Demonstration of a heparin-binding site in serum amyloid P component using affinity capillary electrophoresis as an...

Linear heparin-binding sites in the DNA- and heparin-binding serum protein amyloid P component were investigated using affinity capillary electrophoresis and reversed-phase HPLC in conjunction with affinity chromatography. Peptide fragments were generated from amyloid P component by treatment with Glu-C and Asp-N endoproteinases. This peptide mixture was separated by HPLC before and after passage through a column of immobilized heparin. In addition, the proteolytic digest was separated by capillary electrophoresis in the presence of various amounts of heparin in solution. Migration shift patterns in the presence of heparin were in agreement with one of the components shown by HPLC to interact with immobilized heparin. The identity of this fragment was established by mass spectrometry after preparative HPLC and represents a novel heparin-binding sequence. The results illustrate the potential synergy in the combination of the two high-resolution separation techniques HPLC and CE. HPLC has the advantages of high recovery and preparative power while capillary electrophoresis is noted for highly efficient separations under physiological conditions. The possibility of using unmodified ligands in the study of biological activities of protein substructures while consuming very little material makes CE further attractive.