Synthesis and characterization of highly sensitive heparin probes for detection of heparin-binding proteins.
Three labeled heparin species were synthesized as probes for heparin-binding protein detection. Heparin conjugated with 5([4,6-dichlorotriazin-2-yl]amino)fluorescein can be iodinated to a high specific activity. This probe specifically detected 40 pg histone on a dot blot without affinity purification. Heparin biotinylated on its naturally occurring primary amino groups also detected known heparin-binding proteins in a specific manner. This probe detected lower amounts of collagen I and basic fibroblast growth factor on nitrocellulose membranes than did the iodinated probe, with comparable detection times. To create more attachment sites for biotin, we covalently attached amino groups to the hydroxyl groups of heparin using 3-bromopropylamine hydrobromide. After biotinylation, the amino-rich probe detected heparin-binding proteins at the same or higher sensitivity as the biotinylated native heparin probe, using 100-fold less probe and much shorter detection times. This method of labeling is generally applicable to other polysaccharides, and would be useful when the amount of ligand is limited. We show that these three probes detect essentially the same spectrum of proteins in detergent extract of smooth muscle cell plasma membrane, and expect them to be useful probes for detection of cell-surface heparin receptors.
Stearns
N A
NA
Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Prigent-Richard
S
S
Letourneur
D
D
Castellot
J J
JJ
D000818:Animals; D001710:Biotin; D002352:Carrier Proteins / Q000032:analysis*; D002478:Cells, Cultured; D005452:Fluoresceins; D006493:Heparin / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000032:analysis; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000737:chemistry; D009131:Muscle, Smooth, Vascular / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D012680:Sensitivity and Specificity
D002352:Carrier Proteins; D005452:Fluoresceins; D008024:Ligands; D008565:Membrane Proteins; D015335:Molecular Probes; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D001710:Biotin; D006493:Heparin