Purified human ferroportin (FPN) actively transports iron into liposomes. (A) Fluorescence-based assay of Fe²⁺ transport by FPN. The chemical gradient of Fe²⁺ drives the Fe²⁺ transport by FPN into the liposome. The Fe²⁺ influx is detected by the quenching of calcein fluorescence enclosed in the liposome. (B) Time-resolved FPN transport of Fe²⁺. The representative curves show the fluorescence quenching at different Fe²⁺ concentrations. Addition of Fe²⁺ and calcimycin (Cal) are indicated above the curves. The FPN-mediated Fe²⁺ transport is between 5 and 25 minutes and is saturated at high Fe²⁺ concentrations. Empty liposomes and FPN-containing liposomes with 0 μM Fe²⁺ are used as controls. (C) Michaelis-Menten curve of Fe²⁺ transport. The error bar of each data point is from duplicated experiments. Error of the apparent K_M is calculated from the curve fitting of the duplicates by GraphPad Prism.