Time-Resolved Tryptophan Fluorescence of Fragment 1-86 of Factor X and the Influence of Membrane Binding

Abstract

Membrane binding of the N-terminus of the prothrombinase reaction enzyme, fragment 1-86 of factor X (FX F1-86), to phospholipid vesicles was studied using time-resolved fluorescence spectroscopy of the tryptophan residue Trp41 which is located in the membrane binding part of this protein (Gla domain). Fluorescence lifetimes were determined by the time-correlated single photon counting technique using synchrotron radiation as the excitation source. Different negatively charged lipid vesicles containing either phosphatidylserine or phosphatidylglycerol mixed with phosphatidylcholine were investigated. Pure synthetic lipids (dilauroyl and dioleoyl lipids) as well as natural lipids (from egg yolk or bovine brain) were used in membrane binding studies. Multiexponential data analysis supports the results already found for FX F1-86 in the presence of calcium ions. For all lipid systems we obtained decay times in the range of 0.2, 0.6, 2.6, 5.6 ns. Moreover, a long-lifetime component with minor contribution to the decay amplitude (<5%) was found. The fluorescence lifetimes do not show any wavelength dependence. There was no change in the fluorescence decay times upon membrane binding, which might indicate that a lipid-specific conformational change in the Gla domain of factor X does not take place upon membrane binding.