Abstract:

This work presents the correlation of the enzymatic activity of α-chymotrypsin (α-CT) with the thermodynamics of interaction between α-CT and the cationic gemini surfactant decanediyl-α, ω-bis (dodecyldimethylammonium bromide) (12-10-12). The enzymatic activity was assessed by the rate of 2-naphthyl acetate (2-NA) hydrolysis obtained from UV-Vis absorption spectra. The superactivity of α-CT in the catalytic hydrolysis of 2-NA was obtained by activation with 12-10-12 in a short incubation time; the activated α-CT showed faster denaturation kinetics. The larger superactivities appeared in a bell shape below the critical aggregation concentration (cac_{12-10-12, CT}) of the mixed gemini/α-CT systems in buffered aqueous solution. The results obtained from the variation of the activity with the incubation time highlight that the protein incubated in 12-10-12 has a high catalysis activity and a weakened conformational stability. The mechanism of the superactivity of α-CT in the presence of 12-10-12 has been proposed by combining the results from isothermaltitration calorimetry (ITC), steady state fluorescence, and differential scanning calorimetry (DSC). The superactivity arises from perturbation of the internal structure of α-CT by an interaction between the positively charged 12-10-12 and α-CT, which makes the conformation of α-CT looser than the native one, in the balance of a weak interaction. Such a conformation is favorable for release of the acidic product of 2-NA hydrolysis, whereas it simultaneously leads to instability of the α-CT structure.

Key words: Surfactant, α-chymotrypsin, Superactivity, Isothermal titration calorimetry, Steady state fluorescence, Differential scanning calorimetry