THERMODYNAMIC STUDY OF THE ACID DENATURATION OF BARNASE AND ITS DEPENDENCE ON IONIC-STRENGTH - EVIDENCE FOR RESIDUAL ELECTROSTATIC INTERACTIONS IN THE ACID THERMALLY DENATURED STATE/

We have investigated the acid denaturation of barnase and its dependen ce on ionic strength. From the pH dependence of the protein stability, we have obtained information about the titration properties of the na tive and denatured protein at temperatures ranging from 15 to 60 degre es C in the absence of chemical denaturant. It appears that both the n ative and the denatured state of barnase titrates at higher pH values in the presence of salt. The observation suggests that charge interact ions are present, not only within the native fold but also within the denatured state, and that these interactions contribute to shift the p K(a) values from those of isolated model compounds. Upon addition of s alt these repulsive interactions are shielded, and the electrostatic f ree energy of the native state, as well as the denatured state, is red uced. Accordingly, we suggest that the thermally denatured state of ba rnase is not an extended random coil without residue-residue interacti ons but is sufficiently compact to contain intramolecular charge-charg e repulsions. The results further reveal that the native state of barn ase contains at least one residue with a highly anomalous pK(a) value: At pH 0.3, the difference in degree of protonation between the native and the denatured state is still about 1 mol H+/mol protein.