Interaction of heparin with synthetic antithrombin III peptide analogues.
Heparin-binding proteins may contain specific patterns of basic amino acids, called consensus sequences, that interact with heparin. Small peptides were synthesized that contained consensus sequences (i.e. FAKLNCRLYRKANKSSK) or disrupted consensus sequences (i.e. K136-->A) based on the known sequence of antithrombin III (amino acid residues 123-139). These peptides were then examined in both competitive and non-competitive binding experiments using bioassays, fluorescence spectroscopy, affinity chromatography and n.m.r. spectroscopy. Both the consensus and disrupted-consensus peptide bound to heparin. Peptides with consensus sequences bound specifically to the pentasaccharide antithrombin III-binding site within heparin. In contrast, peptides with disrupted consensus sequences showed no specificity, binding to any sequence within heparin. Proton nuclear Overhauser enhancement spectroscopy demonstrated the proximity of leucine and tyrosine (within the consensus sequence) to the N-acetyl moiety found primarily within the pentasaccharide antithrombin III-binding site of heparin. This experiment confirmed the findings of the other techniques and helped to localize the binding sites in both peptides and heparin. A model is proposed for both specific and non-specific heparin interaction with consensus and disrupted-consensus peptides.
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City.
J
J
Bae
U R
UR
Desai
A
A
Pervin
E E
EE
Caldwell
J M
JM
Weiler
R J
RJ
Linhardt
D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000031:analogs & derivatives* / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D001681:Biological Assay; D002846:Chromatography, Affinity; D016384:Consensus Sequence; D006493:Heparin / Q000378:metabolism*; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D010455:Peptides; D000990:Antithrombin III; D006493:Heparin