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Titration of ionizable groups of D10N variant catalytic domain has been performed by isothermal calorimetric technique and by combined isoelectric focusing-electrophoresis in the range of pH 3–9. pK0iandΔH0irelated to the ionizable groups have been estimated using Lindestrøm–Lang equation. Both experimental techniques are in agreement and confirm the validity of the applied theory. Slight differences in protonation enthalpies of carboxyl groups are caused by Asp10 interaction with the other part of the macromolecule.

The unfolding of α-chymotrypsinogen covalently immobilized on silica beads has been studied by differential scanning calorimetry (DSC). The enzyme undergoes an unfolding transition which, unlike the free protein, cannot be approximated by a single two-state process. After immobilization, the unfolding is characterized by the presence of two partially overlapping transitions, both of them show two-state behavior. The two processes correspond to the separate unfolding of the two domains of the α-chymotrypsinogen molecule. The loss of cooperativity behavior is a consequence of the covalent immobilization. The two domains showed different thermal stability as functions of pH. One of them unfolded with a transition temperature Tm2higher than Tmof the free enzyme, implying stabilization effect of immobilization. However, below pH 4.5, its native structure is lost. The other transition shows a remarkable pH-independent thermal stability from pH 2.5 to 7.0.