Fluorescent-assisted detection of oligosialyl units in glycoconjugates.
A highly sensitive chemical method to detect various types of oligo/polysialic acid (oligo/polySia) units in glycoconjugates, i.e., alpha2 --> 8-linked homo-oligo/polySia [N-acetylneuraminic acid (Neu5Ac), N-glcolylneuraminic acid (Neu5Gc), or 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid], alpha2 --> 8-linked heterodimers of Neu5Ac and Neu5Gc, alpha2 --> 9-linked homooligo/polyNeu5Ac, and alpha2 --> 5-Oglycolyl-linked homooligo/polyNeu5Gc, was developed with an alpha-keto acid-reactive fluorescent labeling reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB). Fluorescent labeled di- or oligoSia was separated and quantitated by fluorometric anion-exchange high-performance liquid chromatography (HPLC). As little as 13 fmol of Neu5Acalpha2 --> 8Neu5Ac was detectable by this method. When alpha2 --> 8-linked oligo/polyNeu5Ac with on average eight Neu5Ac residues was labeled with DMB, DMB derivatives of oligomers with only lower degrees of polymerization of 2 to 7, were detected, due to concomitant partial depolymerization of oligo/polySia chain with the derivatization. For glycoproteins and glycolipids, mild acid hydrolysis was performed to release oligoSia prior to DMB derivatization. The mild acid hydrolysis/fluorometric HPLC method was also applicable to glycoprotein samples blotted on the membrane.
Sato
C
C
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
Inoue
S
S
Matsuda
T
T
Kitajima
K
K
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods; D006001:Glycoconjugates / Q000032:analysis* / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009439:Neuraminidase / Q000737:chemistry; D010655:Phenylenediamines / Q000737:chemistry*; D011145:Polyvinyls / Q000737:chemistry; D012680:Sensitivity and Specificity; D012794:Sialic Acids / Q000737:chemistry*; D005465:Fluorocarbon Polymers
D005456:Fluorescent Dyes; D006001:Glycoconjugates; D006023:Glycoproteins; D008567:Membranes, Artificial; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase; D010655:Phenylenediamines; D011145:Polyvinyls; D012794:Sialic Acids; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C058886:1,2-diamino-4,5-methylenedioxybenzene