Identification of a lysyl residue in antithrombin which is essential for heparin binding.
Identification of lysyl residue(s) in human plasma antithrombin required for binding of heparin was approached using chemical modification with the amino-group reagent pyridoxal 5'-phosphate. Modification of antithrombin with limiting amounts of reagent yields an average incorporation of the phosphopyridoxyl label into 1 lysine/protein molecule (Pecon, J. M., and Blackburn, M. N. (1984) J. Biol. Chem. 259, 935-938). Fractionation of the labeled antithrombin by affinity chromatography on heparin-Sepharose separated a phosphopyridoxylated antithrombin species devoid of heparin binding from modified protein which retained affinity for heparin. To generate peptide maps of the two antithrombin species, the proteins were reductively denatured, S-carboxymethylated, and digested with trypsin. Fractionation of the tryptic digests by reverse-phase high performance liquid chromatography indicated one peak in the chromatogram of the non-heparin-binding species to be clearly different when compared to the chromatogram of the heparin-binding species. The sequence of the unique peptide, determined by automated Edman degradation, was Thr-Ser-Asp-Gln-Ile-His-Phe-Phe-Phe-Ala-Lys-Leu-Asn-Cys-Arg. This peptide corresponds to a tryptic fragment including residues 115-129 in the sequence of antithrombin, with the modified residue identified as Lys-125. Additionally, phosphopyridoxylation of antithrombin in the presence of added heparin indicated that several other lysyl residues were "protected" from modification. Identification of this critical lysine for heparin binding strongly supports previous data which indicate that the heparin-binding domain of antithrombin is located at the NH2 terminus within one of the disulfide cross-linked loops of the protein.
D000595:Amino Acid Sequence; D000991:Antithrombins / Q000378:metabolism*; D001665:Binding Sites; D006493:Heparin / Q000097:blood*; D006801:Humans; D008239:Lysine; D010449:Peptide Mapping; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D014357:Trypsin
D000991:Antithrombins; D011732:Pyridoxal Phosphate; D006493:Heparin; D014357:Trypsin; D008239:Lysine