Methods of Inner Filter Effect Correction in Fluorescence Measurements of Protein–Ligand Association: A Re‐Examination of the Association of Anti‐HIV Drugs to Serum Albumin

Fluorescence‐based methods are widely used to assess protein–ligand and protein–protein interactions. Thanks to their high sensitivity, these techniques allow very low dissociation constants to be determined. However, in some cases, results must be critically revised because some artifacts can occur, one of the most common being the inner filter effect (IFE). In this article, we show how multiple strategies should be used to correct data obtained from fluorescence quenching experiments to avoid overestimation of binding affinities. The extent of the proposed corrections will depend on the spectroscopic properties of the protein and the ligand, i.e., the fluorophore involved in the experiments, as well as on the specific experimental conditions in each case.