Analysis of glycosphingolipids by fluorophore-assisted carbohydrate electrophoresis using ceramide glycanase from Mercenaria mercenaria.
Polyacrylamide gel electrophoresis of fluorophore-labeled saccharides is a simple and sensitive separation method for glycan analysis. This method requires an aldehydic reducing carbon on the saccharide in order to react with the amine group of the fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid or 2-aminoacridone). We have used exoglycosidase-free ceramide glycanase from hard-shelled clam to expose the reducing terminal of the glycan in glycosphingolipid by cleaving the linkage between the glycan and the ceramide. The released glycan was used for fluorophore-assisted carbohydrate electrophoresis, in order to qualitatively determine its monosaccharide composition, chain length, and glycosidic linkages using linkage-specific glycosidases, including a beta 1-3,4 galactosidase from clam.
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
S S
SS
Basu
S
S
Dastgheib-Hosseini
G
G
Hoover
Z
Z
Li
S
S
Basu
D000818:Animals; D049872:Bivalvia / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006028:Glycosphingolipids / Q000032:analysis*; D008969:Molecular Sequence Data; D011817:Rabbits
D005456:Fluorescent Dyes; D006028:Glycosphingolipids; D006026:Glycoside Hydrolases; C051111:ceramide glycanase