Calcium binding to a human factor IXa derivative lacking gamma-carboxyglutamic acid: evidence for two high-affinity sites that do not involve beta-hydroxyaspartic acid.
A derivative of human blood clotting factor IXa beta lacking gamma-carboxyglutamic acid (Gla) residues was prepared by limited proteolysis with chymotrypsin, and subsequently examined for its ability to bind calcium ions. By amino acid analysis, Gla-domainless human factor IXa beta contained 0.3-0.4 moles of beta-hydroxyaspartic acid per mole of protein. Equilibrium dialysis experiments demonstrated that Gla-domainless human factor IXa beta retained two high-affinity calcium binding sites (Kd=52 microM), a finding essentially identical to that observed for Gla-domainless bovine factor IX that contains 0.8-0.9 moles of beta-hydroxyaspartic acid per mole of protein. These data strongly suggest that the beta-hydroxyaspartic acid residue in these proteins does not participate in their high affinity calcium sites.
D015055:1-Carboxyglutamic Acid; D000596:Amino Acids / Q000032:analysis; D001224:Aspartic Acid / Q000031:analogs & derivatives; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D005164:Factor IX / Q000378:metabolism*; D015949:Factor IXa; D005971:Glutamates; D006801:Humans; D013997:Time Factors
D000596:Amino Acids; D005971:Glutamates; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D015055:1-Carboxyglutamic Acid; D005164:Factor IX; D015949:Factor IXa; D002118:Calcium