SALT-INDUCED FORMATION OF THE MOLTEN GLOBULE STATE OF CYTOCHROME-C STUDIED BY ISOTHERMAL TITRATION CALORIMETRY

Although the molten globule state has been proposed as a major interme diate of protein folding, ie has proven difficult to obtain thermodyna mic data characterizing this state. To explore another approach for ch aracterizing the molten globule state, salt-induced formation of the m olten globule state of horse cytochrome c at pH 1.8 was studied by iso thermal titration calorimetry. By titrating the acid-unfolded cytochro me c with sodium perchlorate, an exothermic reaction was observed. The titration curve obtained from the heat was cooperative and agreed wel l with the conformational transition curve measured by CD at 222 nm. T his result indicated that the salt-induced conformation change is well approximated by a two-state transition between the acid-unfolded and molten globule states. The heat for formation of the molten globule st ate estimated by isothermal titration calorimetry was consistent with the enthalpy change for unfolding of the sodium perchlorate-stabilized molten globule state at pH 1.8, which was measured by differential sc anning calorimetry and CD. These results indicate that the heat of tit ration largely reflects the enthalpy change of the conformational tran sition. From these results, we consider that isothermal titration calo rimetry will become a useful approach for investigating the molten glo bule state.