Isolation and biophysical profiling of native chicken spleen ferritin: A highly thermostable H-chain-dominated nanocage.

Chicken spleen, an underutilized poultry by-product with high ferritin content, offers potential as a nanocage-structured iron-storage nanocarrier in food science, yet systematic studies on chicken spleen ferritin (CSF) purification, structure, and thermostability remain lacking. Here, we report the isolation and comprehensive characterization of CSF. CSF was purified through thermoprecipitation, ammonium sulfate fractionation, ion-exchange, and size-exclusion chromatography, achieving a purification yield of 38.8 mg/kg wet tissue. Structural analyses showed that CSF formed a spherical ferritin-like nanocage with a diameter of approximately 12 nm and a predominantly α-helical secondary structure. LC-MS/MS identified chicken ferritin H-chain as the major detectable subunit, while intact-subunit MALDI-TOF MS of apo-CSF revealed a major ferritin-subunit-related peak at m/z 21,182.7, providing intact-mass-level support for an H-chain-dominated subunit profile. ICP-OES analysis indicated that CSF encapsulated ∼1687 iron atoms per cage. After iron-core removal, SEC-MALS analysis of apo-CSF yielded a molecular mass of approximately 497.15 kDa. Apo-CSF further retained Fe2+ oxidation/mineralization capacity and supported iron-core reformation. Thermal profiling showed that CSF retained its cage morphology up to 90 °C and exhibited a denaturation temperature of 96.3 °C. These findings identify chicken spleen as a promising source of thermally resilient native ferritin and provide a foundation for future studies on ferritin-bound iron fortification and cargo-loading applications.