Oligonucleotides with bis-pyrene adduct in the backbone: syntheses and properties of intramolecular excimer forming probe.

2-(N-bis(2-pyrenylethyl)methyl-amino)ethanol (BPAE) was synthesized and its interaction with DNA was examined to explore the efficiency of intramolecular excimer forming probe. As a result the fluorescence of intramolecular excimer of BPAE disappeared with the addition of poly(dA)-poly(dT). This result suggests an unique type of probe with its introduction to oligonucleotide.     

Fluidity of bacterial membrane lipids monitored by intramolecular excimerization of 1.3-di(2-pyrenyl)propane

Intramolecular excimer formation of 1,3-di(2-pyrenyl)propane was used to study the fluidity of liposomes prepared from membrane polar lipids of Bacillus stearothermophilus. On the basis of spectral data, local polarity and polarizability parameters were established suggesting that the probe molecules are located well inside the membranes, but displaced towards the polar head groups of the phospholipid molecules. The excimerization rate is very sensitive to lipid phase transitions and pretransitions of synthetic pure lipid bilayers. In bacterial lipids from cultures grown at 55 and 68 degrees C, thermal profiles of excimer to monomer intensity ratios (I'/I) show a broad transition which is displaced to higher temperatures in response to the increase of the growth temperature; these results correlate well with differential scanning calorimetry data and fluorescence polarization of diphenylhexatriene. Additionally, lipid bilayers of bacteria grown at 68 degrees C exhibit a decreased membrane fluidity, as monitored by both fluorescent probes.     

Use of fluorescent probes that form intramolecular excimers to monitor structural changes in model and biological membranes.

1,3-dipyrenylpropane (PC3P) and bis(4-biphenylmethyl)ether, two molecules that form intramolecular excimers, were embedded in phospholipid vesicles and biological membranes to monitor dynamic properties of membrane lipids. Excimer formation was evaluated from determinations of excimer to monomer emission intensity ratios (ID/IM). ID/IM values of PC3P and bis(4-biphenylmethyl)ether were reduced when cholesterol was added to egg lecithin vesicles. PC3P was sensitive to the temperature-induced crystalline to liquid-crystalline phase transition in dimyristoyl phosphatidylcholine vesicles. For studies of cellular membranes, membranes, PC3P was used exclusively, because of the fluorescence of tryptophan residues of membrane proteins interferes with the responses bis(4-biphenylmethyl)ether. Microviscosities of membrane interiors were calculated from standard curves of IM/ID plotted against solvent viscosity. Microviscosity values of egg lecithin vesicles and biological membranes, especially those obtained with PC3P, were more than an order of magnitude lower than values obtained by other techniques. We concluded that the intramolecular process leading to the formation of the excimer is influenced differently in isotropic solvents than in anisotropic environments, such as lipid bilayers. Although distinguishable ID/IM ratios can be obtained for different biological membranes (mitochondrial, microsomal, and plasma membranes were studied), this parameter may be phenomenological and not simply related to membrane microviscosity. As such, fluorescent probes that form intramolecular excimers are of value in making qualitative comparisons of different membranes and in studying the relative effects of physical changes and chemical agents on membrane structure. These probes may also be valuable for studying structural anisotropy of biological membranes.     

Zeaxanthin ([3R,3'R]-beta, beta-carotene-3-3'diol) as a resonance Raman and visible absorption probe of membrane structure.

