The Effect Of Metal Ions On Human Factor IX Antigenicity

Abstract

Affinity chromatography of an inhibitor to human factor IX (F.IX) separated the antibody into two populations. The ion dependent population of antibodies had an absolute divalent cation (Me++) binding requirement. The non-ion dependent population bound F.IX equally in the presence or absence of Me++. The concentration of Me++ required for ½ the maximum ion dependent antibody binding (½ max) was (in nM) Ca++ 0.40, Mn++ 0.05, Sr++ 0.70 and Mg++ 0.65.Ca++ potentiated the binding of antibody in the presence of excess Mg++. In addition, the ½ max for Ca++ was reduced about four fold. These observations are consistent with separate binding sites on the F.IX molecule for Ca++ and Mg++ and potentiation of Ca++ binding by Mg++. Scat- chard analysis of ion dependent antibody binding indicates about a 10 fold greater affinity of antibody in the presence of Ca++ than Mg++. In the presence of both cations, affinity was at least as high as in the presence of Ca++ alone supporting the presence of separate ion binding sites on the F.IX molecule.