Synthesis of N-acetylneuraminyl-alpha 2,3(6)lactose-malate dehydrogenase conjugate for detecting sialic acid terminal groups on glycoproteins via homogeneous lectin-based enzyme-linked binding assay |
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Authors: | X Guo ME Meyerhoff |
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Affiliation: | Department of Chemistry, University of Michigan, Ann Arbor 48109, USA. |
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Abstract: | An N-acetylneuraminyl-alpha 2,3(6)lactose-malate dehydrogenase (MDH-Lac-Neu5Ac) conjugate is prepared via an isothiocyanate conjugation method using a p-aminophenethylamino derivative of sialyllactose. The newly synthesized conjugate can be utilized as a reagent in a novel homogeneous lectin-based, enzyme-linked, competitive binding assay (1-3) for probing the specific carbohydrate structure and content of intact glycoproteins. The enzymatic activity of the MDH-Lac-Neu5Ac conjugate is shown to be significantly inhibited (35%) by sialic acid-binding lectin, Limax flavus agglutinin (LFA), and this inhibition is reversed by mucin, a glycoprotein possessing sialic acid terminals. The asialo form of mucin, however, binds weakly to LFA, yielding no substantial increase in the MDH-Lac-Neu5Ac activity at comparable glycoprotein concentrations. Use of the newly synthesized conjugate in conjunction with LFA or other lectins capable of binding sialic acid may provide a rapid and convenient way to detect the presence and relative amount of sialic acid terminal groups within intact glycoprotein structures. |
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