色谱

色谱 ›› 2010, Vol. 28 ›› Issue (09): 872-876.DOI: 10.3724/SP.J.1123.2010.00872

• 研究论文 • 上一篇    下一篇

猪肝中单胺氧化酶B的分离纯化

孙嘉怡1,任军2,徐丽1,贾凌云1   

  1. 大连理工大学生命科学与技术学院, 辽宁 大连 116024
  • 收稿日期:2010-04-27 修回日期:2010-06-30 出版日期:2010-09-28 发布日期:2010-09-28
  • 通讯作者: 贾凌云,博士,教授,主要研究方向为生物分离技术.
  • 基金资助:

    国家自然科学基金项目(No. 20775011).

Purification of monoamine oxidase B from porcine liver

SUN Jiayi, REN Jun, XU Li, JIA Lingyun*   

  1. Department of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
  • Received:2010-04-27 Revised:2010-06-30 Online:2010-09-28 Published:2010-09-28

PDF

484

被引次数

1

摘要：

依据单胺氧化酶B(monoamine oxidase B, MAOB)的疏水特性,建立了一种从猪肝中分离纯化MAOB的新方法。用含有1% Triton X-100的膜蛋白裂解液制备粗酶,以饱和度为20%～50%的硫酸铵反抽提进行粗提,再利用自制的配基密度为75.7 μmol/mL的苯基疏水色谱及Sepharose Q High Performance离子交换色谱进一步分离纯化,得到纯化倍数为18.2、酶比活为135 U/mg的MAOB。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示为相对分子质量约60 000的单一蛋白质带。采用高效液相色谱-电喷雾串联质谱对该酶进行鉴定,证实为MAOB。本研究所用分离纯化方法可以有效纯化MAOB, 为MAOB的深入研究提供技术支撑。

关键词: 苯基配基；疏水色谱；阴离子交换色谱；单胺氧化酶B, 猪肝

Abstract:

Monoamine oxidase B (MAOB) was purified from porcine liver by solubilization with lysis buffer containing 1% Triton X-100, precipitation with 20%~50% ammonium sulfate, isolation with hydrophobic chromatography and anion exchange chromatography. The purification fold was 18.2. The specific activity was 135 U/mg. The purified enzyme appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and it had a relative molecular mass of about 60 000. The identification of the enzyme was confirmed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As MAOB is a membrane enzyme, a key step to the successful purification was the use of Phenyl-Sepharose CL-4B with phenyl density of 75.7 μmol/mL. The results showed that this approach could effectively isolate MAOB from porcine liver to yield an enzyme with high purity and specific activity.

Key words: anion exchange chromatography, hydrophobic chromatography, monoamine oxidase B, porcine liver, phenyl ligand