TRANSLATIONAL DIFFUSION OF BOVINE PROTHROMBIN FRAGMENT 1 WEAKLY-BOUND TO SUPPORTED PLANAR MEMBRANES - MEASUREMENT BY TOTAL INTERNAL-REFLECTION WITH FLUORESCENCE PATTERN PHOTOBLEACHING RECOVERY

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca2+-specific manner. The apparent equilibrium dissociation constant is 1-15 mu M, and the average membrane residency time is approximate to 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) cm(2)/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 approximate to 10(-8) cm(2)/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.