PROTEIN HYDRATION DURING GENERATION OF COAGULATION-FACTOR XA IN AQUEOUS-PHASE AND ON PHOSPHOLIPID-MEMBRANES

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 osmoti c 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 ph ospholipid membranes. Osmotic stress was induced on the surface of rea cting proteins with either polyethylene glycol, or dextran of 6000 and 500,000 molecular weight, respectively. These inert polymers are ster ically excluded from the the solvation shells of proteins and thus inc rease the water activity in the excluded spaces. The volume of water t ransferred either to or from the excluded spaces during formation of r eaction intermediates was calculated from the ratio of change in free energy of activation with change in osmotic pressure, Delta G/Delta I I. For aqueous phase-assembled reactions, Delta G values decreased wi th Delta II at ratios of -2.36 +/- 0.38 and -2.26 +/- 0.26 kcal/ mol/a tm for polyethylene glycol and dextran, respectively. These values cor respond to 5488 +/- 883 and 5255 +/- 604 mol of water transferred from the reacting protein surfaces per mol of FXa generated. At a physiolo gic osmotic pressure of 7 atm the work of transfer corresponded to 16 kcal/mol, approximately 70% of Delta G. The observed osmotic effects were independent of the viscosity, temperature, and ionic strength of solutions. For reactions assembled on phospholipid membranes, Delta G increased with hn at a ratio of 0.35 +/- 0.05 kcal/mol/atm, correspon ding to 814 +/- 116 mol of water tansferred from bulk solution to prot ein surfaces, At physiologic osmotic pressure the work of transfer is 2.45 kcal/mol, approximately 12% of Delta G. Results indicate that fo r factor Xa generation in aqueous phase the work of desolvation is a s ignificant component of the free energy of activation. Results also su ggest that phospholipid membranes catalyze the reaction by reducing th e desolvation component of the free energy of activation.