反应详情

The P4 column was calibrated with carbohydrate standards that were obtained as follows. [3H] -Sialic acid was obtained by sialidase treatment (Arthrobacter ureafaciens, Calbiochem, La Jolla, Calif.) 0.3 units per ml, pH 5.5, 30 min, 37° C.) of GlyCAM-1 metabolically labeled with [3H]-glucosamine. N-Acetylglucosamine-3-sulfate (GlcNAc-3S, sodium salt) and galactose-6-sulfate (Gal-6S, sodium salt) were from Sigma. Glucuronic acid monosulfate (GlcU-S, mixture of isomers) was prepared by treatment of glucuronic acid with two molar equivalents of SO3 -trimethylamine (Westerduin, P., Willems, H. A., and van Boekel, C. A. A., Tetrahedron Lett., 31:6915-6918 (1990)) in anhydrous DMF (4 h, 25° C.) under an inert atmosphere. The reaction was neutralized with saturated NaHCO3 and the product was purified by chromatography on DEAE-Sephadex (Aldrich) eluting with a linear gradient of 0-2M pyridine-acetate (pH 5.4). The pyridinium salt was converted to the sodium salt by passage over Bio-Rad 50W-X4 resin (Na+form). Galactose-disulfate and galactosetrisulfate (mixtures of isomers) were prepared by treatment of galactose with five molar equivalents of SO3 -trimethylamine in anhydrous DMF (6 h, 40° C.), followed by neutralization with saturated NaHCO3. The products were purified by anion exchange chromatography, and the resulting pyridinium salts were converted to the corresponding sodium salts as described above. All three synthetic derivatives were characterized by negative fast atom bombardment (FAB-) mass spectrometry. GlcNAc-3S was detected in the fractions eluted from the P4 column with the Elson Morgan reaction (Reissig, J. L., Strominger, J. L., and Leloir, L. F., J. Biol. Chem., 217:959-966 (1955)). The galactose-sulfates were detected with the phenol-sulfuric acid assay (Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F., Anal. Chem., 28:350-356 (1956)). GlcU-S was detected with the carbazole reaction (Bitter, T., and Muir, H. M., Anal. Biochem., 4:330-334 (1956)).

1. customthe product was purified by chromatography on DEAE-Sephadex (Aldrich)
2. washeluting with a linear gradient of 0-2M pyridine-acetate (pH 5.4)
3. customThe products were purified by anion exchange chromatography
4. washeluted from the P4 column with the Elson Morgan reaction (Reissig, J. L., Strominger, J. L., and Leloir, L. F., J. Biol. Chem., 217:959-966 (1955))
5. customGlcU-S was detected with the carbazole reaction (Bitter, T., and Muir, H. M., Anal. Biochem., 4:330-334 (1956))