Fluorescence Anisotropy Of Diphenylhexatriene Research Articles

The effects of dietary ( [formula omitted]) fatty acid supplementation on lipid dynamics and composition in rat lymphocytes and liver microsomes

Rats were fed diets devoid of ( n − 3) fatty acids (olive oil supplementation) or high in ( n − 3) fatty acids (fish oil supplementation) for a period of 10 days. In spleen lymphocytes and liver microsomes derived from animals fed fish oil diets, relatively high levels of ( n − 3) eicosapentaenoic (20:5), docosapentaenoic (22:5) and docosahexaenoic acids (22:6) were obtained compared to minimal levels when fed the olive oil diet. When the average lipid motional properties were examined by measuring the fluorescence anisotropy of diphenylhexatriene, no significant different was found between intact liver microsomes from animals fed the two diets. However, when lipid motion was examined in vesicles of phosphatidylcholine, isolated from the microsomes from fish oil fed animals (21.4% ( n − 3) fatty acids), the fluorescence anisotropy was significantly less than the corresponding phosphatidylcholine from olive oil fed animals (5.6% ( n − 3) fatty acids), indicating a more disordered or fluid bilayer in the presence of higher levels of ( n − 3) fatty acids. Phosphatidylethanolamine ( n − 3) fatty acids were also elevated after fish oil supplementation (41.3% of total fatty acids), compared to the level after olive oil supplementation (21.4%). The major effect of the fish oil supplementation was a replacement of ( n − 6) arachidonic acid by the ( n − 3) fatty acids and when this was ‘modeled’, using liposomes of synthetic lipids, 1- palmitoyl-2- arachidonyl(n − 6) or docosahexaenoyl(n − 3)- phosphatidylcholine , significant differences in lipid motional properties were found, with the docosahexaenoate conferring a more disordered or fluid lipid environment. Thus it appears that although lipid order/fluidity can be significantly decreased by increases in the highly unsaturated ( n − 3) fatty acid levels, alterations in membrane domain organization and/or phospholipid molecular species composition effectively compensated for the changes, at least as far as average lipid motional properties in the intact membranes was concerned.

Calcium-dependent and calcium-independent interactions of prothrombin fragment 1 with phosphatidylglycerol/phosphatidylcholine unilamellar vesicles.

We have measured the phase behavior of mixed dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) in the presence of saturating (greater than 98% occupancy of binding sites) concentrations of bovine prothrombin fragment 1 and 5 mM Ca2+. Binding of fragment 1 in the presence of Ca2+ was verified by an increase in 90 degrees light scattering. Only in the cases of DC15PG/DMPC SUV below their phase transition and of pure DMPC SUV were such light scattering measurements not reversible upon addition of ethylenediaminetetraacetic acid to complex Ca2+. Phase-behavior changes of DC15PG/DMPC SUV as monitored by diphenylhexatriene fluorescence anisotropy occurred in concert with the binding of fragment 1. The major effects of peptide binding on SUV phase behavior were to raise the phase-transition temperature by 2-15 degrees C, depending on vesicle composition, and, in general, to make the phase diagram for these small vesicles closely resemble that of large vesicles. No evidence was obtained for the existence of lateral membrane domains with distinct compositions induced by the binding of prothrombin fragment 1 plus Ca2+. Surprisingly, fragment 1 without Ca2+ also altered the phase behavior of DC15PG/DMPC SUV. Most striking was the effect of fragment 1 (with or without Ca2+) on DMPC SUV phase behavior. Freeze-fracture electron microscopy demonstrated that pure DMPC vesicles were induced to fuse in the presence of fragment 1, while vesicles containing DC15PG remained intact. The rate of DMPC SUV fusion (followed by 90 degrees light scattering) increased with increasing fragment 1 concentration but was not saturable up to 40 microM fragment 1, suggesting a weak, nonspecific interaction between fragment 1 and the neutral phospholipid vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and related functional changes in sarcoplasmic reticulum induced by long-chain fatty acids.

The effect of palmitic and oleic acids on Ca2+-ATPase activity in coupled preparations of sarcoplasmic reticulum isolated from rabbit hind leg muscle have been compared with their effects on vesicles uncoupled with Ca2+ ionophore, A23187. Palmitate at 2 microM X mg protein-1 has no significant effect on enzyme activity and does not uncouple catalytic activity from calcium accumulation within the vesicles. Oleic acid at 1 microM X mg protein-1 uncouples the vesicles, whereas 2 microM X mg protein-1 completely inhibits Ca2+-ATPase activity. Fluorescence anisotropy of diphenylhexatriene is not significantly altered by palmitate, but a large transient increase in motion of the probe is observed with addition of oleic acid. The effects of oleic acid on enzyme activity are not mediated via an effect on the bulk properties of the hydrophobic domain of the membrane lipids.

Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry

Time-resolved decays of fluorescence anisotropy were obtained from frequency-domain measurements of the phase angle difference between the parallel and perpendicular components of the polarized emission and the ratio of the modulated amplitudes. These data were measured at modulation frequencies ranging from 1 to 200 MHz. To demonstrate the general applicability of this method, we describe the resolution of both simple and complex decays of anisotropy. In particular, we resolved single, double, and triple exponential decays of anisotropy and the hindered rotational motions of fluorophores within lipid bilayers. The ease and rapidity with which these results were obtained indicate that frequency-domain measurements are both practical and reliable for the determination of complex decays of anisotropy.

Phase behavior of membranes reconstituted from dipentadecanoylphosphatidylcholine and the Mg2+-dependent, Ca2+-stimulated adenosinetriphosphatase of sarcoplasmic reticulum: evidence for a disrupted lipid domain surrounding protein.

A new method was used for reconstituting active sodium deoxycholate solubilized Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Removal of the detergent by dialysis at the pretransition temperature of the pure lipid (22 degrees C) favored the formation of sheet-like structures with a lipid and protein content close to that of the detergent-solubilized sample. Freeze-fracture electron micrographs revealed the Ca2+-ATPase to be organized in rows corresponding to the typical banded pattern seen in low-temperature freeze-fracture micrographs of pure lipid bilayers. Incubation of the sheetlike structures at a temperature (38 degrees C) above the pure lipid main phase transition (33.5 degrees C) caused closure of the sheets into vesicles displaying homogeneous intramembranous particle distributions, at least for membranes containing less than 150 lipids per Ca2+-ATPase. However, in membranes of higher lipid content, free lipid patches were seen both above and below the lipid phase transition. By use of high-sensitivity differential scanning calorimetry, three classes of excess heat capacity peaks were observed in the vesiculated samples. A broadened "free lipid" peak occurred for samples containing between 550 and 200 lipids per protein (Tm = 33.5 degrees C, as for the order-disorder transition in pure lipid vesicles). Between 200 and 150 lipids per Ca2+-ATPase, a broad shoulder became apparent in the range of 29-32 degrees C. Below 150 lipids per Ca2+-ATPase, a peak at 26-28 degrees C became increasingly prominent with lower lipid content. At a lipid to protein ratio of about 30, no peaks in heat capacity were observed. The temperature dependence of diphenylhexatriene fluorescence anisotropy revealed a similar pattern of membrane phase behavior, except that a phase transition was detected at 33.5 degrees C in all membranes studied. On the basis of these observations, we propose that the Ca2+-ATPase is surrounded by a "lipid annulus" of motionally inhibited lipid molecules that do not contribute to a calorimetrically detectable phase transition. Beyond the annulus, "secondary domains" of disrupted lipid packing account for the peak at 26-28 degrees C and the 29-32 degrees C shoulders. At high lipid to protein ratios, the secondary domains coexist with protein-free, lipid-bilayer patches, which account for the peak at 33.5 degrees C.

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca 2+ flux into ciliary membrane vesicles of Paramecium as measured with the Ca 2+ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca 2+ remained unaffected by temperature. The differences in temperature dependency of Ca 2+ conductance and membrane fluidity indicate that the Ca 2+ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3- pyrene) maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.

Tumor promoter [formula omitted] 13-acetate alters state, fluidity and hydration of 1,2-diacyl- sn-glycero-3-phosphocholine bilayers

Previous results (Castagna et al. (1979) FEBS Lett. 100, 62–66; Fisher et al. (1979) Biochem. Biophys. Res. Commun. 86, 1063–1068) indicated us that the active tumor promoter TPA ( 12-O- tetradecanoylphorbol 13-acetate) decreased fluorescence polarisation of diphenylhexatriene in lymphoblastoid and rat embryo cells. In the present study, experiments aimed at examining the molecular interactions of tumor promoters with cell membrane components are performed with fully hydrated multibilayers of 1,2-diacyl- sn-glycero-3-phosphocholine (DPPC) into which increasing amounts of TPA are inserted. The thermotropic behaviour of both the phospholipid bilayers and the interbilayer water was investigated using the differential scanning calorimetry (DSC) and the approach of Ter-Minassian-Saraga et al. ((1982) J. Colloïd Interface Sci. 81, 369–383). The major effects of the tumor promoter are confined to concentrations up to 20% mol fractions of TPA. In this range of concentrations the incorporation of TPA into liposomes decreases the phase-transition temperature but dit not affect ΔH DPPC . Furthermore TPA increases the hydration of the multibilayers. Above 20% mol fractions of TPA, a different thermal behaviour of the system which might suggest morphological rearrangements was observed. The lipid state in TPA-treated liposomes was monitored by fluorescence polarisation using diphenylhexatriene as a lipophilic fluorescent probe and the phase-transition temperature was calculated. The phase transition temperatures determined by both methods were in good agreement. The lowering of this temperature and the decay of fluorescence anisotropy of diphenylhexatriene were parallel. Those effects are consistent with the ‘fluidising’ effect of TPA on DPPC.

Effect of lipid membrane structure on the adenosine 5'-triphosphate hydrolyzing activity of the calcium-stimulated adenosinetriphosphatase of sarcoplasmic reticulum.

