Sulfation of silk fibroin by sulfuric acid and anticoagulant activity

Silk fibroin (Bombyx mori) was sulfated with an aqueous sulfuric acid solution. The sulfated fibroin was characterized by Fourier transform infrared spectroscopy (FTIR), amino acid analysis, and gel filtration chromatography (GFC). Maximum yield was obtained at around 2-4 h, and it decreased at 6 h and more. The molecular size decreased and dispersed with sulfation, and the molecular weight was estimated at around 10,000 by GFC using protein standards. The amino acid composition indicated that the crystal region of the fibroin molecule remained in sulfated fibroin until a sulfation reaction time of 4 h. The incorporation of sulfate groups was confirmed by FTIR and the amount of sulfate groups introduced for 4 h sulfation was estimated in 0.3 mmol/g by acidimetric titration. The efficiency of sulfation was calculated at 15.7%. Blood coagulation was prevented by 20 mg of sulfated fibroin in 1 mL of blood, while original fibroin did not show the effect. This result indicates that sulfate group introduction results in addition of anticoagulant function to silk fibroin. Although a variety of polymer backbones have been used for synthesis of sulfated polymers as anticoagulant materials, no reports are available concerning a sulfated polymer based on a protein backbone. Sulfated fibroin is a new type of anticoagulant material having a protein backbone. (C) 2003 Wiley Periodicals, Inc. J Appl Polym Sci 87: 2377-2382, 2003.