Binding of bovine factor V-a to phosphatidylcholine membranes

The interaction of bovine factor V-a with phosphatidylcholine membranes was examined using four different fluorescence techniques: 1) changes in the fluorescence anisotropy of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to monitor the interaction of factor V-a with 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), 2) changes in the fluorescence anisotropy of N-(lissamine rhodamine B sulfonyl) diacyl phosphatidylethanolamine (Rh-PE) incorporated into SUVs prepared from 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), 3) changes in the fluorescence anisotropy of fluorescein-labeled factor V-a (labeled in the heavy chain) upon interaction with POPC SUVs, 4) fluorescence energy transfer from fluorescein-labeled factor V-a to rhodamine-labeled POPC SUVs. In the first two sets of experiments, labeled lipid vesicles were titrated with unlabeled protein, whereas, in the latter two types of experiments, labeled factor V-a was titrated with vesicles, For the weak binding observed here, it was impossible from any one binding experiment to obtain precise estimates of the three parameters involved in modeling the lipid-protein interaction, namely, the dissociation constant K-d, the stoichiometry of binding i, and the saturation value of the observable R(max) from any one experiment, However, a global analysis of the four data sets involving POPC SUVs yielded a stable estimate of the binding parameters (K-d of similar to 3.0 mu M and a stoichiometry of similar to 200 lipids per bound factor V-a). Binding to DMPC SUVs may be of slightly higher affinity. These observations support the contention that association of factor V-a with a membrane involves a significant acidic-lipid-independent interaction along with the more commonly accepted acidic-lipid-dependent component of the total binding free energy.