Substrates for the assay of alpha-L-iduronidase.
Procedures are described for the preparation of two disaccharides, 4-O-alpha-L-iduronosyl-2,5-anhydro[3H]mannitol and 3-O-alpha-L-iduronosyl-2,5-anhydro[3H]-talitol, from heparin and dermatan sulfate, respectively. These disaccharides lend themselves to an easy assay of alpha-L-iduronidase which is based on the fractionation of the liberated neutral anhydro[3H]mannitol or anhydro[3H]talitol from the unreacted substrate by adsorption of the latter to Dowex 1. Investigation of the reaction conditions showed that the alpha-L-iduronidase activity (enzyme from human fibroblasts and Helix pomatia) was optimal at pH 3.6 in acetate buffer containing 0.01 M NaCl with iduronosyl-2,5-anhydro[3H]mannitol as substrate. For iduronosyl-2,5-anhydro[3H]talitol the pH optimum was 4.0 with the H. pomatia enzyme. The KM for iduronosyl-2,5-anhydro[3H]mannitol was 0.23 mM with human fibroblasts and 0.04 mM with Helix enzyme; a KM value of 0.02 mM was determined for iduronosyl-2,5-anhydro[3H]talitol with the Helix alpha-L-iduronidase.
D002478:Cells, Cultured; D003871:Dermatan Sulfate; D004187:Disaccharides / Q000138:chemical synthesis; D005347:Fibroblasts / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006372:Helix, Snails / Q000201:enzymology; D006493:Heparin; D006801:Humans; D007067:Iduronic Acid / Q000031:analogs & derivatives / Q000138:chemical synthesis; D007068:Iduronidase / Q000378:metabolism*; D008353:Mannitol / Q000031:analogs & derivatives; D013402:Sugar Alcohols
D004187:Disaccharides; D013402:Sugar Alcohols; D003871:Dermatan Sulfate; D007067:Iduronic Acid; D008353:Mannitol; C042890:altritol; D006493:Heparin; D006026:Glycoside Hydrolases; D007068:Iduronidase