Reevaluation of ANS Binding to Human and Bovine Serum Albumins: Key Role of Equilibrium Microdialysis in Ligand – Receptor Binding Characterization

Irina M Kuznetsova,Konstantin K Turoverov,Olga I Povarova,Anna I Sulatskaya,Rajagopal Subramanyam

doi:10.1371/journal.pone.0040845

Irina M Kuznetsova, Konstantin K Turoverov + Show 3 more

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https://doi.org/10.1371/journal.pone.0040845

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Abstract

In this work we return to the problem of the determination of ligand–receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand–receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS – human serum albumin (HSA) and ANS – bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand–receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.

Highlights

Correct determination of ligand-receptor binding stoichiometry and binding constants is very important both for fundamental investigations and practical aspects of molecule medicine, pharmaceutics and at the development of biosensor systems of high social significance [1,2,3,4,5,6]
Results that can be Obtained on the Basis of Equilibrium Microdialysis The concentration of bound component can be determined by absorption spectrophotometry if the concentration the dye in reference solution equals to its concentration in sample solution
We showed that fluorescence quantum yield of the dye bound to the sites of the different binding modes can be determined on the basis of the fluorescence intensity of the solutions prepared by equilibrium microdialysis

Summary

Introduction

Correct determination of ligand-receptor binding stoichiometry and binding constants is very important both for fundamental investigations and practical aspects of molecule medicine, pharmaceutics and at the development of biosensor systems of high social significance [1,2,3,4,5,6]. The determination of binding parameters significantly enriches tried-and-true method based on fluorescence of extrinsic dyes which is widely used in molecular and cellular biology for investigation of protein’s folding, structural changes induced by different agents, interaction with each other, aggregation, amyloid fibril formation etc. In recent years it is more and more extensively used for practical diagnostics due to significant advances in molecular medicine. In this work we showed how problems that cannot be solved by fluorescence can be solved by spectrophotometric determination of the concentration of bound dye if sample and reference solutions are prepared by equilibrium microdialysis. All said above we illustrated by ANS–serum albumins interaction

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