Department of Biochemistry, Faculty of Chemistry, University of Belgrade, 11000 Belgrade, Serbia; Institute for Chemistry in Medicine, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia.
Laboratory for Molecular Microbiology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, 11000 Belgrade, Serbia; Department for Food Microbiology, Faculty of Agriculture, University of Belgrade, 11000 Belgrade, Serbia.
J Biosci Bioeng. 2021 Mar;131(3):234-240. doi: 10.1016/j.jbiosc.2020.10.010. Epub 2020 Nov 11.
Trypsin is a serine protease with important applications such as protein sequencing and tissue dissociation. Preserving protein structure and its activity during freeze-thawing and prolonging its shelf life is one of the most interesting tasks in biochemistry. In the present study, trypsin cryoprotection was achieved by altering buffer composition. Sodium phosphate buffer at pH 8.0 led to pH shift-induced destabilization of trypsin and formation of a molten globule, followed by significant activity loss (about 70%). Potassium phosphate and ammonium bicarbonate buffers at pH 8.0 were used with up to 90% activity recovery rate after 7 freeze-thaw cycles. The addition of non-ionic surfactants Tween 20 and Tween 80 led to up to 99% activity recovery rate. Amide I region changes, corresponding to specific secondary structures in the Fourier transform infrared (FTIR) spectrum, were modest in the case of Tween 20 and Tween 80. On the other hand, the addition of Triton X-100 led to the destabilization of α-helicoidal segments of trypsin structure after 7 freeze-thaw cycles but also increased protein substrate availability.
胰蛋白酶是一种丝氨酸蛋白酶，具有重要的应用，如蛋白质测序和组织解离。在冷冻和解冻过程中保持蛋白质结构及其活性，并延长其保质期，是生物化学中最有趣的任务之一。在本研究中，通过改变缓冲液组成实现了胰蛋白酶的冷冻保护。在 pH 8.0 的磷酸钠缓冲液中，会导致 pH 诱导的胰蛋白酶不稳定性和形成熔融球蛋白，随后活性显著丧失（约 70%）。在 pH 8.0 的磷酸钾和碳酸氢铵缓冲液中，在经过 7 次冷冻-解冻循环后，可达到高达 90%的活性回收率。添加非离子表面活性剂吐温 20 和吐温 80 可使活性回收率达到 99%。在傅里叶变换红外（FTIR）光谱中酰胺 I 区域的变化对应于特定的二级结构，在吐温 20 和吐温 80 的情况下变化不大。另一方面，在经过 7 次冷冻-解冻循环后，添加 Triton X-100 会导致胰蛋白酶结构的α-螺旋片段不稳定，但也增加了蛋白质底物的可用性。
