The Endothermic Effects during Denaturation of Lysozyme by Temperature Modulated Calorimetry and an Intermediate Reaction Equilibrium

Abstract

To gain insight into the thermodynamics of protein denaturation, the complex heat capacity, Cp* (= Cp‘ − iCp‘ ‘) of lysozyme−water system has been measured at pH 2.5 in the 293−368 K range by using temperature-modulated scanning calorimetry (TMSC), a technique in which the thermally reversible enthalpy changes are measured separately and simultaneously with the thermally irreversible enthalpy changes. The plot of Cp‘ against the temperature T shows a broad peak, which is similar to that observed in Cp,DSC, measured here and elsewhere by differential scanning calorimetry (DSC), a technique which gives the sum of both the reversible and irreversible contributions in the apparent heat capacity value. This peak in Cp,DSC has been generally attributed to endothermic heat absorption on reversible and irreversible unfolding processes and irreversible thermal denaturation. It is shown that the observed Cp‘ peak results from heat absorption when the equilibrium constant between the native lysozyme state and a conformationally different intermediate state increases with T. The plot of Cp‘ versus T is subdivided into four regions, corresponding to the dominance of a certain process. Thermal denaturation of lysozyme was found to occur according to a scheme, native state ↔ unfolded (intermediate) state → denatured state. This conclusion is consistent with the general view that the first step of denaturation of small one-domain globular protein like lysozyme is a reversible conformational (unfolding) transition, and the second step is irreversible denaturation. It is shown that when kept isothermally at T > 341 K, i.e., within the transition temperature range, Cp‘ of lysozyme decreases. This decrease is exponential in time and corresponds to a rate constant, which varies according to the Arrhenius-type equation, with a preexponential factor of 5 × 1020 s-1 and energy of 167 kJ/mol. The overall kinetics of the denaturation reaction is of the first order.