The Ca2+ ion binding of factor VIIa and the derivative lacking the γ-carboxyglutamic acid domain, des(1–38) factor VIIa, was investigated using intrinsic protein fluorescence and Tb3+ ion phosphorescence methods. Binding of Ca2+ ions giving rise to a decrease in the intrinsic protein fluorescence (approximately 50% at saturating conditions) is seen with both proteins. Each of the saturation curves is in accordance with the formation of a 1:1 complex of factor VIIa-Ca2+ (K D∼30 μM) and des(1–38) factor VIIa-Ca2+ (K D∼40 μM)). Yet another Ca2+ ion binding site reveals itself in each protein in Tb3+ ion phosphorescence experiments. Ca2+ ion competition studies have showed 1:1 complexes (K D′s∼2 mM). The results are interpreted in terms of two different Ca2+ ion binding sites, one in the EGF-1 domain and one in the Gly-209-Gin-221 loop of the serine proteinase part.
