Accurate Kd via Transient Incomplete Separation

23 July 2019, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Current methods for finding the equilibrium dissociation constant, Kd, of protein-small molecule complexes have inherent sources of inaccuracy.

We introduce “Accurate Kd 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 Kd value.

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 Kd of protein-small molecule complexes.

Keywords

Protein-small molecule complex
Equilibrium dissociation constants
Transient incomplete separation
Fluorescence
Mass spectrometry

Supplementary materials

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