文献类型：期刊文献

中文题名：联苯胺亲和层析纯化尿激酶

英文题名：Purification of Urokinase by Affinity Chromatography

作者：曹学君[1];唐蒙[1];邬行彦[1];戴干策[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室

年份：1998

卷号：24

期号：5

起止页码：515

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;国家哲学社会科学学术期刊数据库;Scopus;北大核心:【北大核心1996】；CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：联苯胺;亲和层析;尿激酶;GMA

外文关键词：benzidine; glycidyl methacrylate; affinity chromatography; urokinase

摘要：利用甲基丙烯酸缩水甘油酯与乙二醇甲基丙烯酸酯经悬浮聚合反应合成亲水性亲和载体骨架，骨架上的环氧基与氨水反应，继之与丁二酸酐反应，最后与配基联苯胺偶联制备亲和载体，配基密度为５４μｍｏｌ／ｇ湿胶。亲和载体吸附尿激酶的最佳条件为ｐＨ４．６和２．５ｍｏｌ／ＬＮａＣｌ；测定了吸附等温线，算得解离常数Ｋｄ为３１６ＩＵ／ｍＬ，最大吸附量ｑｍ为２．３８３×１０４ＩＵ／ｍＬ湿胶，洗脱条件为含０．５ｍｏｌ／ＬＮａＣｌ，ｐＨ１０．０，０．１ｍｏｌ／Ｌ碳酸氢钠缓冲液。将比活为３００ＩＵ／ｍｇ的尿激酶粗品进行层析，得到比活为６．３×１０３ＩＵ／ｍｇ的洗脱液，收率为８９．１％，纯化倍数为２１。
A number of benzidine GMA EDMA affinity adsorbents were prepared. Glycidyl methacrylate was copolymerized with ethylene dimethacrylate to form the copolymer with epoxy functionality, which was reacted with ammonia to give the aminated resin. The aminated resin reacted with succinic anhydride, forming carboxyl resin. finally, the carboxyl resin was coupled with benzidine to form the affinity medium. The density of the ligand in the affinity adsorbents was found to be about 54μmol/g wet gel. The optimal pH and the optimal salt concentration for urokinase adsorption were 4.6mol/L, and 2.5mol/L NaCl respectively. The optimal conditions for elution were pH10.0,0.1mol/L carbonate buffer containing 0.5 mol/L NaCl. The isotherms of urokinase adsorption on the medium was determined, from which the dissociation constants K d were: 316IU/mL and adsorption capacity of 2.383×10 4IU/mL were calculated. A crude urokinase solution with specific activity 300 IU/mg protein was applied to the affinity chromatoraphy column(1cm×12cm),resulting in a product of a purity 6.3×10 3IU/mg protein with 89.1% recovery yield and 21 fold purification factor.