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 27degreesC, pH = 7.0. The enthalpy of ionization (DeltaH(ion)) is obtained by acid-base titration at 25 and 50degreesC in 100 mM NaCl. Enthalpy of binding is obtained by equilibrium dialysis at 27 and 37degreesC 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 (DeltaH(bin)) and also a modified differential relation is applied for estimation of ionization enthalpy (DeltaH(ion)). 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 (DeltaG(H2O)degrees) is the best parameter for determination of protein 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 -118.62kJ mol(-1) which was obtained by differential relation between enthalpies of unfolding and conformation. (C) 2002 Elsevier Science B.V. All rights reserved.