Probing site-selective binding of rhodamine B to bovine serum albumin

Abstract

With the objective of probing site-selective binding mechanisms of rhodamine dye with serum albumin, the interactions between rhodamine B (RB) and bovine serum (BSA) have been investigated by the complementary methods of absorption spectroscopy, steady-state and time-resolved fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and cyclic voltammetry, respectively. The results from fluorescence analysis indicated that the site-selective binding of RB with BSA originated from the complex formation with high association constant. The conformational changes of BSA after binding with RB were characterized by decreased absorbance and CD signal of BSA, and significant weakened electrochemical signal of RB. The site-selective binding of RB with BSA was confirmed by analysis of time-resolved fluorescence spectra, which exhibited biexponential decay with significantly enhanced lifetime when compared to the lifetime of RB alone. Employing ligand displacement cum fluorescence measurements using known site-specific binding ligands, such as phenylbutazone and ibuprofen, further confirmed that RB selectively bound with BSA at site I and II, which induced the unfolding of BSA. In addition, a reliable mechanism was proposed to explain the interactions of RB with BSA, which were mediated by concentrations-dependent site-selective binding of dye molecules with protein in residues of different subdomains. These results provide further information for better understanding the interaction mechanisms of rhodamine dyes with serum albumin.