Momeni Lida, Mahmodian Sheida, Shareghi Behzad, Saboury Ali Akbar, Farhadian Sadegh
Department of Biology, Faculty of Science, University of Payam Noor, Iran.
Department of Biology, Faculty of Science, University of Shahrekord, Shahrekord, P. O. Box.115, Iran.
Int J Biol Macromol. 2017 Jun;99:343-349. doi: 10.1016/j.ijbiomac.2017.02.090. Epub 2017 Feb 27.
Docking and spectroscopic techniques were performed to probe the stabilizing effect of sucrose on the dynamics, structure and activity of trypsin. The thermodynamic folding properties, melting temperature (T), enthalpy change (ΔH°) and entropy change (ΔS°) were measured by thermal stability studies to understand the picture of trypsin folding. Sucrose acted as an enhancer for trypsin stability. Fluorescence spectroscopy revealed the static model of the quenching. The number of binding sites was 1. The Absorption, Fluorescence and circular dichroism spectral analysis illustrated that sucrose could protect the native structural conformation of enzyme and prevent the enzyme unfolding. Fluorescence spectroscopy and the molecular docking technique simulation displayed that the hydrogen bonding and Vander Waals forces played a main role in stabilizing the trypsin-sucrose complex, and the number of direct H-bonds between sucrose and trypsin was low; thus, the direct interactions had little contribution in the stabilizing effect and the indirect interactions caused by the preferential hydration were resulting from a molecular mechanism principally causing the stabilizing effects of sucrose.Upon sucrose conjugation, the k/K value of the enzyme was increased. T of the trypsin-sucrose complex was increased due to the higher H-bond formation and the lower surface hydrophobicity after sucrose modification. Sucrose acted as enhancers for trypsin stability and activity. The result shows the ability of sucrose to protect the native structural conformation of trypsin. These results explicitly describe that stabilizing sucrose is preferentially excluded from the surface of trypsin, since water has a higher tendency toward favorable interactions with functional groups of trypsin than with sucrose.
采用对接和光谱技术来探究蔗糖对胰蛋白酶动力学、结构和活性的稳定作用。通过热稳定性研究测量了热力学折叠性质、解链温度（T）、焓变（ΔH°）和熵变（ΔS°），以了解胰蛋白酶折叠情况。蔗糖可作为胰蛋白酶稳定性的增强剂。荧光光谱揭示了猝灭的静态模型。结合位点数量为1。吸收光谱、荧光光谱和圆二色光谱分析表明，蔗糖可保护酶的天然结构构象，防止酶解折叠。荧光光谱和分子对接技术模拟显示，氢键和范德华力在稳定胰蛋白酶 - 蔗糖复合物中起主要作用，且蔗糖与胰蛋白酶之间的直接氢键数量较少；因此，直接相互作用对稳定作用贡献不大，而优先水合作用引起的间接相互作用是导致蔗糖产生稳定作用的主要分子机制。蔗糖结合后，酶的k/K值增加。由于蔗糖修饰后形成更多氢键且表面疏水性降低，胰蛋白酶 - 蔗糖复合物的T增加。蔗糖可作为胰蛋白酶稳定性和活性的增强剂。结果表明蔗糖具有保护胰蛋白酶天然结构构象的能力。这些结果明确表明，稳定化的蔗糖优先被排除在胰蛋白酶表面，因为水与胰蛋白酶功能基团形成有利相互作用的倾向高于与蔗糖的相互作用。
