Holoferritins Rendered Iron-Free

Holoferritins (i.e. iron containing ferritin) were rendered iron-free by dialysis against sodium hydrosulfite (dithionite), Na₂S₂O₄, and complexation with 2,2′-bipyridyl at pH 6.0. Protein concentrations were determined spectrophotometrically using a molar absorptivity of 24000 cm⁻¹ M⁻¹ at 280 nm for the 24-mer apoprotein (iron-free protein) (35 (link)). All chemicals were reagent grade and used without further purification. Mops (3-(N-morpholino)-propanesulfonic acid) buffer was purchased from Research Organics (Cleveland, OH) and FeSO₄·7H₂O from J. T. Baker (Phillipsburg, NJ). Sodium dithionite, Na₂S₂O₄ ferrozine and 2,2′-bipyridyl were purchased from Sigma-Aldrich (St. Louis, MO). Ferrous sulfate stock solutions were freshly prepared immediately before each experiment in a dilute HCl solution at pH 2.0.