Adsorption of bovine prothrombin to spread phospholipid monolayers.
The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.
Ellison
E H
EH
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Castellino
F J
FJ
D000327:Adsorption; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium; D002417:Cattle; D066298:In Vitro Techniques; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids / Q000737:chemistry*; D011312:Pressure; D011516:Prothrombin / Q000737:chemistry*; D013499:Surface Properties
D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; D011516:Prothrombin; D002118:Calcium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine