Binding of bovine factor Va to phosphatidylcholine membranes.
The interaction of bovine factor Va 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 Va 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 phosphati-dylethanolamine (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 Va (labeled in the heavy chain) upon interaction with POPC SUVs, 4) fluorescence energy transfer from fluorescein-labeled factor Va 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 Va 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 Kd, the stoichiometry of binding i, and the saturation value of the observable Rmax from any one experiment. However, a global analysis of the four data sets involving POPC SUVs yielded a stable estimate of the binding parameters (Kd of approximately 3.0 microM and a stoichiometry of approximately 200 lipids per bound factor Va). Binding to DMPC SUVs may be of slightly higher affinity. These observations support the contention that association of factor Va 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.
Koppaka
V
V
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill 27599, USA.
Lentz
B R
BR
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D004735:Energy Transfer; D015943:Factor Va / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism*; D008567:Membranes, Artificial; D010713:Phosphatidylcholines / Q000378:metabolism*
D005456:Fluorescent Dyes; D008081:Liposomes; D008563:Membrane Lipids; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D015943:Factor Va