Direct enthalpy measurements of factor X and prothrombin association with small and large unilamellar vesicles.
Isothermal titration calorimetry was used to determine the enthalpy for the calcium-dependent protein conformation change and subsequent interaction 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/mol for factor X and prothrombin, respectively. The range of values for prothrombin arose from use of different protein preparations and may be due to non-ideal behavior of this protein when calcium was added. The apparent enthalpy of association (delta H(assoc)) of both factor X and prothrombin with phosphatidylserine (PS)/phosphatidylcholine (PC) large unilamellar vesicles (LUVs, 120 nm diameter) was shown to be near 0 kcal/mol. In comparison, delta H(assoc) for interaction with PS/PC small unilamellar vesicles (SUVs, 40 nm diameter) was -9 +/- 3 and -7 +/- 2 kcal/mol for factor X and prothrombin, respectively. This difference 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 considerable difference in the manner of calcium binding to LUVs versus SUVs, little difference in the quantity of calcium bound to SUVs and LUVs was detected by equilibrium dialysis. In any event, the results indicate 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 result from protein-induced changes in the phospholipid packing/calcium interaction, possibly related to changes in how calcium is bound to the phospholipid.
Plager
D A
DA
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Nelsestuen
G L
GL
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002151:Calorimetry / Q000379:methods; D002417:Cattle; D003956:Dialysis; D005170:Factor X / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D017186:Titrimetry
D008081:Liposomes; D011516:Prothrombin; D005170:Factor X; D002118:Calcium