Calcium binding and putative activity of the epidermal growth factor domain of blood coagulation factor IX

The first epidermal growth factor (EGF)-like domain of human Factor IX and two chimeric analogs of this domain and EGF were synthesized unambiguously and purified to homogeneity. The synthetic EGF-like domain and its analogs showed the correct mass ions by the fission ionization mass spectrometry and similar disulfide pairings as those found in EGF, but failed to exhibit any putative EGF activity in the receptor and mitogenic assays. However, in NMR titration experiments, the EGF-like domain and one of its analogs were found to bind Ca2+ but not Mg2+. Our results therefore show that the EGF-like domain of Factor IX has the ability to bind calcium ion, shares the structural motif of EGF but does not retain the active determinants responsible for the EGF activity.