An active Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase) isolated from rabbit skeletal muscle sarcoplasmic reticulum membranes has been incorporated into dilauroyl-, dimyristoyl-, dipentadecanoyl-, dipalmitoyl-, and palmitoyloleoylphosphatidylcholine bilayers by using a newly developed lipid-substitution procedure that replaces greater than 99% of the endogenous lipid. Freeze--fracture electron microscopy showed membranous vesicles of homogeneous size with symmetrically disposed fracture-face particles. Diphenylhexatriene fluorescence anisotropy was used to define the recombinant membrane phase behavior and revealed more than one transition in the membranes. Enzymatic analysis indicated that saturated phospholipid acyl chains inhibited both overall ATPase activity and Ca2+-dependent phosphoenzyme formation below the main lipid phase transition temperature (Tm) of the lipid-replaced membranes. At temperatures above Tm, ATPase activity but not phosphoenzyme formation was critically dependent on acyl chain length and thus bilayer thickness. No ATPase activity was observed in dilauroylphosphatidylcholine bilayers. Use of the nonionic detergent dodecyloctaoxyethylene glycol monoether demonstrated that the absence of activity was not due to irreversible inactivation of the enzyme. Increased bilayer thickness resulted in increased levels of activity. An additional 2-fold rise in activity was observed when one of the saturated fatty acids in dipalmitoylphosphatidylcholine was replaced by oleic acid, whose acyl chain has a fully extended length comparable to that of palmitic acid. These results indicate that the Ca2+-ATPase requires for optimal function a "fluid" membrane with a minimal bilayer thickness and containing unsaturated phospholipid acyl chains.

A model for the effect of lipid oxidation on diphenylhexatriene fluorescence in phospholipid vesicles

We have determined by means of a standard spectrophotometric assay that lipid oxidation occurred at a significant rate in large, multilamellar vesicles containing egg phosphatidylcholine under normal experimental conditions. We have also observed that the fluorescence intensity of the vesicle-associated probe, 1,6-diphenyl-1,3,5-hexatriene, decreased with time in vesicles containing such oxidizing lipids. The spectrophotometric data utilized to monitor lipid oxidation were found to fit an apparent first-order kinetic model. The loss of diphenylhexatriene fluorescence intensity in oxidizing liposomes was analyzed in terms of a first-order event superimposed (and thus presumably dependent) upon the ongoing formation of oxidized lipid. These and other data were used to conclude that the oxidation-induced loss of diphenylhexatriene fluorescence intensity was due to chemical modification of the fluorophore rather than to excited-state quenching or ground-state complex formation. Finally, the loss of fluorescence intensity in oxidizable membranes was found to alter drastically the ‘microviscosity’ parameter as derived from diphenylhexatriene fluorescence anisotropy and relative intensity measurements.

Electrostatic interactions at charged lipid membranes Kinetics of the electrostatically triggered phase transition

Charged lipid membranes of dimyristoylmethylphosphatidic acid were mixed rapidly in a stopped-flow cell with protons or Ca 2+ to compensate the charges and thereby trigger the ordered-fluid phase transition. The kinetics of the transition was studied by following the time development of the fluorescence anisotropy of diphenylhexatriene. A relaxation process was observed with a characteristic time in the range 10–200 ms. By comparison with existing theories of non-equilibrium relaxation it was concluded that the relaxation process is governed by a nucleation step.

Lateral pressures in biomembranes estimated from the dynamics of fluorescent probes

A theoretical model is proposed which states that the time-independent fluorescence anisotropy of the rod-shaped molecule diphenylhexatriene incorporated into lipid bilayers is a direct result of forces constraining the diphenylhexatriene molecule. These forces are postulated as equating with the lateral pressure operating within the bilayer independently of the probe molecule. Insertion into the model of experimental observations (recorded in the literature) on anisotropy of diphenylhexatriene in lipid bilayers as a function of temperature yielded values of lateral pressure, which decreased with temperature, and sharply at the temperature defining the transition from gel phase to fluid phase. The values so predicted for the mid-point of the transition and for the entirely fluid phase, respectively, compared favourably with estimates of the lateral pressures in these physical states, that have been reported elsewhere and arrived at either from theories describing lipid chain behaviour or from lipid monolayer compression experiments. Previously documented effects on anisotropy induced by incorporation of cholesterol into fluid lipid bilayers have been interpreted as reflections of rises in intramembranal lateral pressure.

Light-scattering effects in the measurement of membrane microviscosity with diphenylhexatriene

Data from several membrane systems are presented to confirm an empirical means of correcting diphenylhexatriene fluorescence for depolarization caused by sample turbidity. The depolarization proportionally constants obtained are not equal, but are shown to vary with (a) the physical state of the membrane, (b) the cholesterol content of the membrane, (c) the protein content of the membrane, and (d) the method of membrane preparation or isolation. It is concluded that depolarization corrections must always be considered when diphenylhexatriene fluorescence anisotropy is used to compare the fluidities within different membrane bilayers.