Detection of iron-containing proteins contributing to the cellular labile iron pool by a native electrophoresis metal blotting technique

Abstract

The labile iron pool (LIP) plays a role in generation of free radicals and is thus the target of chelators used for the treatment of iron overload. We have previously shown that the LIP is bound mostly to high molecular weight carriers (MW>5000). However, the iron does not remain associated with these proteins during native gel electrophoresis. In this study we describe a new method to reconstruct the interaction of iron with iron-binding proteins. Proteins were separated by native gradient polyacrylamide gel electrophoresis and transfered to polyvinilidene difluoride membrane under native conditions. The immobilized iron-binding proteins are then labeled by 59Fe using a ‘titrational blotting’ technique and visualized by storage phosphorimaging. At least six proteins, in additon to ferritin and transferrin, are specifically labeled in cellular lysates of human erythroleukemic cells. This technique enables separation and detection of iron-binding proteins or other metal–protein complexes under near-physiological conditions and facilitates identification of weak iron–protein complexes. Using a new native metal blotting method, we have confirmed that specific high molecular weight proteins bind the labile iron pool.