波叶青牛胆(Tinospora crispa)胰蛋白酶抑制剂的分离纯化和部分性质研究
桑玉英
学位类型硕士
导师曾英
2000
学位授予单位中国科学院昆明植物研究所
学位授予地点中国科学院昆明植物研究所
学位专业植物学
关键词波叶青牛胆 胰蛋白酶抑制剂 分离纯化
摘要从防己科植物波叶青牛胆(Tinospora crispa)茎中,通过对提取液的3.0% TCA处理、80 ℃热变性、硫酸铵沉淀、固相胰蛋白酶亲和层析、FPLC及电泳制备等,分离纯化到一种胰蛋白酶抑制剂TCTI,在SDS/PAGE和IEF上均显示单一谱带。对TCTI的生化特性进行了研究,表明TCTI具有很高的热稳定性,在100 ℃处理60分钟后仍可保留93%的抑制活性。TCTI对酸碱也很稳定,在pH12.0的溶液中透析3h,期间更换溶液4次,结果仍然保持全部活力,在pH2.0的溶液中做同样处理后,能保持85%的剩于活力。经SDS/PAGE测定,TCTI的分子量约为21KDa。TCTI与胰蛋白酶的摩尔抑制比为1:2,属于双头抑制剂类型。TCTI的抑制比活力较大豆胰蛋白酶抑制剂的高。另外,对用电泳检测胰蛋白酶抑制剂的方法进行了扩展改进,使常规PAGE-明胶电泳在检测分子量相近而电荷密度不同的蛋白酶抑制剂样品时,具有较大的优势,而且快速灵敏。
其他摘要A trypsin inhibitor protein (TCTI) was isolated from stems of Tinospora crispa plants. First, the sample extract was precipitated in 3.0% TCA and denatured at 80 ℃. The supernatant was salted out, exhaustively dialyzed and subjected to affinity chromatography by using trypsin-Sepharose 4B, Then, the eluate was loaded onto an S HyperD~(TM) 10 FPLC column. The combined eluate with high specific activity from FPLC was run on native PAGE. One protein band with trypsin inhibitory activity determined by gelatin-PAGE was take out and pooled. Last, through electroelution TCTI was purified to one band monitored by SDS/PAGE and IEF. TCTI was very stable to heat. When heated up to 100 ℃ for 60 min, it still had 93% residual activity. TCTI showed no loss of activity after dialyzed in a buffer of pH 12.0 for 3h. In a similar way, TCTI remained 85% residual activity after pH 2.0. The size of TCTI was determined to be roughly 21 KDa by SAS/PAGE. The mole ratio 1:2 of TCTI to trypsin suggested that on TCTI molecule had two active sites. TCTI showed a much higher specific activity than soybean trypsin inhibitor. Besides, a fast method for the detection of trypsin inhibitors using native-PAGE was developed. The procedure includes electrophoresis of the trypsin inhibitors in the native-PAGE containing gelatin, incubation of the gels in a solution obtaining trypsin and washing the gels. This methods is especially more sensitive than SDS-PAGE containing gelatin for detecting trypsin inhibitors with similar molecule weight but different charge density.
页数49
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/540
专题昆明植物所硕博研究生毕业学位论文
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桑玉英. 波叶青牛胆(Tinospora crispa)胰蛋白酶抑制剂的分离纯化和部分性质研究[D]. 中国科学院昆明植物研究所. 中国科学院昆明植物研究所,2000.
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