The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products.
The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca2+. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (TM) of 54.5 +/- 0.5 degrees C and a calorimetric enthalpy (delta Hc) of 105 +/- 15 kcal/mol, in the absence of Ca2+. In the presence of Ca2+ concentrations sufficient to saturate its sites on Factor IX, the Tm value is increased to 57.0 +/- 0.5 degrees C and the delta Hc is virtually unchanged. When the activation intermediate, Factor IX alpha, is similarly analyzed in the absence of Ca2+, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca2+, Factor IX alpha displayed thermograms characterized by a TM of 51.0 +/- 0.5 degrees C and a delta Hc of 109 +/- 10 kcal/mol. The activated product, Factor IXa alpha, in the absence of Ca2+ (the values in the presence of saturating Ca2+ are given in parentheses), undergoes thermal denaturation with a TM of 54.5 +/- 0.5 degrees C (57.0 +/- 0.5 degrees C) and a delta Hc of 158 +/- 10 kcal/mol (156 +/- 10 kcal/mol). Similarly, the terminal-activation product, Factor IXa beta, displays a TM of 51.5 +/- 0.5 degrees C (54.0 +/- 0.5 degrees C) and a delta Hc of 85 +/- 5 kcal/mol (126 +/- 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a TM of 54.0 +/- 0.5 degrees C (55.0 +/- 0.5 degrees C) and a delta Hc of 102 +/- 10 kcal/mol (118 +/- 10 kcal/mol), whereas its activated form, Factor Xa beta, possesses a TM of 55.0 +/- 0.5 degrees C (55.0 +/- 0.5 degrees C) and a delta Hc of 92.0 +/- 5 kcal/mol (136 +/- 10 kcal/mol). These studies indicate that, for many of these proteins, Ca2+ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D005164:Factor IX / Q000032:analysis*; D005170:Factor X / Q000032:analysis*; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature
D005164:Factor IX; D005170:Factor X; D002118:Calcium