A heparin-binding peptide from human serum amyloid P component characterized by affinity capillary electrophoresis.
Affinity capillary electrophoresis (CE) was used for a detailed characterization of the binding between heparin and a peptide isolated from the heparin-binding serum protein amyloid P component (SAP). The peptide corresponds to a tryptic fragment (T3) comprising amino acids 14-38 of SAP. By including ligands in the electrophoresis buffer various glycosaminoglycans could be screened for binding of T3 using one sample aliquot. The binding was found to be highly specific for heparin and heparin fragments down to tetramers and appeared strongest at a slightly alkaline pH while no binding could be demonstrated with heparan sulfate, chondroitin sulfate, desulfated heparin, mannose 6-phosphate and phosphotyrosine. The T3-heparin complexes were sufficiently stable to perform quantitative measurements of the binding using preequilibration of samples prior to a CE-mediated separation of bound and free T3-peptide. Plots based on quantitation of analyte peaks corresponding to free and complexed T3 yielded a dissociation constant of 1.5 microM for the interaction with heparin. The results indicate that a specific subfraction of the heparin molecules is active in binding interactions with the peptide. The affinity CE approach proved to be useful for these studies because of its sensitivity to complex formation involving charged ligands and the possibility of achieving separations under native conditions. Also advantageous is the low sample consumption and the ability to analyze unlabeled reactants in solution.
Heegaard
N H
NH
Department of Autoimmunology, Statens Serum Institute, Copenhagen, Denmark. nhe@ssi.dk
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D002352:Carrier Proteins; D004220:Disulfides; D010446:Peptide Fragments; D000683:Serum Amyloid P-Component; D006493:Heparin