DEMONSTRATION OF A HEPARIN-BINDING SITE IN SERUM AMYLOID-P COMPONENT USING AFFINITY CAPILLARY ELECTROPHORESIS AS AN ADJUNCT TECHNIQUE

Linear heparin-binding sites in the DNA- and heparin-binding serum pro tein amyloid P component were investigated using affinity capillary el ectrophoresis and reversed-phase HPLC in conjunction with affinity chr omatography. Peptide fragments were generated from amyloid P component by treatment with Glu-C and Asp-N endoproteinases. This peptide mixtu re 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 he parin in solution. Migration shift patterns in the presence of heparin were in agreement with one of the components shown by HPLC to interac t with immobilized heparin. The identity of this fragment was establis hed by mass spectrometry after preparative HPLC and represents a novel heparin-binding sequence. The results illustrate the potential synerg y 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 se parations under physiological conditions. The possibility of using unm odified ligands in the study of biological activities of protein subst ructures while consuming very little material makes CE further attract ive.