ORIGINAL PAPER A Fluorescence Analysis of ANS Bound to Bovine Serum Albumin: Binding Properties Revisited by Using Energy Transfer Denisio M. Togashi & Alan G. Ryder Received: 22 October 2007 / Accepted: 19 November 2007 / Published online: 21 December 2007 # Springer Science + Business Media, LLC 2007 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 iso- therm 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 (>10 4 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 (~10 4 M -1 ) is located over 34 Å away from the both tryptophans. Keywords Bovine serum albumin . 1-anilino-8-naphthalene sulfonate . Ligand binding . Energy transfer Introduction Serum albumin, in humans, accounts for about 60% of the total serum content. It is the most abundant of the plasma proteins. The main features that distinguish the primary structure of serum albumin from other extracellular proteins are the presence of only one cysteine group (Cys-34), and low tryptophan content. The secondary structure of serum albumin consists of approximately 67% of α-helix. There are nine loops and 17 disulphide bridges, which make a heart-shaped 3D structure revealed by X-ray crystallogra- phy studies [1]. The 3D structure is commonly represented by three homologous domains (I, II, and III) which are divided into two sub-domains (A and B) [2]. Human and Bovine Serum Albumins (HSA and BSA, respectively) are probably the most studied serum albumin proteins. Al- though, they are approximately 76% homologous, the main difference between the two proteins is that in HSA there is only one tryptophan amino acid (Trp-214), whereas in BSA there are two tryptophan units (Trp-134 and Trp-212) [2]. The principal function of serum albumin is to transport a wide variety of fatty acids, hormones, metal ions, and metabolites. The binding characteristics in serum albumin are derived from molecular forces such as hydrogen bonding, electrostatic, and hydrophobic interactions present in the different binding sites [2, 3]. J Fluoresc (2008) 18:519–526 DOI 10.1007/s10895-007-0294-x D. M. Togashi (*) : A. G. Ryder Nanoscale Biophotonics Laboratory, Department of Chemistry, National University of Ireland, Galway, Galway, Ireland e-mail: denisio.togashi@nuigalway.ie D. M. Togashi : A. G. Ryder National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Galway, Ireland