From molecular disparity to material performance: Enhancing wool keratin film properties by hierarchical separation of KAPs and IFPs.

The high-value utilization of wool keratin relies on controllable dissolution and regeneration. However, traditional regenerated wool keratin is a mixture of keratin-associated Proteins (KAPs) and intermediate filament proteins (IFPs), whose structural and behavioral differences are often overlooked, leading to poor process control, low sol stability, and limited material performance. This study developed a two-step hierarchical extraction to separate high-enriched KAPs and IFPs. Comparative analysis revealed that KAPs (10-28 kDa) exhibit a higher density of disulphide bonds, while IFPs (45-100 kDa) are enriched in α-helical secondary structure and exhibit higher crystallinity. These structural distinctions result in distinct dissolution and aggregation behaviors: KAPs readily undergo irreversible precipitation upon denaturant removal, whereas IFPs demonstrate superior solution stability. Notably, enriched IFPs formed uniform films without external plasticizers or crosslinkers. The resulting films showed a dry tensile strength of 26. 47 ± 5.21 MPa and a wet elongation at break of 109 ± 30%. This work clarifies the differential roles of KAPs and IFPs across the structure-dissolution-film-forming continuum, providing a foundation for the precise design of high-performance keratin-based biomaterials. This study systematically clarifies the differential roles of KAPs and IFPs in wool keratin regeneration, from their inherent molecular structures to their dissolution behaviors and final film-forming properties. It provides a theoretical basis for the precise regulation and high-value utilization of regenerated keratin resources.