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 lectin-saccharide interactions. The method, based on the formation of the conjugate of an appropriate glycoenzyme with the specific carbohydrate-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 monosaccharides derived from D-mannose. The inhibition effect of the saccharides on the formation of Concanavalin A-glycosylated enzyme precipitate was compared with their influence on the enzyme sorption on conjugate Concanavalin A-bead cellulose support. The amount of the interacting enzyme was estimated either indirectly from its concentration in a supernatant that was determined spectrophotometrically (Con A was in a free or immobilized form) or directly in the immobilized form linked to Con A-sorbent using the flow microcalorimetric method. The results obtained, using different methods, agreed in general.
Mislovicová
D
D
Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic. chemmisl@savba.sk
Vikartovská
A
A
Gemeiner
P
P
D002151:Calorimetry; D002482:Cellulose / Q000378:metabolism; D003208:Concanavalin A / Q000378:metabolism*; D005434:Flow Cytometry; D005949:Glucose Oxidase / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D008358:Mannose / Q000378:metabolism; D013053:Spectrophotometry; D043324:beta-Fructofuranosidase
D003208:Concanavalin A; D002482:Cellulose; D005949:Glucose Oxidase; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D008358:Mannose