EQUILIBRIUM AND KINETIC-STUDIES ON REVERSIBLE AND IRREVERSIBLE DENATURATION OF MICROCOCCAL NUCLEASE

The effects of pH and temperature on the thermal denaturation of micro coccal nuclease were investigated. The ranges employed were between pH 3.30 and pH 9.70 and between 10 degrees C and 85 degrees C, respectiv ely. The reversible denaturation involved in the whole process was cle arly discriminated from the irreversible one. The former took place wi th a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enz yme exhibited its maximum activity. The latter probably led to aggrega tion because the successive long incubation after complete deactivatio n caused precipitation. A reasonable scheme explaining the process inv olving both denaturations was proposed and the kinetic analysis on the irreversible deactivation was performed. It was revealed that the irr eversible deactivation involved two types of reactions whose activatio n energies were relatively small: 22.2 kJ mol(-1) and 18.8 kJ mol(-1). The presence of sucrose suppressed the reversible denaturation withou t significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change a nd addition of sucrose on the denaturation were discussed thermodynami cally, especially in terms of the entropy change. (C) 1994 John Wiley & Sons, Inc.