Flavobacterium heparinum 2-O-sulphatase for 2-O-sulphato-Delta4,5-glycuronate-terminated oligosaccharides from heparin

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Abstract
The glycosulphatase which hydrolyses the 2-O-sulphate of the disaccharide, 4-deoxy-2-O-sulphato-α-l-threo-hex-4-enopyranosyl uronic acid-(1 → 4)-2-deoxy-2-sulphamido-6-O-sulphato-d-glucose (ΔUA-2S → GlcNS6S), has been isolated from the soluble fraction of disrupted Flavobacterium heparinum. The activity was purified 3300-fold by chromatography on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and blue-Sepharose CL-6B. From sodium dodecylsulphate/polyacrylamide gel electrophoresis, the enzyme was homogeneous and of 62000 Mr. A novel assay was devised using the de-N-sulphonated [1-3H]alditol, 4-deoxy-2-O-sulphato-α-l-threo-hex-4-enopyranosyl uronic acid-(1 → 4)-2-amino-2-deoxy-6-O-sulphato-d-[1-3H]glucitol (ΔUA-2S → [1-3H]GlcNH2-ol-6S). This alditol was shown by 13C-NMR to be desulphated in the analogous manner to the original reducing trisulphated disaccharide. The purified 2-O-sulphatase was completely free of heparinase I, heparinase II (heparitinase), chondroitinases AC, chondroitinase B, the Δ4,5-glycuronidase for heparin Δ4, 5-disaccharides, the 6-O-sulphatase and the 2-sulphamidase. It was optimally active over the range pH 5.5–6.5 and was practically unaffected by Na, K, Ca or Mg ions. Inorganic phosphate inhibited the activity. The Km value for the alditol substrate was 1.22 mmol dm−3. Using 13C-NMR, the 2-O-sulphatase was found to hydrolyse the analogous esters of higher Δ4, 5-oligosaccharides from heparin. This contrasts with the findings of other authors [Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973) J. Biol. Chem. 248, 6408–6415].