1,1,1,3,3,3- Hexafluoroisopropanol Induced Thermal Unfolding and Molten Globule State of Bovine -Lactalbumin: Calorimetric and Spectroscopic Studies Agnita Kundu Nand Kishore Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India Received 8 July 2003; accepted 11 November 2003 Abstract: The thermal denaturation of -lactalbumin was studied at pH 7.0 and 9.0 in aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) by high-sensitivity differential scanning calorimetry. The conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. The most obvious effect of HFIP was lowering of the transition temperature with an increase in the concentration of the alcohol up to 0.30M, beyond which no calorimetric transition was observed. Up to 0.30M HFIP the calorimetric and van’t Hoff enthalpy remained the same, indicating the validity of the two-state approximation for the thermal unfolding of -lactalbumin. The quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. Spectroscopic observations confirm that -lactalbumin is in the molten globule state in the presence of 0.50M HFIP at pH 7.0 and 0.75M HFIP at pH 9.0. The results also demonstrate that -lactalbumin in the molten globule state undergoes a noncooperative thermal transition to the denatured state. It is observed that two of four tryptophans are exposed to the solvent in the HFIP induced molten globule state of -lactalbumin compared to four in the 8.5M urea induced denatured state of the protein. It is also observed that the HFIP induced molten globule states at the two pH values are different from the acid induced molten globule state (A state) of -lactalbumin. © 2004 Wiley Periodicals, Inc. Biopolymers 73: 405– 420, 2004 Keywords: bovine -lactalbumin 1,1,1,3,3,3-hexafluoroisopropanol; differential scanning calo- rimetry; circular dichroism; fluorescence; molten globule INTRODUCTION Ever since Levinthal proposed 1 that protein folding cannot be a random process there has been consider- able interest in the exact nature of protein folding. Different models such as the hydrophobic collapse model, the framework model, and the funnel model have been proposed. 2,3 The common feature of these models is the prediction of the presence of an inter- mediate, although they vary in their structural features in the respective models. Different experiments in recent years have proved that the hypothesis of the Correspondence to: N. Kishore; email: nandk@iitb.ac.in This article includes supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0006-3525/ suppmat/2004/73/v73.4.405.html Biopolymers, Vol. 73, 405– 420 (2004) © 2004 Wiley Periodicals, Inc. 405