Characterization of BSA binding sites for small molecules by Fluorescence spectroscopy and Isothermal titration calorimetry

DIEGO I. CATTONI; SERGIO B. KAUFMAN; F. LUIS GONZÁLEZ-FLECHA

Montevideo, Uruguay

Simposio; 6th International Conference on Biological Physics, 5th Southern Cone Biophysics Congress; 2007

Sociedad Argentina de Biofísica

<!-- @page { size: 21.59cm 27.94cm; margin: 2cm } TD P { margin-bottom: 0cm } P { margin-bottom: 0.21cm } --> Albumin is the most abundant protein in the circulatory system and is responsible for the osmotic blood pressure regulation as well as blood pH. Beyond this the most outstanding characteristic of albumin is undoubtedly its ability to reversibly bind to a vast amount of different ligands, including fatty acids, bilirubin, metal ions, and multiple drugs.  In this work we will characterize the binding of small molecules by calorimetric and spectroscopic techniques using 1-Aniline-8-naphtalenesulfonate (ANS) as a tool [1]. This probe is widely use as a probe for hydrophobic regions, because of its low quantum yield in water and a large increase when its located in hydrophobic environments. ANS binding to albumin was analyzed following the Adair formalism. As it was indicated in previous work [1], albumin shows 5 binding sites and the mean number of occupied sites shows a sigmoid dependence on the free ligand concentration which may indicate cooperativeness or the existence of sites with different affinity. To further study this, the interaction with a series of specific ligands [2] was analyzed and the thermodynamic parameters that characterize the microscopic interaction between these ligands and BSA were calculated from the ITC experiments.