联苯胺亲和层析纯化尿激酶

Purification of Urokinase by Affinity Chromatography

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.