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

Sergey Krylov, Department of Chemistry, York University, Toronto, Canada (skrylov@yorku.ca)
Nicolas Sisavath, Department Of Chemistry, York University, Toronto, Canada
Jean Luc, Department Of Chemistry, York University, Toronto, Canada
J. C., Sciex, Concord, Canada
Victor Galievsky, Department Of Chemistry, York University, Toronto, Canada
Jiayin Bao, Department Of Chemistry, York University, Toronto, Canada
Sven Kochmann, Department Of Chemistry, York University, Toronto, Canada
Alexander Stasheuski, Department Of Chemistry, York University, Toronto, Canada

Current practical methods for finding the equilibrium dissociation constant, K_d, of protein–small molecule complexes are mainly biosensoric and calorimetric. They have inherent sources of inaccuracy; immobilization of molecules on sensors and heat of side reactions often lead to errors in K_d as large as 2–3 orders of magnitude. We introduce “Accurate Constant via Transient Incomplete Separation” (ACTIS), a non-biosensoric and non-calorimetric approach for finding K_d, which appears to be free of inherent sources of inaccuracy. Conceptually, in ACTIS, 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 K_d. In this work, we prove ACTIS validity theoretically, verify its accuracy by computer simulation, and demonstrate its practical use. Owing its suggested accuracy, ACTIS has a potential to become a reference-standard method for finding K_d of protein–small molecule complexes.

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Abstract Reference & Short Personal Biography of Presenting Author

Prof. Sergey N. Krylov obtained his M.Sc. degree in Physics in 1987 and Ph.D. degree in Biophysical Chemistry in 1990, both from Lomonosov Moscow State University. His postdoctoral training was with Prof. Norman J. Dovichi at the University of Alberta. In year 2000, he accepted a position of Associate Professor at York University in Toronto, where he was promoted to Professor in 2006. In 2003-2013, he held Canada Research Chair, Tier II, in Bioanalytical Chemistry. In 2015 he was appointed York Research Chair in Bioanalytical Chemistry, Tier I. He is a founder and Inaugural Director of the Centre for Research on Biomolecular Interactions at York University. He is recognized internationally for his pioneering work in the fields of kinetic analysis of affinity interactions, and methods for selection and applications of oligonucleotide aptamers. He is the author of over 170 peer-reviewed papers. His research contributions have been recognized with a number of awards including Petro Canada Young Investigator Award, Premier’s Research Excellence Award, President's Research Excellence Award (York University) and two recognitions from the Chemical Society of Canada: McBryde Medal (2007) and Maxxam Award (2014). For more information see http://www.yorku.ca/skrylov/index.html