Thermal denaturation of Streptomyces subtilisin inhibitor, subtilisin BPN', and the inhibitor-subtilisin complex

Abstract

The thermal unfolding of the microbial proteinase inhibitor Streptomyces subtilisin inhibitor (SSI) [Sato, S., & Murao, S. (1973) Agric, Biol. Chem. 37, 1067-1074), the bacterial proteinase subtilisin BPN' (EC 3.4.21.14), and the complex formed by these two proteins has been studied by differential scanning calorimetry (DSC). The thermal denaturation of SSI at pH 7.00 is fully reversible while those of subtilisin BPN' and its complex with SSI are not. The DSC data show that dimeric SSI remains dimeric as the temperature is raised until it unfolds and that it then dissociates during the unfolding process. The apparent specific heat of denatured SSI decreases rapidly with increasing temperature, a behavior not previously observed for proteins. The shape of the DSC curves observed with the enzyme-inhibitor complex suggests that the two components of the complex undergo their unfolding transitions more or less independently. The enthalpies of unfolding of mixtures of enzyme and inhibitor in various molar ratios indicate a substantially large enthalph of interaction than that deduced from fluorescence titrations (Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 1195-1202).