The binding distance of fluorescein to bovine serum albumin (BSA) and the hydrodynamic radius of BSA in urea-water mixtures were determined by a fluorescence quenching technique and dynamic light scattering measurements, respectively, and utilized to investigate the interaction between urea and the protein and its influence on the conformation of the protein in aqueous solution, together with the analysis of the fluorescence spectra of BSA and fluorescein in the ternary mixtures of BSA-urea-water and fluorescein-urea-water and the quaternary mixtures of BSA-flurescein-urea-water. The results showed that the three domains of BSA were of different stability in urea-water mixtures, i.e., domain III was unstable even at a low urea content and domain I and II were unfolded above urea concentration of 3.0 and 4.0 mol•L－1, respectively. Domain II of BSA was found to be of more compact conformation in the mixtures up to the subdenaturating concentration of urea, which was attributed to the effect of “protein stiffening” resulting from the binding with urea.

Key words: bovine serum albumin, urea, denaturation, fluorescence quenching, dynamic light scattering