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法(PAGE)的生物样品中蛋白质结合金属离子分布的两种凝胶电泳法。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。另一种是用荧光金属探针分离和检测小体积样品中蛋白质结合的金属离子，灵敏度在ppt范围内。该探针具有螯合部分(多齿多氨基羧酸盐)、荧光素(荧光素)和间隔基(氨基苯甲基)，与金属离子形成发射和动力学惰性络合物。成功地检测到了金属离子：镁、铜、铁、镍、钴、锰、镉、汞。蛋白质经BN-PAGE分离后，洗脱凝胶组分中的金属离子，然后将铜离子衍生化成金属探针络合物，在第二次PAGE中进行荧光检测。在这种基于PAGE的方法中，定量地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.