When zeaxanthin ([3R,3R']-beta, beta-carotene-3,3'diol) is inserted into phospholipid dispersions and the latter heated through their gel-liquid crystal phase transitions, large changes are noted in the resonance Raman and absorption spectra of the carotenoid molecule. By analogy with the data of Carey and co-workers (J. Raman Spectrosc. 6:282) who studied the aggregation of zeaxanthin in acetone-water solutions, it is suggested that the carotenoid aggregates in the phospholipid gel state while forming a monomer in liquid crystal phases. The alterations in both the visible absorption and resonance Raman data have been used to monitor phospholipid phase behavior in dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, (DSPC) one-component systems and binary mixtures. The phase diagram obtained for the binary system, as constructed from visible absorption and resonance Raman data, is compared with that of Shimshick and McConnell (Biochemistry. 12:2351) obtained from electron spin resonance (ESR) studies. Although the agreement between absorption and ESR data is generally satisfactory, onset temperatures for phase separation at low DSPC mole fractions deduced from resonance Raman measurements are several degrees lower than those from the other methods. Nevertheless, the use of zeaxanthin as a resonance Raman and visible absorption probe behavior will be useful in some situations where ordinary Raman spectroscopic data cannot be obtained easily. The advantage of the resonance Raman approach is illustrated in a study of the phase behavior of a phospholipid extract of a cel- mutant of Neurospora crassa. A phase separation region is observed with onset and completion temperatures of -19 and -6 degrees C, respectively.     

Design, synthesis, and in vitro evaluation of new naphthylnitrobutadienes with potential antiproliferative activity: toward a structure/activity correlation

On the grounds of previous encouraging results on the antitumor activity of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1), we have designed and synthesized two new molecules [(1E,3E)-1,4-bis(4-carboxy-1-naphthyl)-2,3-dinitro-1,3-butadiene (2) and methyl (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (3)] characterized by a common naphthylnitrobutadiene array but with different structural properties, with the aim of approaching to some structure-activity correlation. When 2 and 3 were analyzed in vitro for their inhibition of cell proliferation and pro-apoptotic properties, the carboxyderivative 2 did not furnish appreciable results. In contrast, 3 (which contains only one of the two naphthylnitroethenyl moieties of the original compound 1) showed remarkable activities in the range of micromolar concentrations (in six over eight cell lines its IC(50)s are in the 1-3 microM range), with a significant improvement compared to 1. In particular, 3 proved able to bind to DNA, to upregulate p53, to block cells in the G2/M phase of their cycle, and to induce apoptosis. Thus, very interestingly, the performance of 3 with respect to 1 shows that a single 1-(1-naphthyl)-2-nitroethene moiety is able to ensure better (on four out of eight of the cell lines tested) or comparable levels of activity. This result suggests that the 'molecular-simplification strategy' could furnish a useful instrument for future design in our antitumor research.     

Evidence for the formation of microdomains in liquid crystalline large unilamellar vesicles caused by hydrophobic mismatch of the constituent phospholipids.

The excimer-to-monomer fluorescence emission intensity ratio (IE/IM) of the fluorescent probe 1-palmitoyl-2-[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC, 1 mol%) was measured at 30 degrees C as a function of the thickness of fluid liposomal membranes composed of phosphatidylcholines (PCs) with homologous monounsaturated acyl chains of varying lengths N (= number of carbon atoms). Upon decreasing N from di-24:1 PC to di-14:1 PC, the rate of excimer formation was sigmoidally augmented from 0.02 to 0.06. This increase in IE/IM can arise either from enhanced lateral mobility or from the lateral enrichment of PPDPC into domains, or both. Direct evidence for partial lateral segregation of PPDPC being involved is provided by experiments where 1.6 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N- (5-fluoresceinthiocarbamoyl) (DPPF) was included together with PPDPC into the bilayers. Notably, because of spectral overlap DPPF can function as a resonance energy transfer acceptor for pyrene excimer. Fluorescence intensity ratio (F/Fo) measured at 480 nm for PPDPC/DPPF (yielding F) and PPDPC (yielding Fo) containing membranes as a function of N reveals a sharp maximum for di-20:1 PC, i.e., the quenching of pyrene excimer fluorescence by DPPF is least efficient in this lipid and is enhanced upon either decrease or increase in N. This is compatible with colocalization of DPPF in PPDPC enriched domains when N not equal to 20, whereas in di-20:1 PC these probes appear to be effectively dispersed. The driving force for the enrichment of PPDPC in thin (N < 20) and thick (N > 20) PC matrices is likely to be hydrophobic mismatch of the effective ¿lengths of the matrix phospholipids and the fluorescent probes. We also measured fluorescence polarization (P) for 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as the IE/IM for the intramolecular excimer forming probe 1,2-bis[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a function of N. In brief, neither the fluorescence polarization data and nor the measurements of intramolecular chain dynamics using bisPDPC concur with enhanced lateral diffusion as the sole cause for the increase in the IE/IM for PPDPC in thin membranes. Our findings suggest hydrophobic mismatch as the cause of microdomain formation of lipids in fluid, liquid crystalline bilayers, while simultaneously allowing for a high rates of lateral diffusion. Such hydrophobic mismatch-induced compositional fluctuations would also offer one plausible explanation for the chain length diversity observed for biological membranes.     

