α-l-Iduronidase, β-d-glucuronidase, and 2-sulfo-l-iduronate 2-sulfutase: preparation and characterization of radioactive substrates from heparin

Radioactive disaccharide substrates for α-l-iduronidase, β-d-glucuronidase, and 2-sulfo-l-iduronate 2-sulfatase have been prepared from heparin by deaminative cleavage followed by reduction with NaBT₄. Six disaccharides were isolated from this reaction mixture and identified. Acid hydrolysis of the major disaccharide, O-(α-l-idopyranosyluronic acid 2-sulfate)-(1→4)-(2,5-anhydro-d-mannitol-1-t 6-sulfate (IdAs-Ms), produced 48% of O-(α-l-idopyranosyluronic acid)-(1→4)-(2,5-anhydro-d-mannitol-1-t 6-sulfate) (IdA-Ms) and 25% of O-(α-l-idopyranosyluronic acid)-(1→4)-2,5-anhydro-d-mannitol-1-t. The most-sensitive substrate for determining a-l-iduronidase activity was IdA-Ms which, when incubated with leucocyte and skin-fibroblast homogenates prepared from patients having a deficiency of a-l-iduronidase (Mucopolysaccharidosis Type I; MPS-I), was hydrolysed to yield 2,5-anhydro-d-mannitol-1-t 6-sulfate at a rate 50-times less than that found for normal control-preparations. Similarly, O-(β-d-glucopyranosyluronic acid)-(1→4)-(2,5-anhydro-D-mannitol-1-t 6-sulfate) was degraded by whole-cell homogenates prepared from β-d-glucuronidase-deficient (Mucopolysaccharidosis, Type VII) fibroblasts, to yield 2,5-anhydro-d-mannitol-1-t 6-sulfate at a rate 60-times less than that found for MPS-I and normal control-preparations. IdAs-Ms was degraded by 2-sulfo-l-iduronate 2-sulfatase at a rate more than 45-times greater than that found for O-(a-l-idopyranosyluronic acid 2-sulfate)-(1→4)-2,5-anhydro-d-mannitol-1-t. C-6 Sulfation of the anhydro-d-mannitol-1-t residue is an important structural determinant in the mechanism of action of both a-l-iduronidase and 2-sulfo-l-iduronate 2-sulfatase on disaccharide substrates.