COMPARISON OF THE MEMBRANE-BINDING KINETICS OF BOVINE PROTHROMBIN ANDITS FRAGMENT-1

Total internal reflection fluorescence microscopy has been used to com pare the membrane binding characteristics of fluorescein-labeled bovin e prothrombin and fluorescein-labeled bovine prothrombin fragment 1. T he Ca2+-dependent association of these proteins with quartz-supported planar membranes composed of mixtures of phosphatidylserine (2-10 mol %) and phosphatidylcholine was examined. Equilibrium binding measureme nts showed that the apparent equilibrium dissociation constants increa sed with decreasing molar fractions of phosphatidylserine and that the dissociation constants were somewhat lower for intact prothrombin. Ki netic measurements, using fluorescence photobleaching recovery, showed that the measured dissociation rates were approximately equivalent fo r prothrombin and fragment 1 and did not change with the protein solut ion concentration or the molar fraction of phosphatidylserine. The kin etic data also implied that the surface binding mechanism for both pro teins is more complex than a simple reversible reaction between monova lent proteins and monovalent surface sites. Measured equilibrium and k inetic constants are reported and compared for prothrombin and fragmen t 1 on planar membranes.