Quantitation and characterization of low molecular weight iron complexes in mammalian cells in conditions of iron overload and deficiency that may comprise components of the ‘labile iron pool’

Abstract

We have determined that when cultured mammalian cells are exposed to 59Fe‐labeled 1–2 uM Fe(II)‐ascorbate for 3h, and cell lysates are applied to native PAGE, two radiolabeled peaks are shown. One is ferritin, the other of low molecular weight and filterable through a 10 kDa membrane. In the studies reported here, we determined the proportion of 59Fe in the unknown relative to ferritin in several cultured cell lines with and without 24h pretreatment with ferric ammonium citrate (10 ugFe/ml), or desferrioxamine (DFO) to increase and decrease iron availability. The ratio of 59Fe in the unknown to that in ferritin varied inversely with iron status. The unknown peak was present not just in cytoplasm but also in the lysosomes. After elution from native PAGE gels, two components were separated by size exclusion HPLC on Biosep 2000 coupled with ICP‐MS. This was also the case on an open Biogel‐P2 column, where Mr values of 1.8 and 0.6 kDa were obtained. Treatment of filtrates with proteinase K caused a shift in elution of the 1.8 kDa component to 0.6 kDa (detectable in native PAGE), indicating presence of peptide. Lectin analysis demonstrated a lack carbohydrate. Iron in the unknown components was directly available to DFO upon cell lysis, and could be chased out with non‐radioactive Fe. We hypothesize that these low molecular weight iron‐peptide complexes are part of the 'labile iron pool' in mammalian cells.