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$\beta$1-4GlcNAc- and Neu5Ac$\alpha$2-3(6)Gal$\beta$1-4Glc-MDH conjugates. Gal$\beta$1-4GlcNAc-MDH (MDH-LacNAc) also serves as the substrate for rat liver Gal$\beta$1-4GlcNAc$\alpha$2-6 sialyltransferase to prepare a Neu5Ac$\alpha$2-6Gal$\beta$1-4GlcNAc-MDH conjugate (MDH-LacNAc-Neu5Ac). These conjugates are then used in ELBAs to probe the terminal carbohydrate structures of model glycoproteins. The Neu5Ac$\alpha$2-3(6)Gal$\beta$1-4Glc-MDH, when used with Limax flavus agglutinin (LFA), exhibits very distinct dose-responses toward bovine submaxillary mucin and its neuraminidase-treated asialoform. In fact, the EDZ$\sb{50}$ value of mucin is about 3 orders of magnitude lower than that of asialomucin. The MDH-LacNAc conjugate used in conjunction with Toxin RCA$\sb{60}$, also yields a very sensitive response toward asialofetuin, a glycoprotein with Gal$\beta$1-4GlcNAc terminal residues. When fetuin was used, the ED$\sb{50}$ increased more than 10 fold. However, the inhibition of MDH-LacNAc-Neu5Ac conjugate by sialic acid binding lectins was not high enough to allow optimal use of this conjugate in competitive binding assays. 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$\sb{50}$ values of five macromolecules. The applicability of the assay method is further extended to study GAG-peptide interactions. A variety of small synthetic peptides (8-13 amino acid residues) derived from the heparin-binding domains of protamine and type IV collagen are used as model peptide species. Relative GAG-binding affinities of these macromolecules/peptides compare favorably to previous literature values, and data obtained via a new electrode-based titration method.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105098/1/9635527.pdfDescription of 9635527.pdf : Restricted to UM users only