Biochemical Neuropharmacology Laboratory, Kazan Federal University, Kremlievskaya Str. 18, 420111 Kazan, Russia.
Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin Str. 4, 119334 Moscow, Russia.
Molecules. 2022 Oct 13;27(20):6861. doi: 10.3390/molecules27206861.
The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes.
蛋白质的功能结构源于略微稳定的折叠构象。由于 pH 值、离子强度、温度、压力和电场等物理化学条件的变化，或由于存在扰乱稳定和不稳定相互作用之间微妙平衡的共溶剂，化学和物理试剂会导致蛋白质可逆解折叠、不可逆变性和劣化。对于大多数蛋白质来说，变性是一个复杂的过程，涉及到几个可逆的瞬态中间体，最终导致不可逆的步骤。了解蛋白质的稳定性和变性过程对于开发作为工业催化剂、生物制药、分析和医学生物试剂以及安全的工业食品的酶是必不可少的。在极端条件下运行的电泳技术是分析解折叠转变、捕获瞬态中间体以及深入了解变性过程机制的便捷工具。此外，电泳迁移率过渡曲线的定量分析可以估计蛋白质的构象稳定性。这些方法包括在冷、热和静水压力下以及在非离子变性剂或稳定剂（如多元醇和重水）存在下进行的聚丙烯酰胺凝胶电泳和毛细管区带电泳。最后，在暴露于物理条件的极端条件后，在标准条件下的电泳提供了关于不可逆过程、缓慢构象漂移和缓慢复性过程的信息。由于在精细化学、生物制药和纳米医学中有多种应用的酶技术的惊人发展，我们重新审视了这些电泳方法的潜力。本专题综述以作者在胆碱酯酶和对氧磷酶方面获得的已发表和未发表的结果为例进行了说明，这两种酶在生理和毒理学上都很重要。
