title: Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins.
abstract: A procedure for determination of apparent affinity constants K(D)(app) between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10(-7) to 9×10(-6)M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.
authors.affiliation: Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: D
authors.initials: D
authors.lastname: Mislovičová
authors.forename: J
authors.initials: J
authors.lastname: Katrlík
authors.forename: E
authors.initials: E
authors.lastname: Paulovičová
authors.forename: P
authors.initials: P
authors.lastname: Gemeiner
authors.forename: J
authors.initials: J
authors.lastname: Tkac
mesh_terms: D001665:Binding Sites; D003208:Concanavalin A / Q000737:chemistry*; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry; D005949:Glucose Oxidase / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D007118:Immunoassay / Q000592:standards*; D007700:Kinetics; D008358:Mannose / Q000737:chemistry; D008958:Models, Molecular; D010047:Ovalbumin / Q000737:chemistry; D011485:Protein Binding; D014168:Transferrin / Q000737:chemistry; D043324:beta-Fructofuranosidase / Q000737:chemistry
chemical_list: D006023:Glycoproteins; D014168:Transferrin; D003208:Concanavalin A; D010047:Ovalbumin; D005949:Glucose Oxidase; D043324:beta-Fructofuranosidase; D005087:Glucan 1,4-alpha-Glucosidase; D008358:Mannose