Abstract
Thermal denaturation for the wild-type of tryptophan synthase alpha-subunits from E. coli and one of its mutant proteins was followed by CD measurements at various pHs in the alkaline region and the results from van't Hoff analyses of the thermal denaturation curves were compared with those from calorimetry. Although the far-u.v. CD spectra of the thermally denatured proteins differed from those of the completely denatured states in 3.2 M guanidine hydrochloride, the titration curves by denaturants at higher temperatures were not sigmoidal but straight lines, indicating that the cooperative structure of the proteins has been completely destroyed by heating. The ratio of calorimetric enthalpy change to van't Hoff enthalpy change obtained from calorimetric study was unity, indicating that the thermal denaturation of the proteins was a two-state system. The unfolding heat capacity change (delta Cp) of the wild-type protein from van't Hoff analysis of the thermal denaturation curves by CD measurement was estimated to be 2.45 kcal/mol X deg, which was similar to that from calorimetry. The values of unfolding enthalpy change at denaturation temperatures were lower by about 15 kcal/mol compared to those from calorimetry.
Published Version
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