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

Alexey V Gayer,Boris P Yakimov,Gleb S Budylin,Evgeny A Shirshin

doi:10.18287/jbpe20.06.020303

Abstract

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.

Highlights

Quantitative characterization of binding affinities is a central task of biochemistry and pharmacology [1, 2]
A number of papers has been devoted to the discussion of fluorescence quenching technique for binding parameters estimation
We focused on the case of tryptophan fluorescence quenching in proteins upon binding and tried to suggest a simple algorithm for fluorescence quenching technique (FQT) data processing for the curves measured at typical experimental conditions

Summary

Introduction

Quantitative characterization of binding affinities is a central task of biochemistry and pharmacology [1, 2]. It requires determination of binding sites number and affinity constants for each site. Since there is still a confusion in terminology concerning different types of quenching, we will first introduce it according to Ref. In the systems where fluorophore and quencher are different molecules, two types of quenching could be distinguished: collisional quenching (in which the diffusion is required to bring the quencher close enough to fluorophore) and bindingrelated (often called “static”) quenching

Methods

Results

Conclusion