Fluorescence emission from adsorbed bovine serum albumin and albumin-bound 1-anilinonaphthalene-8-sulfonate studied by TIRF

Abstract

Total internal reflection fluorescence (TIRF) spectroscopy was used to analyse the conformational changes of bovine serum albumin (BSA) upon adsorption to silica surfaces. The intrinsic fluorescence emitted by BSA tryptophans and the fluorescence of BSA-bound ligand 1-anilinonaphthalene-8-sulfonate (ANS) were analysed prior to and after the removal of nonadsorbed protein. It was found that the irreversibly adsorbed BSA emits a blue-shifted intrinsic fluorescence (11 nm) and that the surface-adsorbed BSA-bound ANS fluorescence emission is red-shifted (12 nm). It was concluded that the conformational change in adsorbed BSA involves the whole BSA molecule: tryptophans become exposed to a less polar environment while the binding site polarity is increased. The conformation of irreversibly adsorbed BSA is not similar to the unfolded conformation of heat-denatured BSA. The red-edge excitation of adsorbed BSA-bound ANS indicates a slower spectral relaxation in the adsorbed protein as compared with the protein in solution. The fluorescence intensity contributions from irreversibly adsorbed species (BSA with and without bound ANS) suggest some changes in either ANS-BSA binding affinity and/or ANS fluorescence enhancement.