Development of a highly sensitive chemical method for detecting α2→8-linked oligo/polysialic acid residues in glycoproteins blotted on the membrane

A highly sensitive chemical method for detecting alpha 2-->8-linked oligo/polysialic acid (oligo/polySia) chains was developed, including (i) periodate oxidation, reduction with sodium borohydride, and subsequent acid hydrolysis, giving rise to C-7 analogues and intact C-9 compounds from nonreducing terminal and internal sialic acid residues, respectively; (ii) fluorescent labeling of these C-7 and C-9 compounds with 1,2-diamino-4,5-methylenedioxybenzene (DMB); and (iii) quantitation of these DMB derivatives on fluorometric high-performance liquid chromatography. As little as 1 ng of internal sialic acid residues of oligo/polySia chains, the existence of which indicates the presence of oligo/polySia structure, was detectable by this method. This fluorometric C-7/C-9, analysis was successfully applied to glycoproteins blotted onto a slit of polyvinylidene fluoride membranes and suggested the presence of some novel oligoSia-containing glycoproteins in pig embryonic brains. (C) 1998 Academic Press.