Effect of salt concentration on the pK_a of acidic residues in lysozyme

We determined the pK_a values of acidic residues in hen lysozyme by comparing the pH dependency of stability between wild type and mutant lysozymes in which a negative charge is eliminated. In the comparison of the stability between wild type and a mutant lysozyme, the difference in pH titration curve between them could be expressed as a two-state process involving protonation of a single acidic residue. The results strongly indicated that the Aune and Tanford theory of protein denaturation [Aune, K.C. and Tanford, C. (1969) Biochemistry 8, 4579-4585] is applicable to protein stability in solution. On the other hand, the pK_a values of acidic residues in the presence of low (5 mM) or high (400 mM) salt concentration were determined by means of two-dimensional NMR. We found that the pK_a values obtained from the pH dependency of stability were close to those from the NMR experiment under the high salt condition. Moreover, by comparing pK_a values at high salt and low salt concentrations, we could evaluate the dependency of two electrostatic interactions (salt bridge and charge-helix dipole interaction) on salt concentration.