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 Ca(2+)-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 microM) similar to that of unlabeled protein (1.1 +/- 0.1 microM). 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 microM) 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 microM. 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 microM gave average association and dissociation kinetic rates of approximately 10(5) M-1 s-1 and approximately 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.
Pearce
K H
KH
Department of Chemistry, University of North Carolina, Chapel Hill 27599.
Hiskey
R G
RG
Thompson
N L
NL
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008081:Liposomes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D012542:Scattering, Radiation; D012996:Solutions
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011498:Protein Precursors; D012996:Solutions; C029733:prothrombin fragment 1; D011516:Prothrombin; D005990:Glycerol; D002118:Calcium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D019793:Fluorescein