Bulaj G, Otlewski J
Institute of Biochemistry, University of Wroclaw, Poland.
Eur J Biochem. 1994 Aug 1;223(3):939-46. doi: 10.1111/j.1432-1033.1994.tb19071.x.
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 Gden = 44.8 kJ/mol, the denaturation temperature = 65.7 degrees C, the enthalpy change of denaturation delta Hden at the denaturation temperature Tden = 607 kJ/mol and the heat capacity change of denaturation delta Cp,den = 12.4 kJ.mol-1.K-1. Fast phases of both unfolding 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 Gden by 7.1 kJ/mol, Tden by 2.6 K and delta Hden by 80 kJ/mol, but does not induce any substantial structural change in the trypsinogen molecule, as revealed by 1H-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 Hden by 134 kJ/mol and Tden by 2.5 K, which may be attributed to the additional energy required to disrupt the rigidified activation domain in the complexed trypsinogen.
热变性和化学变性已被用于探究胰蛋白酶原与钙离子及配体络合时其热力学稳定性的变化，这些配体会诱导酶原向类胰蛋白酶形式发生构象转变。在pH 5.8条件下，无配体胰蛋白酶原的化学和热变性曲线具有协同性，并得出了以下稳定性参数：变性自由能变化ΔGden = 44.8 kJ/mol，变性温度 = 65.7℃，在变性温度Tden下的变性焓变ΔHden = 607 kJ/mol，以及变性热容量变化ΔCp,den = 12.4 kJ·mol⁻¹·K⁻¹。胰蛋白酶原的解折叠和重折叠的快速阶段都在秒级时间尺度上进行，且符合单一指数规律。在pH 5.8时，钙离子使构象稳定性ΔGden增加7.1 kJ/mol，Tden增加2.6 K，ΔHden增加80 kJ/mol，但如¹H-NMR光谱所示，不会在胰蛋白酶原分子中诱导任何实质性的结构变化。胰蛋白酶原与二肽Ile-Val和/或碱性胰蛋白酶抑制剂（库尼茨）络合产生的类胰蛋白酶形式的特点是，ΔHden增加134 kJ/mol，Tden增加2.5 K，这可能归因于破坏络合的胰蛋白酶原中刚性化的激活结构域所需的额外能量。
