Ferrous ammonium sulfate

Standard buffer consisted of 25 mM sodium bicarbonate (Fisher), 20 mM Tris-base (Fisher), and 10 mM NaCl (EMD), prepared in high purity water and adjusted to pH 7.4 using trace-grade HCl (Fisher). Stock solutions of transferrin (Athens Research and Technology) (76 μM) and bovine serum albumin (Sigma) (1.2 mM) were prepared in the standard buffer. A stock solution of Fe^III citrate (70 μM) was prepared by mixing 40 mM ferrous ammonium sulfate (Fisher) with 120 mM sodium citrate (Fisher), and adjusting the pH to 5 using citric acid (Acros Organics) (final concentrations obtained using deionized water). Iron solutions were mixed with select protein samples, transferrin:iron at molar ratio 1:1 and albumin:iron at molar ratio 0.058:1. Protein and iron solutions were mixed, incubated for 1 hr, then passed through a 0.2 μm filter (VWR). transferrin ± Fe and albumin ± Fe solutions were mixed 1:1 with each other or diluted 1:1 with standard buffer. A solution of alcohol dehydrogenase (Sigma) was prepared (10 mg/mL) in standard buffer, and mixed 1:1:1 with Fe^III transferrin (prepare 1:1 Fe^III citrate) and albumin solutions (in this sample, only transferrin had Fe^III citrate). Ferritin (Sigma; 2 mg/mL) was prepared in standard buffer except lacking bicarbonate. These solutions were then passed though the Superdex 200 GL column as described above.

Ascorbic acid was from EMD Chemicals (Gibbstown, NJ). Sodium nitrite, ferrous ammonium sulfate, sodium EDTA, and potassium iodide were obtained from Fisher Chemicals (Fairlawn, NJ). Hemoglobin was from Sigma (St Louis, MO). All buffers used were treated with chelating resin using the batch method to minimize the level of catalytic transition metals [24 (link)].