US EPA

1493125 

Journal Article 

A fluorescence analysis of ANS bound to bovine serum albumin: Binding properties revisited by using energy transfer 

Togashi, DM; Ryder, AG 

2008 

Yes 

Journal of Fluorescence
ISSN: 1053-0509
EISSN: 1573-4994 

18 

2 

519-526 

English 

18097738 

10.1007/s10895-007-0294-x 

WOS:000253896400032 

http://://WOS:000253896400032
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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 angstrom) to at least one of two tryptophan residues, while the
lowest affinity binding site (similar to 10(4) M-1) is located over 34 angstrom away from the
both tryptophans. 

bovine serum albumin; 1-anilino-8-naphthalene sulfonate; ligand binding; energy transfer