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

Previous work has shown that bovine prothrombin fragment 1 binds to su bstrate-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 membra ne residency time is approximate to 0.25 s(-1). In the present work, f luorescence pattern photobleaching recovery with evanescent interferen ce patterns (TIR-FPPR) has been used to measure the translational diff usion coefficients of the weakly bound fragment 1. The results show th at 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 ca rried out for fragment 1 on solid-like PS/PC planar membranes. The dis sociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also use d to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diff usion coefficient was approximate to 10(-8) cm(2)/s, which is consiste nt with that measured by conventional fluorescence pattern photobleach ing recovery. This work represents the first measurement of a translat ional diffusion coefficient for a protein weakly bound to a membrane s urface.