Structural Changes in Bovine Factor X Associated with Activation

Abstract

Bovine Factor X is converted to Xa in the presence of Ca (II) by the coagulant protein of RVV. To monitor structural transitions in Factor X during conversion, the ultraviolet absorption, fluorescence emission, and circular dichroism spectra of Xa and Factor X were compared. The U.V. difference spectrum in the aromatic region comparing Xa and Factor X is characterized by differences due to tryptophan and tyrosine perturbations. The activation of Factor X yielded a time-dependent increase in this spectrum which was linear for about 60 min. and which paralleled the development of activated Factor X activity. The binding of Ca (II) to Factor X is associated with a red-shifted tryptophan difference spectrum; however, this perturbation makes only a small contribution to the total perturbation observed during Factor X activation. Solvent perturbation studies in 20% glycerol suggest that an average of 3.1 tryptophan residues and 9.0 tyrosine residues are exposed to solvent in Factor X; an additional 0.5 tryptophan residue and tyrosine residue become exposed to solvent during activation of Factor X. The activation of Factor X by the venom protein is associated with a small red shift in the intrinsic tryptophan fluorescence emission spectrum. Far- and near-U.V. circular dichroism spectroscopy detected no difference between Factor X and Xa. In summary, the activation of Factor X to Xa appears associated with exposure of tryptophan and tyrosine side chains previously buried within the protein and with minimal changes in the secondary structure. These results suggest that conversion of Factor X to activated Factor X involves functionally important, but structurally subtle, changes in the three-dimensional structure.