Circular dichroism analysis of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin

Analysis of the far-ultraviolet circular dichroism spectrum of bovine blood coagulation factor IX reveals the presence of approximately 14% helical structures 26% -sheets, 20% -turns, and 40% coils. These values are essentially the same for the activation products of this zymogen, factor IXa and factor IXa. Similar analysis for bovine factor X permits calculation of these secondary structural as approximately 11% helices, 31% -structures, 22% -turns, and 36% random structures. Bovine prothrombin contains approximately 12% helical structures, 35% -structures, 24% -turns, and 29% coils. None of these values is substantially altered as a result of increase of thepH from 7.4 to 10.5, or upon addition of Ca²⁺ to a concentration of at least 20 mM. Analysis of the near-ultraviolet spectra of factor IX and prothrombin suggests that several aromatic amino acid residues and the disulfide bond present in their -carboxyglutamic acid-containing regions are exposed to solvent and are perturbed by the abovepH adjustment and Ca²⁺ addition. Similar effects are observed in the case of factor X; in addition, the Trp residue at the amino terminus of the heavy chain appears to be influenced by the abovepH alteration. The results reported in this paper show that these vitamin K-dependent blood coagulation proteins are similar in their ordered secondary structures, which are dominated by -sheets and -turns. Their overall secondary structures are not influenced by Ca²⁺ binding and are stable to alkalinepH changes. However, these same environmental alterations appear to be effective probes of aromatic residues in the -carboxyglutamic acid regions.