色谱

色谱

• 研究论文 • 上一篇    下一篇

三步柱色谱法从江浙蝮蛇蛇毒中分离纯化类凝血酶

边六交,杨晓燕,刘莉

  

  1. Center of Gene-Engineering, College of Life Science, Northwest University, Xi’an 710069, China
  • 收稿日期:2005-04-18 修回日期:2005-06-24 出版日期:2006-03-30 发布日期:1987-06-25
  • 通讯作者: 边六交

Three-Step Column Chromatographic Method for Separation and Purification of Thrombin-Like Enzyme from Venom of Agkistrodon halys pallas

BIAN Liujiao, YANG Xiaoyan, LIU Li

  

  1. Center of Gene-Engineering, College of Life Science, Northwest University, Xi’an 710069, China
  • Received:2005-04-18 Revised:2005-06-24 Online:2006-03-30 Published:1987-06-25
  • Contact: BIAN Liujiao

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摘要：

建立了一种用CM Sepharose CL-6B阳离子交换、DEAE Sepharose Fast Flow阴离子交换和Sephadex G-75凝胶排阻三步柱色谱从江浙蝮蛇蛇毒中分离纯化类凝血酶的方法。在实验室小柱分离方案的基础上,对该纯化工艺进行了放大。当上样量达实验室小柱的25倍时,所得类凝血酶的质量指标与实验室小柱基本一致。采用该法所得的蝮蛇类凝血酶经Shim-pack Diol-300高效凝胶排阻柱测得其相对分子质量约为33500,用Shim-pack VP-ODS反相色谱柱检测其纯度约为96%。从江浙粗蛇毒中提取类凝血酶时,类凝血酶的总质量收率约为0.3%,总活性收率约为64%,比活可达2000 U/mg。

关键词: 分离纯化 , 江浙蝮蛇, 类凝血酶, 三步柱色谱, 蛇毒

Abstract:

A three-step column chromatographic method, utilizing CM Sepharose CL-6B, DEAE Sepharose Fast Flow and Sephadex G-75 in sequence, for the separation and purification of thrombin-like enzyme from crude venom of Agkistrodon halys pallas was developed. Based on the separation results on lab-scale chromatographic column, this isolation and purification process was amplified according to the diameter of the chromatographic columns used in lab-scale. It was found that when the amount of the sample loaded on the large-scale columns was 25 times as that loaded on the lab-scale columns, the quality level of the thrombin-like enzyme obtained with the large-scale columns was almost the same as that obtained with lab-scale columns. The relative molecular mass of thrombin-like enzyme obtained was about 33500, and its purity was about 96%. When using this method to isolate thrombin-like enzyme from crude venom of Agkistrodon halys pallas, the total protein yield and total activity yield of thrombin-like enzyme were about 0.3% and 64% respectively, and specific activity was more than 2000 U/mg.

Key words: Agkistrodon halys pallas, separation and purification , snake venom, thrombin-like enzyme, three-step column chromatographic method