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首页> 外文期刊>RSC Advances >Kinetically-controlled mechanism-based isolation of metabolic serine hydrolases in active form from complex proteomes: butyrylcholinesterase as a case study
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Kinetically-controlled mechanism-based isolation of metabolic serine hydrolases in active form from complex proteomes: butyrylcholinesterase as a case study

机译：基于动力学控制的机制，从复合蛋白质素中的活性形式中代谢丝氨酸水解酶的分离：丁二醇酯酶作为案例研究

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In this work an activity-based probe containing a carbamate group was designed to isolate human butyrylcholinesterase (hBChE), a metabolic serine hydrolase (mSH), from complex proteomes. The method took advantage of the native interaction mechanism of mSHs with carbamate pseudo-substrates for temporarily capturing the enzyme on a resin functionalized with the carbamate probe and releasing the enzyme in active form after removal of the contaminating proteins. The isolation relied on the possibility of manipulating the carbamylation and decarbamylation kinetics favoring the former during the capture and wash steps and the latter in the release step. The designed probe captured and released all the active hBChE isoenzymes present in plasma with high selectivity (up to ～2000-fold purification) and reasonable yields (17% to 36%). The parameters affecting the performance were the incubation time used in the load and elution steps, the plasma to resin volumetric ratio, the elution temperature and the nature and concentration of the eluting agent. The carbamate resin could be prepared either by coupling a fully synthesized probe with an activated resin or by building the probe onto the resin by a step-by-step procedure, without major differences in performance between the two routes. The prepared resins allowed to process up to about 8.5 mL of plasma per g of resin with constant performance. Since the method was based on the general catalytic cycle of mSHs, we expect this approach to be applicable to other enzymes of the family, by selecting a suitable target-selective feature to link to the carbamate group.
机译：在这项工作中，设计了含有氨基甲酸酯基团的基于活性的探针，以分离来自复杂蛋白质蛋白的代谢丝氨酸水解酶（HBCHE），代谢丝氨酸水解酶（MSH）。该方法利用了用氨基甲酸酯伪衬底的MSH的天然相互作用机制，用于临时捕获用氨基甲酸氨基甲酸氨酸探针官能化的树脂上的酶，并在除去污染蛋白后以活性形式释放酶。分离依赖于在捕获和洗涤步骤和后一步中操纵捕获和洗涤步骤的碳酸氨基化和脱氨酰胺动力学的可能性。设计的探针捕获并释放出血浆中存在的所有活性HBCHE同工酶，具有高选择性（高达〜2000倍纯化）和合理的产率（17％至36％）。影响性能的参数是在负载和洗脱步骤中使用的孵育时间，等离子体为树脂体积比，洗脱剂的洗脱温度和性质和浓度。可以通过将完全合成的探针与活化树脂偶联或通过逐步逐步制备氨基甲酸酯树脂，或者通过逐步的方法将探针建立在树脂上，而两条路线之间的性能具有重大差异。制备的树脂使得每G具有恒定性能的每G树脂高达约8.5ml血浆。由于该方法基于MSH的一般催化循环，因此我们预计该方法通过选择合适的靶选择性以链接到氨基甲酸酯组来适用于家族的其他酶。

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