Solvation of human serum albumin in aqueous solutions of urea, methyl-, N,N'-dimethyl-, and ethylurea was studied by density measurements. From the densities at constant molality and constant chemical potential, the preferential solvation parameters of human serum albumin were determined. In all solutions the denaturant is preferentially bound. From preferential solvation data, Gibbs free energies of transfer from water to alkylurea solutions were calculated. Since the enthalpies of transfer were determined previously the entropies of transfer would also be obtained. Based on these and model compound data an attempt is made to identify various interactions involved in solvation. The total binding of denaturant to protein could also be calculated from preferential solvation data. The following major binding sites have been identified: ionic groups on the surface of protein molecules, peptide groups and aromatic side-chains. The correlation between the total number of sites and the number of urea molecules bound is satisfactory.
