Kinetics and thermodynamics of the interaction of 1-anilino-naphthalene-8-sulfonate with proteins.
Although 1-anilino-naphthalene-8-sulfonate (ANS) has been widely used in protein folding and binding studies, the detailed mechanism of this interaction is not fully understood. In this work the binding of ANS was analyzed at pre-equilibrium and equilibrium conditions using bovine serum albumin (BSA) as model. We employed a combined approach including the analysis of fluorescence, near-UV circular dichroism and isothermal titration calorimetric data. Experiments at equilibrium with these techniques identify three ANS molecules bound at hydrophobic cavities in BSA. Pre-equilibrium fluorescence analysis unambiguously indicated that the binding of ANS at hydrophobic cavities of BSA occurs at two different and independent classes of sites with similar affinities and quantum yields, two features that are undetectable by the equilibrium analysis. The binding of ANS to the first site is thermodynamically favored by similar contributions of the enthalpic (DeltaH = -22 kJ/mol) and entropic terms (-TDeltaS = -17 kJ/mol), while the binding to the second site is enthalpically driven (DeltaH = -31 kJ/mol; -TDeltaS = -0.6 kJ/mol). Complementary information from molecular docking showed three ANS molecules bound at hydrophobic cavities in BSA subdomains IIA and IIIA with binding affinities in the order of those found experimentally and three additional ANS molecules bound at water exposed sites.
Cattoni
Diego I
DI
Laboratorio de Biofísica Molecular, Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires - CONICET, Junín 956, 1113-Buenos Aires, Argentina.
Kaufman
Sergio B
SB
González Flecha
F Luis
FL
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D012710:Serum Albumin, Bovine; C027132:1-anilino-8-naphthalenesulfonate