An improved method for the measurement of total lipid-bound sialic acids after cleavage of alpha 2,8 sialic acid linkage with Vibrio cholerae sialidase in the presence of cholic acid, SDS and Ca2+.
In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD3 and GD2) are not involved because their alpha 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of alpha 2,8 linkages by V. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca2+ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.
Maliakal
M A
MA
Laboratory of Glycolipid Immunotherapy, John Wayne Cancer Institute, Santa Monica, CA 90404.
Ravindranath
M H
MH
Irie
R F
RF
Morton
D L
DL
D002118:Calcium / Q000494:pharmacology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005732:Gangliosides / Q000737:chemistry* / Q000378:metabolism; D006017:Glycolipids / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism*; D010084:Oxidation-Reduction; D012794:Sialic Acids; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D013997:Time Factors; D014734:Vibrio cholerae / Q000201:enzymology*
D002793:Cholic Acids; D003902:Detergents; D005732:Gangliosides; D006017:Glycolipids; D012794:Sialic Acids; D012967:Sodium Dodecyl Sulfate; D009439:Neuraminidase; D019826:Cholic Acid; D002118:Calcium