THE GLYCOSYLATED ENZYME-BINDING ASSAY FOR THE STUDY OF THE INTERACTION OF FREE AND IMMOBILIZED LECTINS WITH CARBOHYDRATES

The glycosylated enzymes (invertase and glucose oxidase) were used as the competitive markers for a simple and rapid determination of the le ctin-saccharide interactions. The method, based on the formation of th e conjugate of an appropriate glycoenzyme with the specific carbohydra te-binding lectins and the inhibition of the conjugate formation with a:monosaccharide, was described. This method was used to estimate the relative carbohydrate specificity of Concanavalin A for monosaccharide s derived from D-mannose. The inhibition effect of the saccharides on the formation of Concanavalin A-glycosylated enzyme precipitate was co mpared with their influence on the enzyme sorption on conjugate Concan avalin A-bead cellulose support. The amount of the interacting enzyme was estimated either indirectly from its concentration in a supernatan t that was determined spectrophotometrically (Con A was in a free or i mmobilized form) or directly in the immobilized form linked to Con A-s orbent using the flow microcalorimetric method. The results obtained, using different methods, agreed in general. (C) 1997 Elsevier Science B.V.