BINDING OF BOVINE FACTOR V-A TO PHOSPHATIDYLCHOLINE MEMBRANES

The interaction of bovine factor V-a with phosphatidylcholine membrane s was examined using four different fluorescence techniques: 1) change s 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 unilame llar 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-phosp hatidylcholine (POPC), 3) changes in the fluorescence anisotropy of fl uorescein-labeled factor V-a (labeled in the heavy chain) upon interac tion with POPC SUVs, 4) fluorescence energy transfer from fluorescein- labeled factor V-a to rhodamine-labeled POPC SUVs. In the first two se ts of experiments, labeled lipid vesicles were titrated with unlabeled protein, whereas, in the latter two types of experiments, labeled fac tor 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-prote in interaction, namely, the dissociation constant K-d, the stoichiomet ry of binding i, and the saturation value of the observable R(max) fro m any one experiment, However, a global analysis of the four data sets involving POPC SUVs yielded a stable estimate of the binding paramete rs (K-d of similar to 3.0 mu M and a stoichiometry of similar to 200 l ipids per bound factor V-a). Binding to DMPC SUVs may be of slightly h igher affinity. These observations support the contention that associa tion 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.