Enzyme-based binding assay for studying lectin-glycoprotein and glycosaminoglycan-macromolecule interactions

The continued development of novel homogeneous lectin-based enzyme-competitive binding assays (ELBAs) is described. Isothiocyanate derivatives of di- and tri-saccharides are synthesized using two different organic synthesis methods, and the products obtained are covalently attached to malic dehydrogenase (MDH), via an isothiocyanate conjugation method, to form Gal;Development of another homogeneous enzyme-based binding assay to study the degree of interaction between glycosaminoglycans (GAGs) and various macromolecules/peptides is also described. The method is based on the homogeneous inhibition of a highly positively charged enzyme, acid deoxyribonuclease II (EC 3.1.22.1), by GAG polyanions. Catalytic activity of DNase II is inhibited to nearly 100% by relatively small amounts of GAGs molecules. In the presence of species that bind GAGs, the activity of the enzyme is regained in an amount proportional to the concentration of the species present. The feasibility of this binding assay principle is demonstrated by measuring the ED...