Accessibility of the Fluorescent Reporter Group in Native, Silica-Adsorbed, and Covalently Attached Acrylodan-Labeled Serum Albumins

Fluorescence quenching techniques are used to investigate the accessibility of a model biorecognition element−reporter group system when in buffer, surface-adsorbed, and covalently attached to a silica surface. The site-selective fluorescent reporter group, 6-acryloyl(dimethylamino)naphthalene (acrylodan, Ac), is attached covalently (at cysteine-34) to bovine and human serum albumin (BSA and HSA, respectively) and serves as a surrogate recognition element−reporter group system. Molecular oxygen is used to quench the Ac fluorescence and the accessibility, in the form of bimolecular rate constants (k_q), in each model system is quantified. Although one might expect these systems to exhibit similar behavior, differences in quenching characteristics are observed, such as wavelength dependency of the Stern−Volmer quenching constant (K_SV) for the native proteins in buffer. BSA-Ac exhibits wavelength dependent K_SV values as well as a blue-shifted emission spectrum on O₂ addition. Physisorption of BSA-Ac onto a fused-silica optical fiber lowers the accessibility of Ac to O₂, whereas covalent attachment of BSA-Ac to APTES/glutaraldehyde-modified silica enhances the accessibility of the Ac reporter group to O₂.