Simple methods to determine the dissociation constant, Kd

Abstract

The determination of the dissociation constant (K_d) is pivotal in biochemistry and pharmacology for understanding binding affinities in chemical reactions, which is crucial for drug development and comprehending biological systems. Here, we introduce a single-molecule fluorescence resonance energy transfer–based method for determining K_d, alongside the conventional electrophoretic mobility shift assay method of K_d, offering insights into thermodynamic interactions between proteins and substrates. The single-molecule fluorescence resonance energy transfer approach is highlighted for its ability to accurately measure binding and dissociation kinetics through fluorescence labeling and the intrinsic nature of protein-DNA interactions, representing a significant advancement in the fields of molecular biology and pharmacology.