Evaluating the Number of Ligand Binding Sites on Protein from Tryptophan Fluorescence Quenching under Typical Experimental Conditions

Fluorescence quenching technique is extensively applied for the characterization of intermolecular interactions in the solution that is one of the major problems in biochemistry and pharmacology. Using the Stern-Volmer equation, one can obtain a measure of binding affinity calculated under the assumption of static quenching, while the possibility to determine other binding parameters is under discussion. Several mathematical approaches are known, which allow determining the number of binding sites from fluorescence quenching curves. However, they usually require high concentrations of the ligand to obtain saturating binding curves that could be complicated in a number of experimental conditions. In this paper, we present a simple algorithm, which allows to prove that the number of binding sites in the system is equal to one or not and to verify that the quantum yield of the complex is zero. The advantage of the suggested approach is its applicability at typical conditions used in tryptophan fluorescence quenching experiments for the protein-ligand binding. A web interface for automated processing of fluorescence quenching experiments based on the suggested approach is presented.