Fluorescence analysis of sulfasalazine bound to defatted serum albumin in the presence of denaturating factors

Human serum albumin binding with drug can be altered in several diseases. To estimate how the binding ability of defatted human serum albumin (dHSA) changes with its conformational alteration we estimated the binding of sulfasalazine (SSZ) in the presence of denaturants: guanidine hydrochloride (Gu·HCl) and urea (U). A decrease in fluorescence intensity (RF), a blue shift in the maximum of fluorescence emission and splitting of the emission peak reflect an unfolding of dHSA tertiary structure in the presence of increasing concentration of Gu·HCl and urea. Guanidine hydrochloride at concentration ⩾3.0 M causes the unfolding of the protein. To obtain the same effect 6.0 M urea was required.The binding (Kb) and quenching (KQ) constants were determined from Scatchard and Stern–Volmer equations, respectively. More than one class of binding sites was found for all studied systems.The decrease of binding constant (Kb) is more distinct in the presence of denaturant (from 3.41 * 107 [M−1] to 8.37 * 104 [M−1] for 5 M Gu·HCl and to 18.50 * 104 [M−1] for 5 M urea) than the decrease of quenching constant (KQ) (from 6.26 * 104 [M−1] to 2.64 * 104 [M−1] and to 1.66 * 104 [M−1]) in the same conditions. This means that even a slight destabilization of dHSA tertiary structure reflects its binding ability.The spectroscopic analysis suggests that one of the sulfasalazine binding sites is located in the IIA subdomain. Both hydrogen bonds and π−π stacking interactions are involved in the formation of the complex.