Stability of proteins with multi-state unfolding behavior Ⅱ: Stability of enzymes with multi-state unfolding behavior

Abstract

Based on the model of structural element, a new method used to study the unfolding of enzymes is presented. In this method, it was proposed that enzymes are composed of structural elements and some of them take part in the formation of active center. A least-square fitting can be used to analyze the contribution of each structural element to the enzyme. Average structural element free energy change G element 0 (H 2 O)> is the probability sum of the free energy change of each structural elements. For the enzyme with multi-state unfolding behavior species fractions of enzyme are given. As example, guanidine hydrochloride induced unfolding of hen egg white lysozyme was measured by using fluorescence. The results show that lysozyme has two structural elements, structural element 2 is more stable than 1, and average structural element free energy change G element 0 (H 2 O)> is 48.47 kJ/mol. The variation of enzyme activity with the concentration of guanidine hydrochloride can be attributed to the unfolding of structural element 1, β-sheet domain of lysozyme. In addition, the unfolding of structural element 2, α-helix domain of lysozyme only occurred in the range from 3.8 to 5.0 mol/L of guanidine hydrochloride. Using the model, we can study the unfolding of enzyme by not only spectral measurement, but also the measurement of enzyme activity.