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 interfa ce. Fluorescence spectroscopy was implemented to determine the equilib rium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The incre ase 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 phosphatidyl serine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/P S monolayers, dc(s)/d pi was independent of the initial surface pressu re (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 pr othrombin 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 monola yers of similar phospholipid composition. This approach, employing spr ead monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentrat ion in protein-monolayer suspensions, offers significant advantages fo r the investigation of protein-membrane interaction. The equilibrium c haracteristics that describe the interaction of prothrombin with the d ifferent phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interac tions are involved in the adsorption of vitamin K-dependent coagulatio n and anticoagulation proteins to model membrane systems.