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 CKLF1-C27/CKLF1-C19 was developed. Samples containing CKLF1-C27/CKLF1-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 10(5) M(-1) by Scatchard analysis. To study structural requirements, CKLF1-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.
Liu
Yi
Y
Department of Pharmacy, Peking University People's Hospital, Beijing 100044, PR China.
Zhang
Shuyu
S
Ling
Xiaomei
X
Li
Yuzhen
Y
Zhang
Yingmei
Y
Han
Wenling
W
Wang
Ying
Y
D001665:Binding Sites; D018925:Chemokines / Q000737:chemistry* / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D062792:MARVEL Domain-Containing Proteins; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D013997:Time Factors
C432676:CKLF protein, human; D018925:Chemokines; D062792:MARVEL Domain-Containing Proteins; D010455:Peptides; D006493:Heparin