Open AccessThe effect of factor Va on lipid dynamics in mixed phospholipid vesicles as detected by steady‐state and time‐resolved fluorescence depolarization of diphenylhexatriene
Author(s) -
WARRT Piet,
VISSER Antonie J. W. G.,
HEMKER H. Coenraad,
LINDHOUT Theo
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb11063.x
Subject(s) - diphenylhexatriene , fluorescence anisotropy , vesicle , chemistry , phospholipid , thermotropic crystal , analytical chemistry (journal) , fluorescence , fluorescence spectroscopy , crystallography , chromatography , membrane , phase (matter) , organic chemistry , biochemistry , optics , liquid crystalline , physics
We have monitored the thermotropic behavior of mixed dimyristoylglycerophosphoserine (Myr 2 Gro P Ser)/dimyristoylglycerophosphocholine (Myr 2 Gro P Cho) and Myr 2 Gro P Ser/dipalmitoylglycerophosphocholine (Pam 2 Gro P Cho) vesicles in the presence of blood‐clotting factor Va, using 1,6‐diphenyl‐1,3,5‐hexatriene as a lipid probe. The Ca 2+ ‐independent interaction of factor Va with these vesicles caused a small increase (1–2°C) in the phase transition temperature, regardless of whether Myr 2 Gro P Che was the lower or higher‐melting component of the mixed vesicles. The major effect of factor Va was to increase the polarization of diphenylhexatriene when the mixed vesicles were in the liquid crystalline phase. The protein did not change the anisotropy in the bilayer gel state. The increase in the polarization value above the transition temperature closely correlated with the amount of phospholipid‐bound factor Va, as verified by a direct binding technique. In addition, we found that the affinity of factor Va for Myr 2 Gro P Ser/Myr 2 Gro P Cho and Myr 2 Gro P Ser/Pam 2 Gro P Cho greatly increased at temperatures above the transition temperatures. Time‐dependent fluorescence anisotropy measurements of diphenylhexatriene embedded in vesicles in the liquid crystalline state give fluorescence decay curves which can best be fitted by two exponential functions with two rotational correlation times and a constant term. Vesicles composed of Myr 2 Gro P Ser exhibit more ordering than Myr 2 Gro P Cho vesicles. However, the order parameter of mixed vesicles composed of 40% Myr 2 Gro P Ser and 60% Myr 2 Gro P Cho (mol/mol) approached that of Myr 2 Gro P Cho. Factor Va dramatically increased the longer rotational correlation time of diphenylhexatriene embedded in mixed vesicles in the liquid crystalline state from 3.7 ns to about 17 ns. The second rank‐order parameter increased only slightly, but the calculated steady‐state anisotropy increased by twofold. These results indicate that the acidic phospholipid‐dependent binding of factor Va to mixed vesicles has an ordering effect on the acyl chains of the acidic phospholipids in the outer layer, but leaves the bulk of the phospholipids, mainly phosphatidylcholine, unaltered. None of the factor‐Va‐induced alterations in the anisotropy parameters point to the occurrence of lateral phase separation.