A novel method for evaluation of carbohydrate-binding activity: enzyme-linked carbohydrate-binding assay (ELCBA)

A highly sensitive method for detection of the carbohydrate-binding activity of proteins is described. The method is based on interactions of carbohydrate-binding proteins, immobilized on a solid phase, with an enzyme-labeled soluble polysaccharide (peroxidase conjugated glycosaminoglycans-heparin, chondroitin sulfate or hyaluronic acid. Binding capacity was measured spectrofotometrically after enzymatic reaction with chromogenic substrate. The reliability of the assay was tested by use of two heparin-binding proteins-i) fibronectin (soluble) and ii) heparin-binding protein purified from the human brain (water-insoluble). Binding of heparin was dependent on metal ions, detergents and urea. The assay is believed to be applicable for the identification and characterization of a variety of carbohydrate(glycosaminoglycan)-binding proteins, especially, when traditional methods can not be applied (e.g., when proteins are water-insoluble).