Sulfation of silk fibroin by chlorosulfonic acid and the anticoagulant activity

Silk fibroin (Bombyx mori) was sulfated using chlorosulfonic acid in pyridine. FT-IR spectra showed introduction of sulfate group by this reaction; NMR spectra indicated that sulfation occurred mainly at tyrosine and serine residues. Molecular size decreased and dispersed with sulfation. The molecular weight was estimated in around 20,000 by GPC using protein standards. Amino acid composition suggested that sulfated fibroin came from H-chain of fibroin; the crystal region of fibroin molecule remained in sulfated fibroin. The amount of sulfate groups increased with overall reaction time. The maximum amount was estimated in 1.0 mmol/g by acidimetric titration. Sulfation efficiency was calculated as 66.7%. Blood coagulation was prevented by 0.5 mg of sulfated fibroin in 1 ml of blood, while original fibroin did not show any effect. Anticoagulant activity of sulfated fibroin strongly depends on the amount of sulfate groups introduced. These results indicate that sulfate group introduction results in addition of anticoagulant function to silk fibroin. Sulfated fibroin is a new type of anticoagulant material having a protein backbone.