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

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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.