Kinetics of heat- and acidification-induced aggregation of firefly luciferase

Abstract

The general approach to analysis of the kinetics of protein aggregation registered by the turbidimetric method has been elaborated. The terminal part of the kinetic curves is analyzed using a theoretical equation connecting the derivative of the apparent absorbance (A) with respect to time (dA/dt) and A (t is time). This analysis allows the limiting value of A at t--> infinity (A(lim)) and the order of aggregation with respect to protein (n) to be calculated. Approach proposed was applied to analysis of thermal and acidification-induced aggregation of firefly luciferase. In both cases the A(lim) value is a linear function of the protein concentration. The terminal part of the kinetic curves of thermal aggregation follows the first-order kinetics (n=1), whereas the kinetics of acidification-induced aggregation are characterized by the value of n higher than unity (n=1.29). The mechanism of nucleation-dependent aggregation has been discussed.