Preparation of bioactive hepcidin. (A) The HPLC profile of purified ¹²⁵I-hepcidin. Synthetic human hepcidin was renatured, labeled using ¹²⁵I, as described in “Methods,” and subjected to HPLC purification (broken line: ¹²⁵I radioactivity; solid line: 210 nm UV absorbance). More than 98% of the peptide was present in the main ¹²⁵I peak. (B) Hepcidin significantly (*P < .01) decreases the saturation of transferrin (Tf) with iron when mice were injected with the renatured peptide. Results in panel A are typical of 3 or 4 experiments, while those in panel B are mean ± SD (3-4 experiments).