Analysis of the interactions between the peptides from secreted human CKLF1 and heparin using capillary zone electrophoresis

The Chemokine-like factor 1 (CKLF1) is a novel human cytokine and exhibits chemotactic activities on leukocytes. Two peptides named CKLF1-C27 and CKLF1-C19. were obtained from secreted CKLF1. In this study. a selective high-performance analytical method based on capillary zone electrophoresis (CZE) to investigate interactions between heparin and CKLF1C27/CKLF1-C19 was developed. Samples containing CKLF1-C27/CKI.F1-C19 and heparin at various ratios were incubated at room temperature and then separated by CZE with Tris-acetate buffer at. pH 7.2. Both qualitative and quantitative characterizations of the binding were determined. The binding constants of the interactions between CKLF1-C27/CKLF1-C19 and heparin were calculated as (3.38 +/- 0.49) x 10(5) M-1 and (1.10 +/- 0.02) x 105 M-1 by Scatchard analysis. To study structural requirements. CKI.FI-C19pm and CKLF1-C19km have been synthesized. and their interactions with heparin have been studied by CZE. We found that the Pro or Lys to Ala substitution within the residues of CKLF1-C19 (CKLF1-C19pm or CKLF1-C19km) strongly decreased or abolished its interaction with heparin. suggesting that the residues of Pro affect: the affinity of CKLF1-C19 for heparin, and the residues of Lys of CKLF1-C19 play the important role for the interaction of CKLF1-C19 and heparin, respectively. The methodology presented should be generally applicable to study peptides and heparin interactions quantitatively and qualitatively. Copyright (C) 2008 European Peptide Society and John Wiley & Sons. Ltd.