一种新型提取SOD的方法——串联凝胶过滤层析法

目的:建立串联凝胶过滤层析法,一步即可从猪血中提取纯酶。方法:猪血中粗酶液的分离采用经典的有机溶剂(乙醇-氯仿)的方法进行。后在室温下用葡聚糖G100-G50进行柱层析,层析液用pH 7.6,0.02 mol/L^-1的磷酸盐缓冲液。首次过柱就可生产出一部分纯酶。接着收集不纯的酶液,再进行第2次串联层析,就可把所有的酶液彻底纯化。结果:2次之和的酶液总产率达70%,比活性达4700 u·mg^-1。结论:经聚丙烯酰胺凝胶电泳及SDS-聚丙烯酰胺凝胶电泳鉴别,该产品是高纯度的,并且高产率。

Objective: To establish tandem gel-filtration chromatography method on large yield of copper/zinc superoxide dismutase. Method: The isolation of crude Cu,Zn-SOD was performed with organic solvents(ethanol-chloroform) by routine manner.Then tandem column chromatography was used to purity SOD on Sephadex G100-G50 at room temperature with pH 7.6,0.02 mol·L^-1 phosphate buffer.There were three elution peaks on the elution curve.By detecting the SOD activity of fractions,results showed that pure enzyme was in fraction 2,while impure enzyme was in fraction 1.The enzyme solution of fraction 1 ran on Sephadex G100-G50 again to purity the SOD enzyme. Results: The SOD enzyme we got had high purity with specific activity at 4700 u·mg^-1,the total yield of SOD enzyme reached to 70%. Conclusion: SOD enzyme is of high purity and large yeild which identified using polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis.