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).
Department of Biophysics and Biochemistry, State University, Dniepropetrovsk, Ukraine.
M I
MI
Dolzhenko
E A
EA
Lepekhin
V A
VA
Berezin
D001921:Brain / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry; D007700:Kinetics; D008722:Methods; D010544:Peroxidases; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry
D002241:Carbohydrates; D002352:Carrier Proteins; D006025:Glycosaminoglycans; D011506:Proteins; D006820:Hyaluronic Acid; D006493:Heparin; D002809:Chondroitin Sulfates; D010544:Peroxidases