Abstract
A heparin-binding peptide within antithrombin III (ATIII) was identified by digestion of ATIII with Staphylococcus aureus V8 protease followed by purification on reverse-phase high pressure liquid chromatography using a C-4 column matrix. The column fractions were assayed for their ability to bind heparin by ligand blotting with 125I-fluoresceinamine-heparin as previously described (Smith, J. W., and Knauer, D. J. (1987) Anal. Biochem. 160, 105-114). This analysis identified at least three fractions with heparin binding ability of which the peptide eluting at 25.4 min gave the strongest signal. Amino acid sequence analysis of this peptide gave a partially split sequence which was consistent with regions encompassing amino acids 89-96 and 114-156. These amino acids are present in a 1:1 molar ratio which is consistent with a disulfide linkage between Cys-95 and Cys-128. High affinity heparin competed more effectively for the binding of 125I-fluoresceinamine-heparin to this peptide than low affinity heparin. Chondroitin sulfate did not block the binding of 125I-fluoresceinamine-heparin to the peptide. These data strongly suggest that the isolated peptide represents a native heparin-binding region within intact ATIII. Computer generation of a plot of running charge density of ATIII confirms that the region encompassing amino acid residues 123-141 has the highest positive charge density within the molecule. A hydropathy plot of ATIII was generated using a method similar to that of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). This plot indicates that amino acid residues 126-140 are exposed to the exterior surface of the molecule. Based on these data, we suggest that the region corresponding to amino acid residues 114-156 is a likely site for the physiological heparin-binding domain of ATIII. We also conclude that the proposed disulfide bridges within the protein are suspect and should be re-examined (Petersen, T. E., Dudek-Wojiechowska, G., Sottrup-Jensen, L., and Magnussun, S. (1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen, D., Wiman, B., and Verstaeta, M., eds) pp. 43-54, Elsevier Scientific Publishing Co., Amsterdam).
Highlights
(ATIII) wasidentified by digestion of ATIII with vates anumber of serine proteases involved in the coagulation Staphylococcus aureus V 8 protease followed by puri- cascade [1,2,3,4]
Based on these data,we suggest that theregion corre- chloride treatment, and which contain lysines that may be sponding to amino acid residues 114-156 is a likely essential for heparin binding, point to amino acid residues site for the physiological heparin-binding domain of ATIII
The digest was applied to a C-4 column equilibrated in HPLC-grade water 0.1% trifluoroacetic acid and ATZZZ and 125Z-F-HRH-We have previously shown that '%IF-HRH interacts with ATIII whether in solution or onnitrocellulose [1].As an added proof, we have confirmed this using two additional methods
Summary
Demonstration of the Specificity of the Interaction between digested with S. aureus V8 protease a t 1 mg/ml for 24 h at room temperature. ATIII were immobilized on nitrocellulose as described under "Materials and Methods." The dot blots were incubated with serial dilutions of high affinity and low affinity heparin for 1 h at 37 "C in a volume of 1ml. '251-F-HRH(5 ng as uronate) was added to each dot blot in the presence of high affinity and low affinity heparin for an additional 30 min. Analytical digests were performed by digesting 200pgof ATIII with 200pgof V8 protease as described under "Materials and Methods." This sample was analyzed using HPLC and generated a highly reproducible chromatogram (Fig. 5A). The fractions from this chromatogram were analyzed for their ability to bind 1251-FHRH by dot blotting on nitrocellulose. The recovery of each amino acid in pmol is shown in parenthesis
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