A fluorescence analysis of ANS bound to bovine serum albumin: binding properties revisited by using energy transfer.
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 (>10(4) M(-1)) are located at a reasonably close distance (18-27 A) to at least one of two tryptophan residues, while the lowest affinity binding site (approximately 10(4) M(-1)) is located over 34 A away from the both tryptophans.
Togashi
Denisio M
DM
Nanoscale Biophotonics Laboratory, Department of Chemistry, National University of Ireland, Galway, Galway, Ireland. denisio.togashi@nuigalway.ie
Ryder
Alan G
AG
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004735:Energy Transfer; D005453:Fluorescence; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D012710:Serum Albumin, Bovine