Abstract

In this study Firefly Luciferase from Lampyris turkestanicus and some of its representative mutants with surface charge modifications have been used for kinetic studies using pH-Jump experiments with fluorescence detected Stopped-Flow apparatus. All Kinetic Curves were analysed by Biokine analysis software and were fitted to single exponential function. Accordingly, the rate constants of unfolding reactions were obtained.  The rate constants of unfolding reactions were converted to free energy differences of transition state (∆G‡) using transition state theory formula. According to our study, substitution of Glu by Arg (R mutant) results in decreasing of rate constant of unfolding reaction upon pH jumping, while, insertion of other Arg in R mutant (RR mutant) and substitution of Glu by Lys (K mutant) elicit to increasing the rate constant and decreasing energy barrier of unfolding reactions of mutants relative to that of wild type luciferase. We concluded that energy barrier of pH-Jumping unfolding is affected by charge balance  at the surface of protein.