猪肝中单胺氧化酶B的分离纯化  

Purification of monoamine oxidase B from porcine liver

作　　者：孙嘉怡[1] 任军[1] 徐丽[1] 贾凌云[1] 

机构地区：[1]大连理工大学生命科学与技术学院,辽宁大连116024

出　　处：《色谱》2010年第9期872-876,共5页Chinese Journal of Chromatography

基　　金：国家自然科学基金项目(No.20775011)

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75.7μmol/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135U/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60000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。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.

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 

分 类 号：O658[理学—分析化学]