Identification of the glycosidically bound sialic acid in mucin glycoproteins that reacts as 'free sialic acid' in the Warren assay

A widely employed colorimetric assay for sialic acids based on periodate oxidation followed by reaction with thiobarbituric acid depends on the formation of a hexos-5-uluronic acid product, the pre-chromogen, by the periodate cleavage of the C6-C7, C7-C8, and C8-C9 bonds in free sialic acid. Glycosidically bound sialic acids are not expected to react in the assay since cleavage cannot occur between C6-C7 to yield the pre-chromogen. However, several investigators have reported the detection of a positive reaction by certain sialoglycoconjugates. In this study, it was found that various mucins but not other classes of sialoglycoconjugates or asialomucins exhibited this phenomenon. Of the mucins tested, ovine submaxillary mucin showed the maximum reactivity followed by the bovine and porcine counterparts. The disaccharide Neu5Acα2→6 GalNAc(OH) released from mucins by alkaline borohydride treatment also reacted, albeit weakly compared to the native mucins, but other sialyl saccharides including 6'-sialyllactose and 6'-sialyl N-acetyllactosamine did not react. The positive reaction of the submaxillary mucins is not due to the presence of 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN), a minor component in submaxillary mucins, or the release of sialic acid by the acidic condition of the assay. It is demonstrated that sialyl residues linked α2→6 to unsubstituted N-acetylgalactosamine (sialyl Tn antigen structure) in mucin glycoproteins is responsible for the positive reaction. Apparently, periodate oxidation of the N-acetylgalactosamine residue leads to the release of sialic acid from the Neu5Acα2→6 GalNAc linked to serine/threonine by an acid-catalyzed β-elimination reaction. The findings provide a basis for the development of a chemical method to estimate sialyl Tn epitopes associated with cancer cells.