苜蓿二胺氧化酶(DAO)的分离纯化

目的]寻求操作简单、高效的苜蓿二胺氧化酶(DAO)纯化方法。方法]苜蓿种子黑暗培养3 d,幼苗依次经硫酸铵沉淀,葡聚糖凝胶G-100(sephadex G-100),DEAE-纤维素以及羟基磷灰石(hydroxyapatite,HAP)层析分离纯化,最后经聚丙烯酰胺凝胶电泳。结果]最佳分段盐析的硫酸铵饱和度为20%~50%。经一系列层析分离后酶液比活力达84.67 U/mg,回收率为26%。经非变性聚丙烯酰氨凝胶电泳后,出现1个蛋白质条带,相对分子质量约为110 k Da。而在变性聚丙烯酰氨凝胶电泳后,出现2个蛋白质条带,相对分子质量约为55和40 k Da。结论]苜蓿DAO是由2个大小不同的亚基组成的异源二聚体。

Separation and Purification of Diamine Oxidase from Alfalfa

Objective]To seek a simple and efficient method for purifying the diamine oxidase from alfalfa.Method] Alfalfa seeds were cul-tured in darkness for 3 days.The seedlings were isolated and purified by ammonium sulfate precipitation, dextran gel G-100 ( sephadex G-100), DEAE-cellulose and hydroxyapatite (HAP),finally, polyacrylamide gel electrophoresis.Result] The best fraction of salting out am-monium sulfate saturation was 20% -50%.After a series of chromatographic separation, the specific activity of the enzyme reached 84.67 U/mg, and the recovery rate was 26%.After the non-denaturing polyacrylamide gel electrophoresis, a protein band appeared with a relative molecular mass of about 110 kDa.But after denaturing polyacrylamide gel electrophoresis, two protein bands appeared at a relative mo-lecular mass of about 55 kDa and 40 kDa. Conclusion] Diamine oxidase from alfalfa is a heterodimer composed of two subunits of different si-zes.