Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells.
Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.
Disciplina de Biologia Molecular, Departamento de Bioquímica, Universidade Federal de São Paulo, SP, Brazil.
Rodrigo Ippolito
RI
Bouças
Edvaldo S
ES
Trindade
Ivarne L S
IL
Tersariol
Carl P
CP
Dietrich
Helena B
HB
Nader
D000818:Animals; D019921:Biotinylation; D002245:Carbon Dioxide / Q000737:chemistry; D002417:Cattle; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005063:Europium / Q000737:chemistry; D005453:Fluorescence; D006025:Glycosaminoglycans / Q000378:metabolism*; D018613:Microscopy, Confocal; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D006025:Glycosaminoglycans; D002245:Carbon Dioxide; C041069:carboxyl radical; C013786:A73025; D005063:Europium; D019809:Streptavidin