dissociation Constant ◽  
Equilibrium Dissociation
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