Localization of the specific binding site for magnesium(II) ions in factor IX

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Abstract
We demonstrated recently that coagulation factor IX has a specific binding site(s) for Mg2+ ions, independent of the Ca2+‐binding sites, and that binding of Mg2+ ions is very important for expression of the functional conformation of this protein. We report here the localization of this Mg2+‐specific binding site. We prepared three Gla‐containing fragments of bovine factor IX, namely GlaEGFNC (residues 1–144+286–296), GlaEGFN (1–83) and the Gla domain peptide (1–46). Fragments GlaEGFNC and GlaEGFN retained the ability to undergo a conformational change upon binding of Mg2+ ions in the presence of excess Ca2+ ions. This change could be detected by a conformation‐specific antibody. Furthermore, the Gla domain peptide was capable of binding Mg2+ ions, as determined by the metal ion‐induced quenching of the intrinsic fluorescence. It appears that the Mg2+‐specific binding site of factor IX is located in the N‐terminal Gla domain.