Heparin-von Willebrand factor binding as assessed by isothermal titration calorimetry and by affinity fractionation of heparins using synthetic peptides.
The ability of proteins to bind heparin, a heterogeneous sulfated glycosaminoglycan, likely depends on the conformational uniqueness of specific binding domains. Based on the motif of a consensus heparin-binding synthetic peptide, a 23-residue sequence (Y565-A587) of human von Willebrand factor (vWF) was previously identified which binds heparin with affinity comparable to that of the native protein (Sobel, M., Soler, D. F., Kermode, J. C., and Harris, R. B. 1992 J. Biol. Chem. 267, 8857-8862). This peptide undergoes a conformational change upon binding heparin. Isothermal titration calorimetry has now been used to further quantify this binding reaction. In experiments done at 25 degrees C, Y565-A587 bound heparin with about the same affinity (Kd = 9.0 x 10(-7) M) as a "core" sequence peptide encompassing residues K569-I580. Binding between these peptides and heparin is overwhelmingly enthalpically favored and is dependent on the formation of productive electrostatic bonds; hydrophobic interactions do not play a significant role in mediating binding. Furthermore, when immobilized on Sepharose in a manner which does not compromise essential cationic residues, the vWF domain peptides are effective affinity ligands. They bind a species of heparin which possesses significantly enhanced affinity for native vWF. The Kd for binding between the high-affinity heparin and Y565-A587 is about threefold lower than that determined with crude, unfractionated heparin. Thus, the vWF peptides are a useful model for studying the physiological role of heparin binding to the native protein.
Tyler-Cross
R
R
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614.
Sobel
M
M
Marques
D
D
Soler
D F
DF
Harris
R B
RB
D000595:Amino Acid Sequence; D002151:Calorimetry / Q000379:methods; D002846:Chromatography, Affinity; D006493:Heparin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D010974:Platelet Aggregation / Q000502:physiology; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology
D010455:Peptides; D014841:von Willebrand Factor; D006493:Heparin