Dipyrenylphosphatidylcholines as membrane fluidity probes. Relationship between intramolecular and intermolecular excimer formation rates.

In the intramolecular excimeric membrane probe, dipyrenylphosphatidylcholine (dipyn PC), pyrene moieties are linked to the terminal carbons of the two acyl chains, each of which contains n carbons. We show here how the probe intramolecular excimer production rate, K, may be determined from the excimer/monomer intensity ratio, rl, by making use of the fluorescence titrations of the related monopyrenyl probe, pyn PC, analyzed according to the milling crowd model. rl and the rate K of dipy10 PC in four model membrane systems were measured over a wide temperature range and both parameters are shown to be sensitive functions of the lateral fluidity of the host matrix. A model for relating the intramolecular and intermolecular excimer formation rates is proposed according to which both processes are limited by the reorientational rate of the pyrene moiety. Above the fluid-gel transition temperature, Tc, the diffusion rate (f) of the monopyrenyl probe (pyn PC) is accordingly related to K by: pE approximately K/(K + 1/2f + tau -1M), where pE is the probability of excimer formation between nearest neighbor pyn PC probes, and tau M is the monomer lifetime. Values of pE derived in this way are found to be consistent with pE values derived from the milling crowd analysis of fluorescence yield titration experiments. K for dipy10 PC in DMPC multibilayers ranges from 0.21 x 10(7) s-1 at 10 degrees C in the gel phase, to 5.7 x 10(7) s-1 at 60 degrees C in the fluid phase, whereas the lateral diffusion coefficient, D, for py10 PC in the same bilayers ranged from 8 to 34 microns2 s-1, when calculated with D = fL2/4, L being the average lipid-lipid spacing of the host membrane. Above Tc and at the same reduced temperature, (T - Tc)/Tc, both f for py10 PC, and K for dipy10 PC were found to have relative magnitudes in the order: DPPC greater than DMPC greater than POPC greater than DOPC. This and the similarity of the activation energies for f and K suggest that the rotation of the the pyrene moiety is the rate-limiting step for both the lateral mobility of py10 PC and intramolecular excimer formation in dipy10 PC.     

Lipid-triggered conformational switch of apolipophorin III helix bundle to an extended helix organization

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic alpha-helices (H1-H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C-apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C-apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene "excimer" fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene-A8C-apoLp-III/DMPC or pyrene-A138C-apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene-A8C/A138C-apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.     

