SURFACE BINDING-KINETICS OF PROTHROMBIN FRAGMENT-1 ON PLANAR MEMBRANES MEASURED BY TOTAL INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY

Total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate the Ca2+-dependent membrane-binding characteristics of fluorescein-labeled bovine prothrombin fragment 1 (F-BF1). Light scattering measurements demonstrated that F-BF1 bound to small unilamellar phosphatidylserine/phosphatidylcholine (25/75, mol/mol) vesicles with an apparent dissociation constant (1.5 +/- 0.2-mu-M) similar to that of unlabeled protein (1.1 +/- 0.1-mu-M). Negatively charged supported planar membranes were constructed by fusing small unilamellar vesicles at quartz surfaces. TIRFM measurements under equilibrium conditions showed that F-BF1 bound to planar membranes with an apparent dissociation constant (0.9 +/- 0.2-mu-M) approximately equal to that on vesicles. Total internal reflection/fluorescence photobleaching recovery (TIR/FPR) curves for F-BF1 on 25 mol % PS planar surfaces were diffusion-influenced at F-BF1 solution concentrations less-than-or-equal-to 5-mu-M. Fluorescence recovery rates from samples of high F-BF1 concentrations were slowed by increasing the solution viscosity with glycerol, thus providing further support for a diffusion-limited effect at low F-BF1 concentrations. Analysis of the reaction-limited fluorescence recovery curves at F-BF1 solution concentrations greater-than-or-equal-to 10-mu-M gave average association and dissociation kinetic rates of almost-equal-to 10(5) M-1 s-1 and almost-equal-to 0.1 s-1, respectively. Kinetic association rates increased significantly with increasing PS, whereas kinetic dissociation rates increased only slightly. Fluorescence recovery curves were nonmonoexponential; possible mechanisms for this behavior are described.