Denaturation and stabilization of Human Serum Albumin: combined effect of drugs

The unfolding pathway of the defatted human serum albumin (HSA) binding ibuprofen and propofol has been studied by using small angle X-ray scattering (SAXS). A set of HSA solutions with urea concentrations between 0.00 and 9.00 M was analyzed and the singular value decomposition method applied to the complete SAXS data set allowed us to distinguish four different states in solution. Besides the Native and Unfolded forms, two intermediates I1 and I2 have been identified and the low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. The I1 structure was characterized by only one domain open (domain I, which does not host a binding site for either of the ligands), while I2 presents only one closed domain (domain III). A direct comparison with the unfolding pathway of the HSA:Ibu complex pointed out that the presence of propofol as a second ligand, located in subdomain IIIB, leads to the appearance of an intermediate with two closed domains (domains II and III) which are those which accomodate the ligands. Moreover, the equilibrium between I2 and the Unfolded form is slightly shifted towards higher urea concentrations. These results suggest that the cobinding significantly hinders the unfolding process.