Kinetics and Thermodynamics of the Interaction of ANS with Proteins

Abstract

1-anilino-naphtalene-8-sulfonate (ANS) is a fluorescent probe widely used in protein folding and conformational transitions studies. The fluorescent features of ANS, a blue shift of the emission maximum and the increase of quantum yield and lifetime, are generally attributed to the binding at hydrophobic sites. Despite the interaction of ANS with proteins has been extensively studied since the early works of Gregorio Weber, few high-resolution structures of proteins complexed with ANS have been resolved. In this work the binding of ANS to BSA was analyzed at equilibrium and pre-equilibrium conditions. The combined analysis of fluorescence, near UV circular dichroism and isothermal titration calorimetric data provided a detailed description of the binding mechanism. Three ANS molecules bound to BSA in 100 mM phosphate pH 7 at 25°C. Pre-equilibrium experiments allowed to determine the affinity and the relative quantum yield at each binding site by fitting a microscopic model to the fluorescence time-course data. This analysis unambiguously indicated that the binding of ANS to BSA occurs at two different and independent binding sites with similar quantum yields and affinities (ΔG° @ -35 kJ/mol). The binding of ANS to the first site is thermodynamically favored by similar contributions of the enthalpic (ΔH = -16.3 kJ/mol) and entropic terms (-TΔS = -19.4 kJ/mol), while the binding to the second site is enthalpically driven (ΔH = -36.6 kJ/mol; -TΔS = 4.3 kJ/mol). Complementary information from molecular docking showed 3 ANS molecules bound at hydrophobic cavities in BSA subdomains IIA and IIIA with binding affinities in the order of those found experimentally. The sulfonate group of ANS was oriented towards clusters of polar residues, a common feature in the reported crystal structures of other ANS-protein complexes. Supported with grants from UBA, CONICET and ANPCyT.