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.
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.
Guo
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Department of Chemistry, University of Michigan, Ann Arbor 48109, USA.
Meyerhoff
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ME
D001212:Asialoglycoproteins / Q000494:pharmacology; D001667:Binding, Competitive; D006023:Glycoproteins / Q000032:analysis*; D007202:Indicators and Reagents; D007700:Kinetics; D007785:Lactose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D037102:Lectins; D008291:Malate Dehydrogenase / Q000138:chemical synthesis* / Q000378:metabolism; D009077:Mucins / Q000494:pharmacology; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D037121:Plant Lectins; D012794:Sialic Acids / Q000138:chemical synthesis* / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D001212:Asialoglycoproteins; D006023:Glycoproteins; D007202:Indicators and Reagents; D037102:Lectins; C042962:Limax flavus agglutinin; D009077:Mucins; D037121:Plant Lectins; D012794:Sialic Acids; D014909:Wheat Germ Agglutinins; C000020:N-acetylneuraminoyllactose; D008291:Malate Dehydrogenase; D019158:N-Acetylneuraminic Acid; D007785:Lactose