Abstract
The energetic contribution of protein solvation-desolvation reactions to generation of coagulation activated factor X (FXa) by the extrinsic pathway protease complex was determined using the technique of osmotic stress. The initial rate of FXa generation by limited proteolysis of human FX was measured in reaction mixtures with human tissue factor (TF) and factor VIIa (FVIIa) assembled either in aqueous phase or on phospholipid membranes. Osmotic stress was induced on the surface of reacting proteins with either polyethylene glycol, or dextran of 6000 and 500,000 molecular weight, respectively. These inert polymers are sterically excluded from the solvation shells of proteins and thus increase the water activity in the excluded spaces. The volume of water transferred either to or from the excluded spaces during formation of reaction intermediates was calculated from the ratio of change in free energy of activation with change in osmotic pressure, delta G*/delta II. For aqueous phase-assembled reactions, delta G* values decreased with delta II at ratios of -2.36 +/- 0.38 and -2.26 +/- 0.26 kcal/mol/atm for polyethylene glycol and dextran, respectively. These values correspond to 5488 +/- 883 and 5255 +/- 604 mol of water transfer...Continue Reading
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