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

Current practical methods for finding the equilibrium dissociation constant, Kd, of protein–small molecule complexes have inherent sources of inaccuracy. Introduced here is “accurate constant via transient incomplete separation” (ACTIS), which appears to be free of inherent sources of inaccuracy. Co...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2019-05, Vol.58 (20), p.6635-6639
Main Authors: Sisavath, Nicolas, Rukundo, Jean‐Luc, Le Blanc, J. C. Yves, Galievsky, Victor A., Bao, Jiayin, Kochmann, Sven, Stasheuski, Alexander S., Krylov, Sergey N.
Format: Article
Language:English
Subjects:
analytical methods
Capillary pressure
Computer simulation
equilibrium dissociation constant
Equilibrium methods
fluorescence
mass spectrometry
Mathematical analysis
Proteins
Separation
Superposition (mathematics)
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Summary:Current practical methods for finding the equilibrium dissociation constant, Kd, of protein–small molecule complexes have inherent sources of inaccuracy. Introduced here is “accurate constant via transient incomplete separation” (ACTIS), which appears to be free of inherent sources of inaccuracy. Conceptually, a short plug of the pre‐equilibrated protein–small molecule mixture is pressure‐propagated in a capillary, causing fast 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 and used to calculate a fraction of unbound small molecule, which, in turn, is used to calculate Kd. Herein the validity of ACTIS is proven theoretically, its accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become a reference‐standard method for determining Kd values of protein–small molecule complexes. Constant focus: Introduced here is a method called accurate constant via transient incomplete separation (ACTIS). This approach serves to measure accurate equilibrium dissociation constants (Kd) of protein–small molecule complexes (PL).
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.201901345