In bovine protein C normal activation by the thrombin-thrombomodulin complex requires binding of calcium to one high affinity binding site, contained in a protein fragment that lacks the gamma-carboxyglutamic acid (Gla) region (Esmon, N. L., De Bault, L. E., and Esmon, C. T. (1983) J. Biol. Chem. 258, 5548-5553). In this work, the calcium binding to and the conformational change induced by calcium in the corresponding Gla-domainless fragment of bovine factor X, prepared by limited proteolysis by chymotrypsin, were compared with the calcium-binding properties of Gla-domainless protein C. Equilibrium dialysis experiments demonstrated that the proteolytically modified factor X has one high affinity calcium ion-binding site with Kd = 180 microM, a value almost identical to the Kd for the binding of calcium to proteolytically modified protein C. Measurements of the rate of disulfide bond reduction by thioredoxin showed that the disulfide bonds of both factor X and protein C lacking the Gla domains were more rapidly reduced in the absence than in the presence of calcium. Thus, calcium binding induces a conformational change in both proteolytically modified proteins. Calcium binding to Gla-domainless protein C is accompanied by a quenching of the intrinsic tryptophan fluorescence and by changes in the CD spectrum, indicative of perturbation of the environment of aromatic amino acids by the metal ion. However, no such changes were observed with the proteolytically modified factor X. This difference may be due to the fact that one tryptophan residue (in position 84) is present in the light chain of the proteolytically modified protein C but none in the light chain of the modified factor X. The light chain of factor X has beta-hydroxyaspartic acid in position 64 which is homologous to the beta-hydroxyaspartic acid in position 71 in the light chain of protein C. Our results are compatible with the hypothesis that beta-hydroxyaspartic acid is involved in the Ca2+ ion binding.