Multi-dimension polyacrylamide gel electrophoresis for detection of metal ions bound to metalloproteins

建立了一种基于聚丙烯酰胺凝胶电泳（PAGE）的方法，该方法由两个凝胶电泳组成，用于获得生物样品中蛋白质结合金属离子的分布。前者是蓝原生（BN） PAGE，在迁移过程中不解离地分离蛋白质。另一种是PAGE，利用荧光金属探针在ppt范围内对小体积样品中的蛋白结合金属离子进行高灵敏度的分离和检测。探针具有螯合部分（多齿聚氨基羧酸酯），荧光团（荧光素）和间隔基团（氨基苯基），可与金属离子形成发射和动力学惰性配合物。一些金属离子、镁^ < 2 + >,铜^ < 2 + >,Fe ^ < 2 + > / ^ < 3 + >,倪^ < 2 + >,公司^ < 2 + >,Mn ^ < 2 + >, Cd ^ < 2 + >和Hg ^ < 2 + >,成功检测到这个新页面。用BN-PAGE对蛋白进行分离后，将凝胶组分中的金属离子洗脱，铜离子衍生化进入金属探针配合物中，在第二页进行荧光检测分离测定。在基于page的方法中，定量测定了铜蓝蛋白和超氧化物歧化酶结合的铜离子，以及转铁蛋白和血凝素结合的铁离子。

A polyacrylamide gel electrophoresis (PAGE)-based method has been developed, consisting of two gel electrophoresis, to obtain distribution of protein-bound metal ions in biological samples. The former is blue-native (BN) PAGE to separate proteins without dissociation during migration. The other is a PAGE for the separation and detection of protein-bound metal ions in small volume samples with high sensitivity in the ppt range using fluorescence metal probe. The probes have a chelating moiety (multidentate polyaminocarboxylate), a fluorophore (fluorescein) and a spacer (aminobenzyl group) to form emissive and kinetic inert complexes with metal ions. Some metal ions, Mg^<2+>, Cu^<2+>, Fe^<2+>/^<3+>, Ni^<2+>, Co^<2+>, Mn^<2+>, Cd^<2+> and Hg^<2+>, were successfully detected by this new PAGE. After separation of the proteins by BN-PAGE, the metal ions in the gel fractions were eluted, followed by derivatization of copper ions into the metal probe complexes to be separated and determined by fluorescence detection in the second PAGE. In this PAGE-based method, the copper ions bound to ceruloplasmin and superoxidedismutase, and iron ions bound to transferrin and hemopexin were quantitatively determined.