Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides

Hyaluronan was partially depolymerized on a large-scale quantity using bact erial hyaluronidase (E.C. 4.2.2.1) fbr preparation of chemically fully O-su lfated oligosaccharides, The hyaluro-oligosaccharide (HAoligo) mixture obta ined by partial digestion was repeatedly applied to low pressure gel permea tion chromatographic separation to purify the size-unified oligosaccharide ranged from 4- to 20-mer, The purity and size of each HAoligo was confirmed by using proton nuclear magnetic resonance (H-1 NMR) spectroscopy, capilla ry electrophoresis (CE) on normal polarity mode, and a newly established se paration method by normal phase chromatography with Amide-80 column. The pu rified HAoligos ranged 4- to 20-mer were applied to chemically fully O-sulf ation, Characterization of chemically fully O-sulfated HAoligos was perform ed by both chemical compositional analyses after hydrolysis and 1H NMR spec troscopy. While the anti-factor IIa activity of 4- to 20-mer O-sulfated HAo ligos was less than 3.1 units/mg, the inhibitory action for hyaluronidase ( bovine testicular hyaluronidase (E.C.3.2.1.35)) of the oligosaccharides ran ged 16- to 20-mer were corresponding to 79% of that shown by fully O-sulfat ed hyaluronan (MW 100 kDa) through both competitive and noncompetitive effe cts.