Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain

Abstract

High‐sensitivity differential scanning calorimetry has been applied to the study of porcine pancreatic carboxypeptidase B, the proenzyme and its 81‐residue activation domain. The thermal study has been carried out over a range of scan rates, ionic strengths and pH values. The thermal unfolding of the isolated activation domain has been found to be reversible and corresponds to that of a typical compact globular structure, with melting temperatures higher than those of the enzyme and proenzyme. Both proteins, on the other hand, undergo an irreversible, highly scan‐rate‐dependent thermal denaturation under all the experimental conditions investigated. The denaturation of the enzyme at pH 7.5 and the proenzyme at pH 7.5 and 9.0 follows the two‐state irreversible model [Sánchez‐Ruiz, J. M., López‐Lacomba, J. L., Cortijo, M. & Mateo, P. L. (1988) Biochemistry 27, 1648–1652]. Thus the kinetic constants and activation parameters of the denaturation process could be obtained and compared to those for other proteins, particularly those of the closely related carboxypeptidase A system. Copyright © 1991, Wiley Blackwell. All rights reserved