CALCIUM-BINDING TO THE FIRST EGF-LIKE MODULE OF HUMAN FACTOR-IX IN A RECOMBINANT FRAGMENT CONTAINING RESIDUES-1-85 - MUTATIONS V46E AND Q50E EACH MANIFEST A NEGLIGIBLE INCREASE IN CALCIUM AFFINITY

The first EGF-like module of human coagulation factor IX contains a si ngle functionally important calcium ion binding site, We have non show n the dissociation constant for this site to be approximately 160 mu M in a recombinant protein fragment consisting of residues 1-85 in huma n fIX, This represents a approximate to 10-fold increase in affinity a s compared with the isolated EGF module (residues 46-85), The Gla modu le (here with Glu instead of Gla) thus increases the affinity of the E GF module calcium ion binding site, Each of two mutations, V46E and Q5 0E, made to investigate whether the extra negative charge would increa se the affinity of the calcium binding site manifested a negligible in crease in affinity. (C) 1998 Federation of European Biochemical Societ ies.