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，该方法由两次凝胶电泳组成，用于获得生物样品中蛋白质结合金属离子的分布。前者是蓝色天然（BN）PAGE，用于分离蛋白质，而不会在迁移过程中解离。另一种是PAGE，用于分离和检测小体积样品中蛋白质结合的金属离子，使用荧光金属探针在ppt范围内具有高灵敏度。探针具有螯合部分（多齿聚氨基羧酸盐）、荧光团（荧光素）和间隔基（氨基苄基）以与金属离子形成发射性和动力学惰性络合物。该聚丙烯酰胺凝胶电泳可有效地检测Mg^<2+>、Cu^<2+>、Fe^<2+>/^<3+>、Ni^<2+>、Co^<2+>、Mn^<2+>、Cd^<2+>和Hg^<2+>等金属离子。通过BN-PAGE分离蛋白质后，洗脱凝胶级分中的金属离子，然后将铜离子衍生成金属探针复合物，以在第二次PAGE中通过荧光检测进行分离和测定。在这种基于PAGE的方法中，定量测定了与铜蓝蛋白和超氧化物歧化酶结合的铜离子以及与转铁蛋白和血液结合蛋白结合的铁离子。

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.