Effect of Mg 2+ on the thermal inactivation and unfolding of creatine kinase

Abstract

The effect of Mg 2+ on the thermal inactivation and unfolding of rabbit muscle creatine kinase has been studied for various temperatures and Mg 2+ concentrations. Increasing the Mg 2+ concentration in the denatured system significantly enhanced the inactivation and unfolding of creatine kinase during thermal denaturation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47°C the increased free Mg 2+ concentration caused the creatine kinase inactivation rate to increase. Increasing the temperature strengthened the effect of Mg 2+ on the thermal inactivation. Control experiments showed that treating native creatine kinase with different concentrations of Mg 2+ did not change the enzymatic activity. The fluorescence emission spectra showed that the emission maximum for creatine kinase red-shifted from 335 to 337 nm during thermal denaturation at 47°C for 10 min, while the presence of 3 mM Mg 2+ caused the enzyme emission maximum to red-shift from 335 to 342.5 nm for the same thermal denaturation conditions. In addition, Mg 2+ also enhanced the unfolding of the equilibrium state and decreased the time required to reach the equilibrium state of creatine kinase at 47°C. The potential biological significance of these results are discussed.