Collect. Czech. Chem. Commun. 2002, 67, 1872-1882

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

Angelika Häfnera, Martin Benešb, Fabienne Mérolac, Guy Duportaild, Friedemann W. Schneidera and Martin Hofb,*

a Institut für Physikalische Chemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
b J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, and Center for Complex Molecular Systems and Biomolecules, 182 23 Prague 8, Czech Republic
c Laboratoire de Chimie Physique, UMR CNRS 8000, Bat. 349, Université de Paris Sud, 91405 Orsay Cedex, France
d Laboratoire de Pharmacologie et Physicochimie, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur, B.P. 24, 67401 Illkirch Cedex, France


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.

Keywords: Protein fluorescence; Factor Xa; Gla domain; Blood coagulation; Lipid-specificity; Prothrombinase; EGF-like domain; Synchrotron radiation; Enzymes.

References: 19 live references.