Li He, Tong Larry, Schopfer Lawrence M, Masson Patrick, Lockridge Oksana
Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA.
Chem Biol Interact. 2008 Sep 25;175(1-3):68-72. doi: 10.1016/j.cbi.2008.04.027. Epub 2008 May 2.
Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30 min to purify BChE from 1 ml plasma. The method uses a microfuge spin column to build a 0.2 ml procainamide affinity column. The eluted BChE contains 3-4 microg of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.
传统的血浆丁酰胆碱酯酶（BChE）纯化方法包括透析和多个色谱步骤。我们描述了一种普鲁卡因酰胺亲和凝胶纯化方案，该方案从1毫升血浆中纯化BChE只需15至30分钟。该方法使用微量离心机旋转柱构建一个0.2毫升的普鲁卡因酰胺亲和柱。洗脱的BChE含有3至4微克经过500倍纯化的BChE，且不含99%的血浆污染蛋白。通过凝胶电泳对BChE进行了进一步纯化。含有BChE的凝胶电泳条带的胰蛋白酶肽段通过胶内消化制备，经反相液相色谱分离并通过质谱鉴定。鉴定出了用神经毒剂梭曼或沙林标记的29个残基的活性位点胰蛋白酶肽段。
