DENATURATION OF FREE AND COMPLEXED BOVINE TRYPSINOGEN WITH THE CALCIUM-ION, DIPEPTIDE ILE-VAL AND BASIC PANCREATIC TRYPSIN-INHIBITOR (KUNITZ)

Thermal and chemical denaturation has been used to probe changes in the thermodynamic stability of trypsinogen upon complexation with calcium ion and with ligands, which induce the conformational transition of the zymogen to the trypsin-like form. Chemical and thermal unfolding curves of ligand-free trypsinogen at pH 5.8 are cooperative and yielded the following stability parameters: the free energy change of denaturation Delta G(den) = 44.8 kJ/mol, the denaturation temperature = 65.7 degrees C, the enthalpy change of denaturation Delta H-den at the denaturation temperature T-den= 607 kJ/mol and the heat capacity change of denaturation Delta C-pden = 12.4 kJ.mol(-1).K-1. Fast phases of both unfold ing and refolding of trypsinogen proceed on a time scale of seconds and fit to a single exponential. At pH 5.8, the calcium ion increases the conformational stability Delta G(den) by 7.1 kJ/mol, T-den by 2.6 K and Delta H-den by 80 kJ/mol, but does not induce any substantial structural change in the trypsinogen molecule, as revealed by H-1-NMR spectra. The trypsin-like form of trypsinogen, generated by complexation of the zymogen with the dipeptide Ile-Val and/or basic pancreatic trypsin inhibitor (Kunitz), is characterized by increase of Delta H-den by 134 kJ/mol and T-den by 2.5 K, which may be attributed to the additional energy required to disrupt the rigidified activation domain in the complexed trypsinogen.