Critical roles of small silk fibroin molecules in the self-assembly and properties of regenerated silk fibroin.

Silk is primarily composed of silk fibroin (SF) and silk sericin (SS), with SF significantly contributing to the mechanical properties of silk fibers. SF consists of the large molecular fibroin heavy chain (Fib-H), small molecular fibroin light chain (Fib-L), and P25 protein. Degumming is a crucial step in both the silk reeling process and the preparation of regenerated silk fibroin (RSF), but it can cause damage to Fib-H. This study investigates how degumming affects small SF molecules and their influence on the properties of silk fibers and RSF. A gradient degumming treatment using various reagents was employed. SS antibody detection indicated that 3 g/L papain and sodium carbonate (NaCO) at concentrations ≥0.2 % almost completely removed SS. SF antibody detection revealed that NaCO degumming severely damaged Fib-H and degraded Fib-L and P25. While tensile tests showed that this damage did not significantly affect the mechanical properties of SF fibers, the loss of small SF molecules reduced the mechanical properties of the RSF membranes and delayed RSF gelation. Atomic force microscopy demonstrated that RSF containing Fib-H of similar molecular weight (100-180 kDa) can self-assemble into nanofibrils when small SF molecules are present, whereas 0.5 % NaCO-degummed RSF lacking these small SF molecules cannot form nanofibrils. By adding additional small SF molecules to the 0.5 % NaCO-degummed RSF, nanofibrils can be formed. This research highlights the critical role of small SF molecules in the properties of RSF and provides a theoretical foundation for the development of RSF-derived materials.