Preferential interactions of urea with lysozyme and their linkage to protein denaturation

The interactions involved in the denaturation of lysozyme in the presence of urea were examined by thermal transition studies and measurements of preferential interactions of urea with the protein at pH 7.0, where it remains native up to 9.3 M urea, and at pH 2.0, where it undergoes a transition between 2.5 and 5.0 M urea. The destabilization of lysozyme by urea was found to follow the linear dependence on urea molar concentration, M u , Δ G u o =Δ G w o −2.1 M u , over the combined data, where Δ G u o and Δ G w o are the standard free energy changes of the N⇌D reaction in urea and water, respectively. Combination with the measured preferential binding gave the result that the increment of preferential binding, δΓ 23=Γ 23 D −Γ 23 N , is also linear in M u . A temperature dependence study of preferential interactions permitted the evaluation of the transfer enthalpy, Δ H ̄ 2, tr o , and entropy, Δ S ̄ 2, tr o of lysozyme from water into urea in both the native and denatured states. These values were found to be consistent with the enthalpy and entropy of formation of inter urea hydrogen bonds (Schellman, 1955; Kauzmann, 1959), with estimated values of Δ H ̄ 2, tr o =ca. −2.5 kcal mol −1 and Δ S ̄ 2, tr o =ca. −7.0 e.u. per site. Analysis of the results led to the conclusion that the stabilization of the denatured form was predominantly by preferential binding to newly exposed peptide groups. Combination with the knowledge that stabilizing osmolytes act by preferential exclusion from peptide groups (Liu and Bolen, 1995) has led to the general conclusion that both the stabilization and destabilization of proteins by co-solvents are controlled predominantly by preferential interactions with peptide groups newly exposed on denaturation.