Enthalpy analysis of horseradish peroxidase in the presence of Ni2+: a stabilization study

The interaction of horseradish peroxidase (HRP) with Ni2+ has been investigated by isothermal titration calorimetry (ITC), potentiometry, equilibrium dialysis, and spectrophotometry. Total enthalpy of cited interaction was measured by ITC at 27 °C, pH=7.0.The enthalpy of ionization (ΔHion) is obtained by acid–base titration at 25 and 50 °C in 100 mM NaCl. Enthalpy of binding is obtained by equilibrium dialysis at 27 and 37 °C based on the Wyman binding potential to evaluate equilibrium constants at two temperatures. The van’t Hoff relation is used for calculation of enthalpy of binding (ΔHbin) and also a modified differential relation is applied for estimation of ionization enthalpy (ΔHion). Denaturation profiles on HRP with and without the presence of Ni2+ using n-dodecyl trimethylammonium bromide (DTAB) as a denaturant, has been studied. Using the Pace theory to evaluate free energy in the absence of denaturant (ΔG°H2O) is the best parameter for determination of protein stability. The results show 4.9 kJ mol−1 higher free energy for stabilization of HRP in the presence of Ni2+. Enthalpy of conformational change of HRP by Ni2+ (3.5 mM) is determined by resolving the contributions of calorimetric enthalpy, binding enthalpy and ionization enthalpy. The enthalpy of stabilization for HRP in the presence of Ni2+ (3.5 mM) is equal to −118.62 kJ mol−1 which was obtained by differential relation between enthalpies of unfolding and conformation.