Ligand binding to proteins – when flawed fluorescence quenching methodology and interpretation become the new norm

Intrinsic protein fluorescence quenching measurements have become a widespread methodology to determine ligand-binding properties of in particular serum albumin. Particularly common is the use of double log equations to extract parameters like binding constant and stoichiometry and/or number of binding sites. In this article we discuss that the methodology has several significant and often unrecognized pitfalls, and the double log equations are improperly derived for their purported use. Using simulations, it is shown that the binding stoichiometry and binding constants obtained using these equations may differ substantially from their true values. In addition, it is illustrated how this methodology, via the use of site markers, is unsuited to determine the binding site of ligands on serum albumin. We thus call for a reassessment of the literature in which this methodology plays a central role in characterizing ligand binding to proteins.