Flavobacterium heparinum 6-O-sulphatase for N-substituted glucosamine 6-O-sulphate

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Abstract
A specific glyco-6-0-sulphatase has been purified to homogeneity from F. heparinum. The enzyme hydrolyses the 6-0-sulphates of 2-deoxy-2-sulphamido-6-0-sulpho-D-glucose (GlcNS-6S), 2-acetamido-2-deoxy-6-0-sulpho-D-glucose (GlcNAc-6S) and 2-amino-2-deoxy-6-0-sulphato-D-glucose (GlcN-6S). The activity was purified 2100-fold by successive chromatography on CM-Sepharose CL-6B, Sepharose CL-4B, hydroxyapatite and blue-Sepharose CL-6B. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed a protein of relative molecular mass 64000. Four novel assays were developed using 35S-labeled and 14C-labeled monosaccharides. The purified enzyme was free of all other known heparin-degrading enzymes. In particular this was the first resolution of the 6-0-sulphatase from the sulphamidase. Optimal activity was at pH 7.5. Enzyme activity was virtually unaffected by Na+ and K+ ions. Enhancements of activity of 12% and 30% were effected by Mg2+ and Ca2+ ions respectively. Inorganic phosphate and sulphate (both 0.005 mol dm-3) inhibited activity by 48% and 50% respectively. The Km value for the free amino substrate GlcN-6S was 1.35 mmol dm-3. In contrast the Km values for the GlcNAc-6S and GlcNS-6S were 54 .mu.mol dm-3 and 16 .mu.mol dm-3 respectively.