对氨基脲敏感的胺氧化酶生物功能化色谱的方法学研究

Investigation on Semicarbazide-Sensitive Amine Oxidase Bio-Functionalized Chromatography

采用毛细管电泳法建立了对氨基脲敏感的胺氧化酶(SSAO)生物功能化色谱模型。将SSAO酶固载到一种仿细胞膜的载体——脂质体上,pH值对固定化酶稳定性影响的实验表明,pH5.0是理想的固载条件;SSAO酶活性检测表明游离态和固定态的酶活性分别为0.0404和0.0347mmolg-1min-1。将有活性的固定化SSAO酶作为假固定相添加到磷酸盐缓冲液中(10mmol/L,pH5)。随着固定化酶的浓度从0.004g/L上升到0.856g/L,其特异性底物苯甲胺的有效淌度从4.06×10-4cm2/(V.s)下降到0.81×10-4cm2/(V.s)。据此,建立的生物功能化色谱模型为研究生物分子间的特异性作用提供了新的技术方法。

A model of semicarbazide-sensitive amine oxidase （SSAO） bio-functionalized chromatography was developed using capillary electrophoresis. SSAO was immobilized on liposome, a carrier that simulated cell membrane. The experiment of the effect of pH value on the stability of immobilized enzyme showed that pH 5.0 was the perfect condition for immobilization. SSAO activity detection displayed that the specific activity of SSAO both in free form and immobilized form were 0. 0404 and 0. 0347 mmol g^-1 min^- 1 respectively. The active immobilized SSAO was added to the PBS（ 10 mmol/L, pH 5）as pseudo-stationary phase. With the concentration of immobilized enzyme increasing from 0. 004 g/L to 0. 856 g/L, the effective mobility of its specific substrate, benzylamine, decreased from 4.06 × 10^-4 （cm^2/V · s） to 0. 81 × 10^-4（ cm^2/V · s）. Subsequently, the established bio-functionalized chromatographic method provides a new technology for investigating interactions among the biological molecules.