Sulfation of silk fibroin by sulfuric acid and anticoagulant activity

Abstract

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.

Homogeneous sulfation of silk fibroin in an ionic liquid

“…Optimization of the homogeneous sulfation condition: Generally, the anticoagulant activity of the sulfated polymer strongly depends on the amount of the incorporated sulfate group [9,10]. Therefore, to investigate the optimal condition, the amount of sulfur bound to SF was adopted to follow the sulfation extent as a function of the reaction temperature and time, and the amount was determined by elemental analysis and expressed as the weight ratio of sulfur to carbon (S/C) ( Table 1).…”

“…The sulfation of polymers is an approach to model the structure of heparin, a highly sulfated polysaccharide and a commonly used anticoagulant. Heparinlike modification has proved to be an effective way to improve the anticoagulant activity of the polymers [3,[9][10][11]. The sulfation was investigated also for purpose of comparing with the previous heterogeneous sulfation which had disadvantage of severe SF hydrolysis [10].…”

Identifying the Sulfate Ion Binding Residues in Proteins

“…At the same time, it can be found that in the area of position [6][7][8][9][10][11][12] th , it has a significant conservative of amino acid Hydropathy. Base on Panek's scale [15], G is a separate class, R, K, E, H are strongly hydrophilic, S, T are weak hydrophilicity.…”

“…In 2003, Tamada has researched sulfation of protein, and modified sulfated silk fibroin by concentrated sulfuric acid. They got silk fibroin sulfated efficiency, and found that the reaction efficiency is not high and is very susceptible to decomposition of the sample [9]. Monigatti et al have done a statistical analysis on Tyrosine which may be sulfated in 5 proteins, and given the analysis regularity of Tyrosine sulfation in 2002 [4].…”

Antheraea assama Silk Fibroin‐Based Functional Scaffold with Enhanced Blood Compatibility for Tissue Engineering Applications

“…There were only a couple of reports by Tamada et al, in which they demonstrated the method of sulfation of B. mori SF and subsequently proved its anticoagulant activity. [20][21] They have proposed that sulfation results in the addition of sulfate groups to hydroxyl moieties of Ser and Tyr residues in SF. However, in the above reports they have not i) used SF in 3D scaffold form, ii) studied its potential for TE applications, or, iii) performed detailed characterization of the biological properties of SF before and after sulfation.…”