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

Abstract Current practical methods for finding the equilibrium dissociation constant, K d , 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 K d . 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 K d values of protein–small molecule complexes.