Fluorescence lifetime and time-resolved polarization anisotropy studies of acyl chain order and dynamics in lipid bilayers

Abstract

The time-resolved fluorescence intensity and anisotropy decays of cis- and trans-parinaric acids and phosphatidylcholines labeled with trans-parinaric acid have been characterized in bilayers formed by several phosphatidylcholines and by dipalmitoylphosphatidylcholine-cholesterol mixtures, at several temperatures. Both a conventional free-running nitrogen flashlamp and the novel synchrotron source at the Stanford Linear Accelerator Center (SLAC) were used as excitation sources for a modified single photon counting fluorescence lifetime apparatus. The measured emission decay kinetics of both isomers of parinaric acid were biexponential in all but one of the lipid systems examined. The fluorescence anisotropy of parinaric acid was large and constant in gel phase lipids, but showed a very rapid (approximately 2 GHz) decay of large amplitude in fluid lipids. In all lipid systems studied, the fluorescence anisotropy decayed to a nonzero asymptote, in striking contrast to the behavior observed in viscous solvent solutions. The asymptotic anisotropy was used to calculate an "order parameter" of the emission transition dipole. The value of the order parameter is quite close to that obtained by deuterium NMR. Cholesterol increased the order parameter measured in fluid dipalmitoylphosphatidylcholine but did not substantially affect the rate of angular relaxation. Experiments conducted with trans-parinaroylphosphatidylcholines yielded results virtually identical with those obtained with trans-parinaric acid.

Calcium Modulates Fatty Acid Dynamics in Rat Liver Plasma Membranes

“…The steady-state anisotropy, rs, is equivalent to [2p/(3 -p)] where p is the polarization. However, it should be noted that the steady-state anisotropy which is calculated according to Eqn ( 5 ) actually depends on both the rate and range of wobbling motion of the probe molecule, as well as on the fluorescence lifetime [41,43]. The steadystate anisotropy, rs, can be expressed as the sum of a kinetic term r b ( t ) and a static term, r W .…”

Binding of phospholipids to the phosphatidylinositol transfer protein from bovine brain as studied by steady-state and time-resolved fluorescence spectroscopy

“…We have followed up on this hypothesis by investigating the binding of parinaroyl-PI and parinaroyl-PC by use of time-resolved fluores-cence spectroscopy. This technique allows one to compare fluorescence lifeitmes, which are very sensitive to changes in environment [12], as well as fluorescence anisotropy decays which are a measure of acyl chain motion [13]. In a similar study on the binding of 1-parinaroyl-PC and 2-parinaroyl-PC to the PC-transfer protein from bovine liver it was demonstrated that each acyl chain was completely immobilized on the protein thereby occupying different binding sites [14].…”

A comparative fluorescence polarization study of cis-parinaroyl-phosphatidylcholine and diphenylhexatriene in membranes containing different amounts of cholesterol

“…The thickness of the DPH molecule approximates that of an acyl chain and its ordering in a membrane is presumed to reflect the order of the acyl chains [17], be it that the precise localization of the probe is unknown. The parinaroyl chromophore is relatively rigid (carbon atoms 9-16) and its movement will depend not only upon the movement and order of the surrounding acyl chains but also on depolarizing motions of the acyl chain in which it is present [16]. Cholesterol increases mainly the order of the first 8-10 carbon atoms of the phospholipid acyl chains [411.…”

“…As a first approach the measurement of the steady-state fluorescence anisotropy (rs) was carried out. It has been shown that in case of DPH [12][13][14][15], parinaric acid and PnPC [16] anisotropy values are determined by two components: The residual anisotropy (too) which is related to the order parameter of the probe and a dynamic term (rf) which depends upon the apparent rotational correlation time and the fluorescence life time of the probe [17]. The relation between these factors has been discussed in detail by van Blitterswijk et al [18].…”

“…Many eukaryotic membranes contain cholesterol which strongly modifies the physical behaviour of the membrane lipids. Time-resolved measurements have revealed for both DPH and parinaric acid that r~ is the term mainly affected by cholesterol in fluid phase lipids [16,18]. With this information in mind we have compared the rs-values for PnPC and DPH in PC-containing liposomes in the presence and absence of cholesterol.…”