INTERPRETATION AND APPLICATIONS OF THERMAL DIFFERENCE SPECTRA OF PROTEINS

Publisher Website
Google Scholar
Abstract
The technique of thermal perturbation difference spectroscopy for examining chromophores in proteins was set on a more acceptable theoretical and experimental foundation. The origins of the thermal effect were analyzed to explain changes in spectral band width and wavelength for simple chromophores in various solvents. Comparison with theoretical curves shows that the effect of heating chromophore solutions is mainly spectral broadening coupled with an almost negligible blue shift. The apparently anomalous behavior of tryptophan and tyrosine in aqueous solvents, where the main effect is a red shift on heating, is traced to H-bonding with water. A model in which tyrosine acts simultaneously as H-donor (Type I) and H-acceptor (Type II) and the latter is the more temperature sensitive, is successful in explaining all available data for a variety of solute derivatives and solvents. The contribution of chromophores in the protein interior was assessed using polyvinyl alcohol films of differing water content. These models simulate the rigidity and low thermal expansivity of the protein interior and confirm that buried chromophores contribute negligibly if at all to thermal difference spectra. Curve-fitting procedures were used for matching protein difference spectra over a wavelength range, to those for mixtures of models, rather than relying on data measured at extrema.