烟草CuZnSOD同工酶的分离纯化与鉴定

Separation,Purification and Identification of CuZnSOD Isoenzymes from Tobacco

利用硫酸铵盐析分级分离、乙醇 -氯仿沉淀除杂、丙酮再沉淀分级得到含铜锌超氧化物歧化酶(CuZnSOD)的粗酶液 ,通过DEAE_52离子交换层析实现了3种CuZnSOD同工酶的拆分 ;进一步通过G_75凝胶层析对3种同工酶分离纯化得到2种电泳纯产品CuZnSODⅠ和CuZnSODⅢ ;SDS-PAGE实验发现CuZnSODⅠ由两个不等亚基组成 ,亚基相对分子质量分别为15050、20950;CuZnSODⅢ存在一种亚基 ,亚基相对分子质量为22950 ;实验检测到2种纯酶分子含有不等量的Cu和Zn,酶学特性实验也证实了2种蛋白酶都是CuZnSOD。

The crude enzyme solution of CuZnSOD from tobacco was obtained by using ammonium sulˉphate to form different levels of precipitates;chloroform and alcohol to isolate the impurities and acetone to further clean-up.Three CuZnSOD isoenzymes were then obtained by DEAE-52ion exchange chroˉmatography.Two of them,CuZnSODⅠand CuZnSODⅢwere obtained when further purified to a PAGE purity by G-75gel chromatography.Fromthe SDS-PAGEexperiment,itwas found that CuZnSODⅠwas comprised of two unequal subunits,their relative molecular mass were15050and20950,respecˉtively;and that CuZnSODⅢhad only one kind of subunit,whose relative molecular mass was22950.Two isoenzymes containing different amount of Cu and Zn were also detected in the experiment.