Interaction of urea with fluorophores bound to protein surfaces

Abstract

The effect of urea on the fluorescence properties of 6-(p-toluidino)-2-naphthalenesulfonate (TNS) and 8-anilino-1-naphthalenesulfonate (ANS) bound to bovine serum albumin (BSA) has been studied by steady-state and time-resolved emission spectroscopy. The urea-induced structural changes of the protein are accompanied by an at least three-fold decrease of the emission yield (Φ_f) of both TNS and ANS. The fluorescence lifetime (τ_f), however, does not change much (ca. 10%). In a homogeneous medium [90% alcohol in water (v/v)], addition of urea leads to a decrease of both Φ_f and τ_f which is shown to be due to polarity-dependent twisted intramolecular charge transfer (TICT) processes. It is suggested that addition of urea leads to the removal of some of the fluorophores bound to proteins. Since the fluorophores are almost non-fluorescent in aqueous media, the overall emission is still accounted for by those fluorophores bound to denatured protein. Thus τ_f and the emission spectra remain more or less unchanged. The decrease in the number of fluorophores bound to proteins during denaturation causes a reduction of Φ_f.