Preparation of cell-affinity adsorbents by immobilization of peptides to Sephadex G-10.
In this study, affinity adsorbents for the binding of activated blood platelets and endothelial cells were prepared from Sephadex G-10 by immobilization of peptides, derived from the cell-adhesive amino acid sequence RGD (arginine-glycine-aspartic acid). Derivatization of Sephadex G-10 was accomplished by sequential coupling of specific dipeptides (RG and DV) (V = valine) and by coupling of the RGD-containing hexapeptide GRGDSP (S = serine, P = proline). Two types of gel were prepared by sequential coupling, designated as G10 (acetone) and G10 (dimethylformamide) (G10(DMF)), containing peptides which had been coupled to the outer side of the beads and throughout the porous beads, respectively. The binding capacity of the prepared Sephadex-derivatives amounted up to 2 x 10(9) human blood platelets per millilitre GRGDV-Sephadex at immobilized peptide concentrations, that were in the nanomole range (per millilitre packed gel) and which differed a factor 10 between G10 (acetone) and G10 (DMF). In a second series of experiments, different amounts of the hexapeptide GRGDSP were coupled to carboxylated Sephadex G-10 and carboxylated Sepharose CL 6B. The binding of human umbilical vein endothelial cells to the resulting materials was studied. Up to 10(6) endothelial cells attached per ml GRGDSP-derivatized hydrogel at peptide concentrations of 15 nmol GRGDSP/ml Sephadex and at +/- 300 nmol GRGDSP/ml Sepharose. Substitution of the arginine residue of the RGD-sequence by glutamine abolished the cell-binding activity of the immobilized peptide towards activated blood platelets but not towards endothelial cells. From the results of this study it can be concluded that small peptides can be coupled to the outer side of the porous Sephadex beads, resulting in high effective ligand densities for cell-affinity applications. In this respect, Sephadex G-10, derivatized according to 'the acetone method' is a good alternative for polystyrene and other solid phase materials.
Besselink
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GA
Department of Chemical Technology, University of Twente, Enschede, The Netherlands.
Beugeling
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Poot
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Van Aken
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WG
Bantjes
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D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002234:Carbodiimides / Q000737:chemistry; D002448:Cell Adhesion; D003432:Cross-Linking Reagents; D003911:Dextrans / Q000737:chemistry*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005782:Gels; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D014471:Umbilical Veins
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D003911:Dextrans; D005782:Gels; D010455:Peptides; D012997:Solvents; D012685:Sepharose; C025614:sephadex