Thermal stability of immobilised alpha-chymotrypsinogen

The unfolding of alpha-chymotrypsinogen covalently immobilized on silica be ads has been studied by differential scanning calorimetry (DSC). The enzyme undergoes an unfolding transition which, unlike the free protein, cannot b e approximated by a single two-state process. After immobilization, the unf olding is characterized by the presence of two partially overlapping transi tions, both of them show two-state behavior. The two processes correspond t o the separate unfolding of the two domains of the alpha-chymotrypsinogen m olecule. The loss of cooperativity behavior is a consequence of the covalen t immobilization. The two domains showed different thermal stability as fun ctions of pH. One of them unfolded with a transition temperature T-m2 highe r than T-m of the free enzyme, implying stabilization effect of immobilizat ion. However, below pH 4.5, its native structure is lost. The other transit ion shows a remarkable pH-independent thermal stability from pH 2.5 to 7.0.