Hydrodynamic and spectroscopic analysis of the denaturation of serum albumin induced by guanidinium chloride and sodium dodecyl sulfate

The combination of highspeed sedimentation equilibria and specific volume estimates under isomolal or isopotential conditions allows the molar mass of proteins, denatured by chaotropic reagents such as guanidinium chloride (GdmCl) or ionic detergents such as sodium dodecyl sulfate (SDS), to be analyzed. The results depend strictly on the procedures applied: dialyzed samples allow the mass of the unliganded protein to be determined, whereas undialyzed samples yield the masses of the protein-ligand complexes. The first alternative is the only way to obtain thermodynamically sound data; it was applied in the case of GdmCl-denatured bovine serum albumin (BSA). In the case of detergents which do not allow equilibrium dialysis to be applied, mixtures of stock solutions of BSA and SDS were used to determine the molar masses of the BSA-SDS complexes and their binding isotherms. Spectroscopic techniques (UV absorption, fluorescence and circular dichroism) were used to monitor protein unfolding in terms of transition curves and free energies of denaturation. The free-energy change obtained for the SDS-induced denaturation of BSA is found to be considerably smaller than that for the denaturation by GdmCl, in agreement with the lower denaturation capacity of SDS and other detergents compared with highly potent chaotropic denaturants such as GdmCl.