猪肝中单胺氧化酶B的分离纯化

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。

Purification of monoamine oxidase B from porcine liver

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.