DIRECT ENTHALPY MEASUREMENTS OF FACTOR-X AND PROTHROMBIN ASSOCIATION WITH SMALL AND LARGE UNILAMELLAR VESICLES

Isothermal titration calorimetry was used to determine the enthalpy fo r the calcium-dependent protein conformation change and subsequent int eraction of blood clotting factor X and prothrombin with phospholipid vesicles. The effect of vesicle size was also determined. The protein conformation change was accompanied by -12 +/- 1 and -7 to -15 kcal/mo l for factor X and prothrombin, respectively. The range of values for prothrombin arose from use of different protein preparations and may b e due to non-ideal behavior of this protein;when calcium was added. Th e apparent enthalpy of association (Delta H-assoc) of both factor X an d prothrombin with phosphatidylserine (PS)/phosphatidylcholine (PC) la rge unilamellar vesicles (LUVs, 120 nm diameter) was shown to be near 0 kcal/mol In comparison, Delta H-assoc for interaction with PS/PC sma ll unilamellar vesicles (SUVs; 40 nm diameter) was -9 +/- 3 and -7 +/- 2 kcal/mol for factor X and prothrombin, respectively. This differenc e appeared complementary to Delta H-assoc for calcium binding to these vesicles. That is, the interaction of calcium was athermic with SUVs and exothermic with LUVs. While such properties might suggest a consid erable difference in the mariner: of calcium binding to LUVs versus SU Vs, little difference in the quantity of calcium bound to SUVs and LUV s was detected by equilibrium dialysis. In any event, the results indi cate that protein binding to LUVs was primarily entropy driven whereas binding to SUVs was primarily enthalpy driven. The exothermic process for calcium-dependent factor X or prothrombin binding to SUVs may res ult from protein-induced changes in the phospholipid packing/calcium i nteraction, possibly related to changes in how calcium is bound to the phospholipid.