Evidence for the extended phospholipid conformation in membrane fusion and hemifusion

Molecular-level mechanisms of fusion and hemifusion of large unilamellar dioleoyl phosphatidic acid/phosphocholine (DOPA/DOPC, 1:1 molar ratio) vesicles induced by millimolar Ca2+ and Mg2+, respectively, were investigated using fluorescence spectroscopy. In keeping with reduction of membrane free volume Vf, both divalent cations increased the emission polarization for 1,6-diphenyl-1,3, 5-hexatriene (DPH). An important finding was a decrease in excimer/monomer emission intensity ratio (Ie/Im) for the intramolecular excimer-forming probe 1, 2-bis[(pyren-1-)yl]decanoyl-sn-glycero-3-phosphocholine (bis-PDPC) in the course of fusion and hemifusion. Comparison with another intramolecular excimer-forming probe, namely, 1-[(pyren-1)-yl]decanoyl-2-[(pyren-1)-yl]tetradecanoyl-sn-gl ycero-3-p hosphocholine (PDPTPC), allowed us to exclude changes in acyl chain alignment to be causing the decrement in Ie/Im. As a decrease in Vf should increase Ie/Im for bis-PDPC and because contact site between adhering liposomes was required we conclude the most feasible explanation to be the adoption of the extended conformation (P.K.J., Chem. Phys. Lipids 63:251-258) by bis-PDPC. In this conformation the two acyl chains are splaying so as to become embedded in the opposing leaflets of the two adhered bilayers, with the headgroup remaining between the adjacent surfaces. Our data provide evidence for a novel mechanism of fusion of the lipid bilayers.     

Effect of poly(ethylene glycol) on phospholipid hydration and polarity of the external phase

The hydration properties of phosphatidylcholine (PC)/water dispersions on the addition of poly(ethylene glycol) were studied by means of ²H-NMR. The quadrupole splittings and their temperature dependences correspond to measurements of PC/water dispersions at low water content. It is concluded that the bound water is partly extracted by poly(ethylene glycol) but the binding properties of the water in the inner hydration shell of about five water molecules are not changed. The ability of some phospholipid/water dispersions to undergo phase transitions to nonlamellar structures upon dehydration is discussed. Dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine do not form nonlamellar structures on addition of purified poly(ethylene glycol), as was demonstrated by means of ³¹P-NMR. Poly(ethylene glycol) decreases the polarity of the aqueous phase and the partition of hydrophobic molecules between the membrane and the external phase is changed. This was demonstrated using the excimer fluorescence of pyrene in a ghost suspension. It is suggested that the changes in polarity and hydration on the addition of poly(ethylene glycol) can contribute to the alterations in the membrane surface observed under conditions of membrane contact and fusion.     

Changes in the lipid dynamics of liposomal membranes induced by poly(ethylene glycol): free volume alterations revealed by inter- and intramolecular excimer-forming phospholipid analogs.

Influence of osmotic shrinkage, swelling, and dehydration on large unilamellar liposomes (LUVs) of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) was investigated using the fluorescent lipid probes 1-palmitoyl-2-[10-(pyren-1-yl)]-decanoyl-sn-glycero-3-phosphocholi ne (PPDPC) and 1,2-bis[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC). Increasing concentrations of poly(ethylene glycol) (PEG, average molecular weight of 6000) producing osmotic gradients delta omega up to 250 mOsm/kg were first added to the outside of LUV labeled with 0.1 mol% of either of the above fluorescent phospholipids. The resulting osmotic shrinkage was accompanied by a progressive reduction in the lateral diffusion of the membrane-incorporated PPDPC, evident as a decrease in the rate of its intermolecular excimer formation. In contrast, under the same conditions the rate of intramolecular excimer formation by bisPDPC increased. Notably, signals opposite to those described above were observed for both of the fluorescent probes upon osmotic swelling of DOPC liposomes with encapsulated PEG. The lateral diffusion of PPDPC became progressively reduced upon membrane dehydration due to increasing concentrations of symmetrically distributed PEG (with equal polymer concentrations inside and outside of the liposomes) when neither shrinkage nor swelling occurs while enhanced excimer formation by bisPDPC was evident. The later results were interpreted in terms of osmotically induced changes in the hydration of lipids. In brief, the removal of water from the phospholipid hydration shell diminishes the effective size of the polar headgroup, which subsequently allows for an enhanced lateral packing of the phospholipid acyl chains. Our findings are readily compatible with membrane free volume Vf changes due to osmotic forces under three different kinds of stress (shrinkage, swelling, and dehydration) applied on the lipid bilayers.     

