Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia.
Colloids Surf B Biointerfaces. 2012 Jun 1;94:163-9. doi: 10.1016/j.colsurfb.2012.01.036. Epub 2012 Feb 1.
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.
本文报道了一种使用酶联凝集素测定法（ELLA）测定刀豆凝集素 A（Con A）与天然含有 d-甘露糖的糖蛋白之间表观亲和力常数 K(D)(app)的方法。比较了三种不同的 ELLA 方案，并使用了三种不同的拟合模型（Liliom、Hill 及其无协同因子模型）进行比较。糖蛋白通过物理吸附固定在免疫测定板的高电荷聚苯乙烯固体表面上，并通过光度法测定与糖蛋白结合的凝集素的量。通过 ELLA 方法定量测定了 Con A 与五种含有甘露糖的糖蛋白（INV、GA、GOx、OVA 和 TRF）的相互作用，表观亲和力常数范围为 2×10(-7)至 9×10(-6)M。根据每种糖蛋白上糖链结构/暴露情况，讨论了 Con A 与糖蛋白之间的相互作用强度。
