新型染料配基对碱性磷酸酶的亲和纯化  

Application of a Novel Synthesized Azo-Dye Ligand for the Affinity Separation of Alkaline Phosphatase

作　　者：王静云[1] 彭孝军[2] 杨冬[3] 包永明[1] 安利佳[1] 郑学仿[1] 

机构地区：[1]大连理工大学化工学院生物工程系 [2]精细化工国家重点实验室 [3]东北林业大学,黑龙江哈尔滨150040

出　　处：《高校化学工程学报》2001年第6期563-567,共5页Journal of Chemical Engineering of Chinese Universities

基　　金：国家自然科学基金资助项目(29676006)

摘　　要：设计合成了一种含有碱性磷酸酶抑制剂——对氨基苯磷酸的新型偶氮染料配基并将其偶联到凝胶Sepharose CL-6B 上,制备出新型的亲和层析介质,分离纯化小牛肠中的碱性磷酸酶。通过考察不同配基密度的层析介质对碱性磷酸酶的选择性,得出配基密度为4.55mg 配基(g 湿胶)-1的层析介质选择性高。用NaCl和Na2HPO4进行阶段洗脱,可使酶的纯化倍数一步达到65倍,活力回收率达到89%。通过测定不同配基浓度下酶的动力学,发现新合成的配基是碱性磷酸酶的竞争性抑制剂,其KI为3.0×10-3mol L-1。Alkaline phosphatase is widely used in molecular biology and immunodiagnostics. A new azo dye affinity ligand comprising a p-Aminophenylphosphonic acid as inhibitor of alkaline phosphatase was synthesized and coupled to an agrose support by chlorotriazine ring to separate and purify alkaline phosphatase from calf intestine. The effects of temperature, time, and ligand's concentration on the coupling reaction between ligand and agrose support were investigated, the result showed that 60°C was the most suitable temperature to the coupling reaction, and the optimal reaction time and ligand's concentration are six hours and 1.0% respectively. The selectivity of affinity media with different density of dye ligand was examined, the result shows that the optimal density of dye ligand on agrose is 4.55 mg&middot (g moist gel)-1. Using NaCl and Na2HPO4 in turn to elute bound alkaline phosphatase, a 65-fold purified enzymatic preparation was obtained in one step with a yield of 89%. The resulting alkaline phosphatase displayed a specific activity in approximate 1000 U&middotmg-1 and was of equivalent purity to commercial product. The kinetics of alkaline phosphatase under different ligand's concentration was measured. It was found that the newly-synthesized affinity ligand to be a competitive inhibitor of alkaline phosphatase, and its K1 is 3.0×10-3 mol&middotL-1.

关 键 词：亲和层析 染料配基 偶氮染料 碱性磷酸酶 纯化 分离 

分 类 号：Q555.7[生物学—生物化学] Q503