牛血清乙酰胆碱脂酶的分离纯化

Purification of Acetylcholinesterase(AChE) from Bovine Serum

以牛血清为原材料,采用硫酸铵分级沉淀、Sephadex G-100凝胶柱和DEAE-纤维素DE23柱层析对牛血清中的乙酰胆碱酯酶进行分离纯化。结果表明,经以上三步分离纯化后,可获得电泳纯的乙酰胆碱酯酶,最终收集的酶液经聚丙烯酰胺(PAGE)凝胶电泳鉴定后,仅出现一条谱带;对Sephadex G-100凝胶和Sephadex G-75凝胶分离纯化乙酰胆碱脂酶的效果进行了比较,发现两者对乙酰胆碱酯酶的分离效果相差不大。

Acetylcholinesterase was purified from bovine serum. Ammonium was added to bovine serum to form 25% saturated solution which was stored in fridge at 4 ℃ overnight, then the precipitation was discarded. Ammonium was added to the supernatant to get the concentration of 35% saturation. After centrifugation at 6 000 r· min^-1 for 15 rain at 4 ℃ precipitate was collected and dissolved in buffer A （100 mM phosphate （pH8.0））. Ammonium was added to the superuatant to increase the concentration to 45% and 60% saturation respectively in the same way above. The enzyme solution from previous step was loaded onto Sephadex G-100 column （1.6 cm×50 cm） and DEAE-cellulose DE23 chromatography column （1.0 cm×40 cm）. After the three purification steps, the final purified AChE was identified to be homogeneous by polyacrylamide gel electrophoresis. There was little difference between the purified effect between Sephadex G-100 gel and Sephadex G-75 gel.