A TIRF titration study of 1-anilinonaphthalene-8-sulfonate binding to silica-adsorbed bovine serum albumin

Abstract

A novel fluorescence titration method was applied to the qualitative study of conformational characteristics of a surface-adsorbed bovine serum albumin (BSA) layer. The probe, 1-anilinonaphthalene-8-sulfonate (ANS), was used as a fluorescent ligand. The selective excitation of a bound dye in the adsorbed protein layer was achieved using the evanescent surface wave created by total internal reflection at the solid/liquid interface. The same protein/ligand pair dissolved in the bulk solution was used as a reference. The apparent affinity of the ligand towards the surface-adsorbed protein is lower as compared with the dissolved protein. The results of the solution and the surface titration experiments were quantitatively compared after the titration results were normalized with respect to the intrinsic BSA fluorescence in the absence of the ligand. It was found that the protein adsorption leads to fluorescence enhancement of those bound ANS molecules which are otherwise nonfluorescent when bound to BSA in the solution. It is shown that ANS molecules, which are bound to the outer binding sites on the protein surface, can serve as a probe of protein-protein and protein-surface contacts in the adsorbed layer.