Postadsorption changes in the emission maximum of acrylodan-labeled bovine serum albumin using total internal reflection fluorescence

Acrylodan (Ac), the thiol reactive analog of the polarity-sensitive fluorescent dye Prodan, was investigated as a site-specific probe for postadsorption conformational changes of bovine serum albumin (BSA) adsorbed to bare and alkyl silane-treated glass coverslips. A commercial fluorometer, adapted with a custom-built total internal reflection fluorescence (TIRF) cell, was utilized to examine the adsorbed Ac-BSA fluorescence as a function of substrate surface wettability as measured by the Wilhelmy plate balance technique. Fluorescence emission of adsorbed Ac-BSA displayed a blue shift and edge excited red shift upon adsorption to all substrates that increased with increasing substrate hydrophobicity. Additionally, center excited emission spectra from unconjugated Ac adsorbed to bare and hydrophobic glass were similar to those for the adsorbed Ac-BSA. These results are consistent with the hypothesis of postadsorption conformational changes, but also suggest a direct probe-surface contact for adsorbed Ac-BSA.