Journal of Fluorescence

, Volume 18, Issue 2, pp 519–526

A Fluorescence Analysis of ANS Bound to Bovine Serum Albumin: Binding Properties Revisited by Using Energy Transfer

Original Paper

DOI: 10.1007/s10895-007-0294-x

Cite this article as:
Togashi, D.M. & Ryder, A.G. J Fluoresc (2008) 18: 519. doi:10.1007/s10895-007-0294-x

Abstract

Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman–Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job’s plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites (>104 M−1) are located at a reasonably close distance (18–27 Å) to at least one of two tryptophan residues, while the lowest affinity binding site (~104 M−1) is located over 34 Å away from the both tryptophans.

Keywords

Bovine serum albumin1-anilino-8-naphthalene sulfonateLigand bindingEnergy transfer

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Nanoscale Biophotonics Laboratory, Department of ChemistryNational University of Ireland, GalwayGalwayIreland
  2. 2.National Centre for Biomedical Engineering ScienceNational University of Ireland, GalwayGalwayIreland