魔竽葡苷聚糖凝胶为亲和层析载体与Sepharose 4B的比较研究——Ⅰ.KGM金属螯和层析分离纯化猪血SOD

COMPARISON INVESTIGATION OF KGM GEL AND SEPHAROSE 4B AS AFFINITY SUBSTRATES I . Purification of porcine red cells SOD by metal Chelate affinity chromatography using KGM gel as substrate

将KGM凝胶和Sepharose 4B在同样条件下活化偶联,制成Cu^(2+)金属螫合亲和胶,亲和纯化猪血SOD,并对这两种亲和胶的层析效果和性能进行了比较。KGM金属螫合胶对猪血SOD吸附量、纯化倍数、纯化SOD的比活力和回收率分别为53000U/ml胶、19倍、12000U/mg蛋白和94.6％,而Sepharose 4B亲和胶对SOD 的吸附量、纯化倍数、纯化SOD的比活力和回收率分别为79920U/ml胶、11倍、10125U/mg蛋白和95.4％。两种亲和胶所纯化的SOD经聚丙烯酰胺凝胶电泳(PAGE)、活性染色及SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)证明其均为电泳纯。KGM金属螯合胶使用六次后,其对SOD吸附量、去Cu量及SOD的回收率均无明显影响。

KGM gel and sepharose 4B were used as substrates for chromatography, epichlorohydrin as activitor and iminodiacetic acid as ligands,then Cu was chelated to form metal chelate affinity column. The Superoxide dismutase (SOD)in porcine red cells was purified to homogeneous in one step affinity chromatography using KGM gel and sepharose 4B. The 53000U SOD was adsorbed by 1ml KGM gel and 79920U SOD was adsorbed by 1ml sepharose 4B. The SOD specific activity is 12000U/mg and 10125U/mg using KGM gel and sepharose 4B as substrates. The purification factor and yield of SOD are 19,94. 6% and 11,95. 4% using KGM gel and sepharose 4B as substrates. After the KGM column had been used six times for chromatography, Its adsorption capacity and recovery for, SOD did not change.