Lateral distribution of a pyrene-labeled phosphatidylcholine in phosphatidylcholine bilayers: fluorescence phase and modulation study

The lateral distribution of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]phosphatidylcholine (PyrPC) was studied in small unilamellar vesicles of 1,2-dipalmitoyl-, 1,2-dimyristoyl-, and 1-palmitoyl-2-oleoyl-phosphatidylcholine (DPPC, DMPC, and POPC, respectively) under anaerobic conditions. The DPPC and DMPC experiments were carried out over temperature ranges above and below the matrix phospholipid phase transition temperature (Tm). The excimer to monomer fluorescence intensity ratio (E/M) was determined as a function of temperature for the three PyrPC/lipid mixtures. Phase and modulation data were used to determine the temperature dependence of pyrene fluorescence rate parameters in gel and in liquid-crystalline bilayers. These parameters were then used to provide information about excited-state fluorescence in phospholipid bilayers, calculate the concentration of the probe within liquid-crystalline and gel domains in the phase transition region of PyrPC in DPPC, and simulate E/M vs. temperature curves for three systems whose phase diagrams are different. From the simulated curves we could determine the relationship between the shape of the three simulated E/M vs. temperature curves and the lateral distribution of the probe. This information was then used to interpret the three experimentally derived E/M vs. temperature curves. Our results indicate that PyrPC is randomly distributed in pure gel and fluid phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of 8-anilino-1-naphthalenesulfonate as a probe of the effect of cholesterol on the phospholipid bilayer structures.

The transport of 8-anilino-1-naphthalenesulfonate in dimyristoyl-L-alpha-lecithin bilayers has been found to be extremely sensitive to the crystalline state of the phospholipid dispersions. Thus this reaction may be used for probing the membrane structures. In binary mixtures of cholesterol and phospholipid the fluorescence enhancement of the dye completely disappears when the mole fraction of cholesterol reaches 33%. At temperatures below and above the phase transition of the lipid bilayers, the rate of the probe transport increases significantly in the binary mixtures. It reaches a maximum at 17 mol % of cholestero. The rate at this cholesterol content approaches the maximum value obtained for the probe transport in pure phospholipis, e.i., the rate at the midpoint of the phase transition. These observations indicate that the effect of cholesterol in the phospholipid dispersion is to maintain the bilayer structure close to the melting temperature of the lipid phase transition. In other words, cholesterol may be an effective buffer for membrane crystalline state when its concentration is near 17 mol %.     

Intramolecular excimer formation of pyrene-labeled lipids in lamellar and inverted hexagonal phases of lipid mixtures containing unsaturated phosphatidylethanolamine

The rates of intramolecular excimer formation of di(1'-pyrenemyristoyl)phosphatidylcholine (dipyPC) in dioleoylphosphatidylethanolamine (DOPE), egg PE/diolein (DG) and dilinoleoyl-PE (DLPE)/1-palmitoyl-2-oleoyl-PC (POPC) were studied at different temperatures and lipid compositions. Both the excimer-to-monomer intensity ratio and the excimer association rate constant were employed to quantify the rate of excimer formation. The latter was calculated from the measured monomer fluorescence lifetime of dipyPC. We observed that the rate of excimer formation was sensitive to either the temperature-induced or lipid composition-induced lamellar-to-inverted hexagonal phase transition of the above lipid systems. As the lipids entered the inverted hexagonal phase, the rate of excimer formation increased at the temperature-induced phase transition for DOPE, but decreased at the composition-induced phase transition for both TPE/DG and DLPE/POPC systems by increasing the DG% and decreasing the PC%, respectively. We conclude that the rate of intramolecular excimer formation of dipyPC in the non-lamellar phase is influenced both by the intra-lipid free volume of the hydrocarbon region and the intra-rotational dynamics of the two lipid acyl chains.     

