The goal of these studies is to understand the relationship which exists between the structure of the phospholipid bilayer and the structure of prothrombin in the presence of calcium ions which results in the catalytically important prothrombin; lipid interaction. The structural homology which exists between the vitamin K-dependent blood clotting factor generalizes the importance of such investigations to our understanding of a number of crucial zymogen activations which are phospholipid surface-catalyzed during clot formation. The experimental studies involve correlation of criteria of phospholipid binding by prothrombin with measurements of the physical state of the phospholipid surface. Particular attention will be paid to characteristics of phospholipid surfaces which bear a net zero electric charge since only in such systems can the effects of metal ions on the structure of prothrombin be readily separated from otherwise potentially important metal ion lipid surfce interactions. Preliminary studies carried out by the applicant demonstrate the feasibility of such an approach. Equilibrium cnstants characterizing the prothrombin: phospholipid surface interaction will be determined as a function of changing mole fraction of membrane component lipids. The temperature dependence of such interactions will be evaluated. Since the model proposed for prothrombin: lipid binding involves interactions of the protein with defect structures located at laterally phase separated regions of the lipid bilayer, and since such regions contain high quache to trans isomer ratios in the lipid polymethylene chains, the expected influence of prothrombin lipid binding on the quache/trans ratio will be examined via laser Raman spectroscopy. Phospholipid phase behavior in the presence and absence of protein and metal ions will be characterized via differential scanning calorimetry. A small observed increase in protein intrinsic fluorescence which accompanies prothrombin fragment: lipid interactions will be examined since it may indicate a lipid-induced conformational change in the protein in order to optimize hydrophobic interactions.
