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(dimethyl amino)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 O-2 addition, Physisorption of BSA-Ac onto a fused-silica optical fiber lowers the accessibility of Ac to O-2, whereas covalent attachment of BSA-Ac to APTES/glutaraldehyde-modified silica enhances the accessibility of the Ac reporter group to O-2.