THERMAL UNFOLDING OF PROTEINS AT HIGH PH RANGE STUDIED BY UV ABSORBENCY

This work describes a methodology to monitor protein unfolding by usin g the well known changes in tyrosine absorbance with the ionization of the side chain phenol group. It can be applied to proteins that are f unctionally active at pH values higher than 9.0 where the current UV d ifferential spectroscopy technique can not be used. The simplicity and facility of the proposed methodology (only two absorbance measurement s have to be acquired) can make it very useful namely for technologica l applications. Thermal unfolding of cutinase and alpha-chymotrypsin w ere followed using this methodology and the thermodynamic stability da ta was obtained assuming a two-state mechanism. The transition from th e folded to the unfolded state was further confirmed by fluorescence m axima for both proteins proving the validity of the methodology based on UV measurements. (C) 1997 Elsevier Science B.V.