EVIDENCE FROM TOTAL INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY FOR CALCIUM-INDEPENDENT BINDING OF PROTHROMBIN TO NEGATIVELY CHARGED PLANAR PHOSPHOLIPID-MEMBRANES

Measurements to test for a proposed Ca2+-independent interaction of prothrombin with membranes containing acidic phospholipids are described. Fluorescein-labeled bovine prothrombin and its amino- and carboxy-terminal peptides, prothrombin fragment 1 and prethrombin 1, were added at various concentrations in the presence or absence of Ca2+ to the aqueous space bathing substrate-supported planar membranes composed of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), POPC/bovine brain phosphatidylserine (bovPS) (70:30 mol/mol), or POPC/ 1.2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) (70:30 mol/mol). Total internal reflection fluorescence microscopy (TIRFM) at the membrane-solution interface showed a significant enhancement by acidic lipids of prothrombin and prothrombin fragment 1 binding in the presence of 5 mM Ca2+, with apparent dissociation constants of 0.4 and 1-mu-M, respectively. TIRFM measurements indicated that bovPS and DOPG also significantly enhanced the binding of fluorescein-labeled prothrombin to the planar membranes in the absence of Ca2+, with apparent dissociation constants (13-30-mu-M) at least an order of magnitude larger than the Ca2+-dependent constant for prothrombin binding. Association of prethrombin 1 but not prothrombin fragment 1 with membranes in the absence of Ca2+ was enhanced by the presence of bovPS in the membranes, which suggests that the Ca2+-independent binding site(s) is (are) in the prethrombin 1 but not the fragment 1 portion of prothrombin.