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

Whereas apomyoglobin is unfolded at pH 2 by HCl in the absence of salt , the addition of anions either from salts or acids stabilizes the mol ten globule state, i.e, a compact denatured state with a significant n ative-like secondary structure but with largely flexible side-chains, To clarify the thermodynamic mechanism responsible for the conformatio nal stability of the molten globule state of apomyoglobin, we studied the salt-induced formation of the molten globule state of horse apomyo globin at pH 2 by isothermal titration calorimetry (ITC). By titrating the acid-unfolded apomyoglobin with NaClO4 or Na2SO4, an exothermic r eaction was observed. The titration curve obtained from the heat was c ooperative and consistent with the conformational transition curve mea sured by circular dichroism at 222 nm, This suggested that the salt-in duced conformation change can be approximated by a two-state transitio n between the acid-unfolded and molten globule states. However, the he at for formation of the molten globule state estimated by ITC was slig htly larger in magnitude than the enthalpy change for unfolding of the salt-stabilized molten globule state at pH 2, suggesting a relatively small contribution of heat other than the conformational change, Thes e results support a view that the conformational transition of apomyog lobin at pH 2 can be represented, as a first approximation, by a two-s tate transition between the molten globule state and the fully unfolde d state.