α-L-艾杜糖苷酶测定的底物。

Substrates for the assay of alpha-L-iduronidase.

作者信息

Thompson J N

出版信息

Clin Chim Acta. 1978 Nov 1;89(3):435-46. doi: 10.1016/0009-8981(78)90407-2.

Abstract

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.

摘要

本文描述了分别从肝素和硫酸皮肤素制备两种二糖的方法，即4-O-α-L-艾杜糖醛酸基-2,5-脱水[3H]甘露糖醇和3-O-α-L-艾杜糖醛酸基-2,5-脱水[3H]木糖醇。这些二糖有助于基于将释放的中性脱水[3H]甘露糖醇或脱水[3H]木糖醇与未反应的底物通过后者吸附到Dowex 1上进行分离来简便地测定α-L-艾杜糖苷酶。对反应条件的研究表明，α-L-艾杜糖苷酶活性（来自人成纤维细胞和苹果螺的酶）在含有0.01 M NaCl的醋酸盐缓冲液中，以艾杜糖醛酸基-2,5-脱水[3H]甘露糖醇为底物时，在pH 3.6时最佳。对于艾杜糖醛酸基-2,5-脱水[3H]木糖醇，苹果螺酶的最适pH为4.0。人成纤维细胞的艾杜糖醛酸基-2,5-脱水[3H]甘露糖醇的KM为0.23 mM，苹果螺酶的KM为0.04 mM；苹果螺α-L-艾杜糖苷酶的艾杜糖醛酸基-2,5-脱水[3H]木糖醇的KM值为0.02 mM。