Prothrombin Association with Phospholipid Monolayers

Surface pressure measurements demonstrated that prothrombin and its fragment 1 region caused minor perturbations of lipid structure upon binding to phospholipid monolayers. These perturbations did not appear to be due to insertion of the protein into the monolayer film. The actual amount of protein bound to the monolayer was measured by recovering the monolayer and the ³H-labeled fragment 1 associated with it. Binding was strictly dependent on calcium and acidic phospholipid. Titration of the protein-monolayer interaction indicated a single class of binding sites with a K_D of 0.15 µM for the interaction of fragment 1 with 20% phosphatidylserine (PS)-80% phosphatidylcholine (PC) monolayers at an initial surface pressure of 21 dyn/cm. A small increase in binding affinity was observed at higher percentages of PS. The fragment 1 binding capacity of PS-PC monolayers was proportional to the PS content between 0 and 20% PS with a stoichiometry of 7.8 ± 0.9 PS residues per bound protein molecule. Above 20% PS, the direct proportionality was no longer observed, indicating that some theoretical binding sites were being sterically excluded. The surface concentration of fragment 1 molecules bound to 100% PS monolayers at saturating protein corresponded to one bound protein per 1077 Ų. This area compared well with 1100 Ų per molecule calculated from surface pressure changes. One protein molecule, therefore, occupied an area greater than that of the required PS residues. At high surface pressures, the protein binding capacity of the monolayers remained relatively unchanged, but the surface pressure changes decreased and, in some cases, became zero. Binding affinity also decreased at higher surface pressures. Comparison of prothrombin binding to monolayers with its binding to bilayer vesicles revealed few, if any, significant differences. Thus, the radius of curvature does not appear to be an important determinant in this protein-lipid interaction, and the two systems appear complementary in the analysis of this association.