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. Fire found that the pK(a) values obtained hom the pH dependency of stability were dose 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.