Unknown low molecular weight iron‐peptide complexes and their possible contribution to the “labile iron pool” in mammalian cells

Abstract

We have conducted studies to re-examine an unknown iron-binding component previously discovered in rat mucosal supernatants following oral 59Fe administration. Cytoplasmic extracts of mammalian cell cultures treated with 1–2uM 59Fe(II)-ascorbate, applied to native PAGE, consistently showed the presence of only two radioactive components, one being ferritin, the other unknown and filterable through 10kDa cutoff membranes. The proportion of 59Fe in the unknown component varied inversely with cellular iron status. Eluted from native PAGE gels, it separated into two 59Fe components in size exclusion HPLC on Biosep 2000 coupled with ICP-MS, which had some coincident absorbance at 280nm. An open, standardized, size exclusion Biogel-P2 column reliably showed two component peaks with Mr of 0.6 and 1.8 kDa. Treatment of filtrates with trypsin made no difference; but proteinase K treatment caused a shift in elution profile of the 1.8 kDa component to 0.6 kDa (still detectable in native PAGE), indicating the presence of peptide. Non-radiolabeled filtrate samples analysed by electron plasmon resonance (EPR) demonstrated a peak of high intensity in the Fe(III) range. Lectin analysis demonstrated a lack carbohydrate. We hypothesize that these low molecular weight iron-peptide complexes are part of the “labile iron pool” contributing to iron fluxing between proteins and compartments within mammalian cells.