Abstract
Total internal reflection fluorescence microscopy has been used to compare the membrane binding characteristics of fluorescein-labeled bovine prothrombin and fluorescein-labeled bovine prothrombin fragment 1. The Ca(2+)-dependent association of these proteins with quartz-supported planar membranes composed of mixtures of phosphatidylserine (2-10 mol%) and phosphatidylcholine was examined. Equilibrium binding measurements showed that the apparent equilibrium dissociation constants increased with decreasing molar fractions of phosphatidylserine and that the dissociation constants were somewhat lower for intact prothrombin. Kinetic measurements, using fluorescence photobleaching recovery, showed that the measured dissociation rates were approximately equivalent for prothrombin and fragment 1 and did not change with the protein solution concentration or the molar fraction of phosphatidylserine. The kinetic data also implied that the surface binding mechanism for both proteins is more complex than a simple reversible reaction between monovalent proteins and monovalent surface sites. Measured equilibrium and kinetic constants are reported and compared for prothrombin and fragment 1 on planar membranes.
Highlights
Prothrombin and fragment 1 and did not change with One method for obtaining quantitative information about the protein solution concentration or the molar fraction the physical dynamics of proteins at membrane surfaces is to of phosphatidylserine
Thesestudies showed that the proteins bound to the planar membranes in a Ca2+-specificmanner and that theequilibrium dissociation constants were approximately equivalent to those measured on phospholipid vesicles
Equilibrium Binding of ProthrombinandFragment 1 to Planar Membranes-TIRFM was used to measure the surface-associated fluorescence on PS/POPC planar membranes that had been treated with solutions of fluorescein-labeled prothrombin or fluorescein-labeled fragment 1
Summary
Proteins-Prothrombin was obtained from bovine plasma and fragment 1 was produced and purified as previously described (1921). Both proteinswere greater than 95% pure as determined by high performance liquid chromatography on Mono Q (prothrombin) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (prothrombin and fragment 1).Protein concentrations were determined by ultraviolet spectrophotometry at 280 nm (prothrombin, c = 1.44 ml mg"cm", molecular mass = 72 kDa; fragment 1, c = 1.05 ml mg" cm", molecular mass = 23 kDa). The laser beam was directed through two lenses to reduce the beam diameter; the focal lengths were223 and 25mm andthe lenses were separated by approximately 248mm This arrangement produced an elliptically shaped evanescent wave with minor and major axes approximately equal to 15 and 120pm, respectively. The chi-squared values for the best fits to different functional forms were compared with an F-statistic [24]
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