Melo_1997_J.Biochem.Biophys.Methods_34_45

This work describes a methodology to monitor protein unfolding by using the well known changes in tyrosine absorbance with the ionization of the side chain phenol group. It can be applied to proteins that are functionally active at pH values higher than 9.0 where the current UV differential spectroscopy technique can not be used. The simplicity and facility of the proposed methodology (only two absorbance measurements have to be acquired) can make it very useful namely for technological applications. Thermal unfolding of cutinase and alpha-chymotrypsin were followed using this methodology and the thermodynamic stability data were obtained assuming a two-state mechanism. The transition from the folded to the unfolded state was further confirmed by fluorescence maxima for both proteins proving the validity of the methodology based on UV measurements.