Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery

• Published in: Biophysical journal 1994-10, Vol.67 (4), p.1754-1766
• Main Authors: Huang, Z., Pearce, K.H., Thompson, N.L.
• Format: Article
• Language: English
• Subjects: 1,2-Dipalmitoylphosphatidylcholine; Animals; Cattle; Diffusion; Kinetics; Liposomes; Mathematics; Peptide Fragments - chemistry; Phosphatidylcholines; Phosphatidylserines; Photochemistry; Protein Binding; Protein Precursors - chemistry; Prothrombin - chemistry; Spectrometry, Fluorescence - instrumentation; Spectrometry, Fluorescence - methods; Structure-Activity Relationship
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(94)80650-2

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1–15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.