Journal of Thermal Analysis and Calorimetry, Vol. 57 (1999) 329-341 ON THE NATURE OF THE TEMPERATURE-INDUCED TRANSITION FROM THE MOLTEN GLOBULE TO THE UNFOLDED STATE OF GLOBULAR PROTEINS G. Graziano, F. Catanzano and G. Barone à à à Abstract In this paper we try to perform a thermodynamic analysis of the temperature-induced transi- tion from the molten globule to the unfolded state of globular proteins. A series of calorimetric in- vestigations showed that this process is not associated with an excess heat capacity absorption peak, and cannot be regarded as a first-order phase transition. This result contrasts with the well- established conclusion that the thermal unfolding of the native tertiary structure of globular pro- teins is a first-order phase transition. First, the theoretical approach developed by Ikegami is out- lined to emphasize that a second-order or gradual transition induced by temperature is expected for globular proteins when the various secondary structure elements do not interact cooperatively. Secondly, a simple thermodynamic model is presented which, taking into account the inde- pendence of the secondary structure elements among each other, is able to rationalize the shape of the experimental DSC profiles. Keywords: differential scanning calorimetry, globular proteins, molten globule state, phase tran- sition Introduction In recent years several studies revealed the existence, under specified conditions, of stable partially unfolded states between the native and unfolded conformations of several globular proteins, as excellently reviewed by Kuwajima and Ptitsyn [1–4]. Such partially unfolded states have common properties: a) the molecule is compact relative to the unfolded form, as measured by intrinsic viscosity and rotational re- laxation (i.e., the hydrodynamic radius is only 10–20% larger than that of the native molecule); b) it contains a significant amount of secondary structure, as indicated by far-UV CD spectra; c) it does not possess a unique tertiary structure because the side-chains are largely flexible, as indicated by NMR chemical shifts, and by near- UV CD spectra of aromatic side-chains. These partially unfolded states are collec- tively called the molten globule state [5]. Investigations by means of NMR tech- 1418–2874/99/ $ 5.00  1999 Akadémiai Kiadó, Budapest Akadémiai Kiadó, Budapest Kluwer Academic Publishers, Dordrecht * Author for correspondence: fax: +39/81/5527771, e-mail: barone@chemna.dichi.unina.it