Structural Characterization of Urea-Induced BSA Denaturation Using Size-Exclusion Chromatography Coupled with Small-Angle X-ray Scattering

This study investigates the urea-induced structural transitions of bovine serum albumin (BSA) in solution using size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS). BSA samples treated with 0 to 8 M urea were subjected to SEC to isolate monomeric species, enabling the acquisition of high-quality SAXS data and ab initio 3D modeling using GASBOR. Key structural parameters-including the radius of gyration (R_g), maximum dimension (D_max), and pair distance distribution function P(r)─were derived from the scattering profiles. Three major conformational states were identified: a compact globular form (0-3 M urea), a partially unfolded intermediate (4 M urea), and a highly disordered conformation (5-8 M urea). These transitions were corroborated by Kratky analysis and structural modeling. At higher urea concentrations, GASBOR yielded representative average structures, while the ensemble optimization method (EOM) revealed conformational heterogeneity, reflecting increased structural flexibility. Overall, SEC-SAXS enabled detailed characterization of BSA unfolding and provided insights into protein dynamics and stability under denaturing conditions.