Extraction of lipid membrane incorporated vitamin E by sucrose polyesters

2H and 31P solid state NMR have been used to probe, at the molecular level, the interaction between structurally different sucrose polyesters and a phospholipid membrane into which alpha-tocopherol and specifically deuterated alpha-[5,7-(2)H(6)] tocopherol has been incorporated. Our results show that at high concentration (>or=10 mol%) sucrose octapalmitate (SOP) and sucrose hexapalmitate (SHxP) deplete bilayer-associated alpha-tocopherol in dipalmitoyl phosphatidalcholine (DPPC) multilamellar dispersions and preferentially sequester the alpha-tocopherol into a fluid sucrose polyesters (SPE) phase located proximal to the membrane surface. It is demonstrated that the ability of SPEs to function as a 'lipophilic sink' depends strongly on sucrose polyester concentration and degree of esterification.     

Interaction of Paclitaxel with Phospholipid Bilayers

Abstract

Paclitaxel is an effective anticancer drug. Recently, paclitaxel encapsulated in liposomes was promoted as a better tolerated pharmaceutical formulation than that currently in use. The data presented in this study show the effects of paclitaxel on phospholipid bilayers. Experiments involving the phospholipid head group probe CAT-16 show significant disordering of the interfacial region. The pretransition was abolished and the main phase transition temperature in paclitaxel loaded liposomes was reduced. 2T II values of 7-NSA and 16-NSA spin probes reporting from the middle and from the core of the phospholipid bilayer, respectively, show that the presence of paclitaxel eliminated the pretransition (from Lβ/ to Pβ/) while inducing a slight reduction in the main (Pβ/ to Lα) phase transition temperature; in the same temperature interval, the central resonance line width δ H O displayed a greater rate of spin label reorientation in paclitaxel loaded bilayers. Further data are presented clearly demonstrating that the presence of paclitaxel in liposomal membrane increases the solubility of hydrophobic compounds. Differential scanning calorimetry was used to confirm that the presence of paclitaxel stabilized the lamellar structure of the bilayer and increased the transition temperature from lamellar Lα phase to hexagonal H II phase of TPE liposomes. The encapsulation of paclitaxel in liposomes depends on phospholipid characteristics; more drug is contained in the bilayer of liposomes containing unsaturated fatty acid chains and phosphorylcholine headgroups, such as DEPC and egg PC.     

Effects of unsaturation on 2H-NMR quadrupole splittings and 13C-NMR relaxation in phospholipid bilayers

Motional order and motional rates in unsonicated phospholipid bilayers were assessed as a function of unsaturation of the phospholipid. A measurement sensitive to motional order was obtained using 2H-NMR of 18:1, 18:1-phosphatidylcholine labelled at positions 9 and 10 with deuterium and included as a probe in phospholipid bilayers of interest at 10 mole percent. Spin lattice relaxation times from magic angle spinning 13C-NMR spectra of phospholipid dispersions of interest were used as a measure of motional rates. Measurements were made of phospholipid bilayers containing from 0 to 8 double bonds per molecule. No large effect of an increase in unsaturation was noted for the 2H-NMR quadrupole splittings or for the 13C-NMR spin lattice relaxation rate.     

Hydrolysis of very-low-density lipoproteins labeled with a fluorescent triacylglycerol: 1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol

The fluorescent triacylglycerol (DPBG) 1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol was incorporated into plasma very-low-density lipoproteins (VLDL) to form DPBG-VLDL. In the presence of albumin, the addition of milk lipoprotein lipase to DPBG-VLDL hydrolyses DPBG together with the VLDL triacylglycerol and pyrenyl fatty acids are transferred to albumin. As a consequence the monomer fluorescence increases while that of the excimer decreases [Mantulin, W. W., Massey, J. B., Gotto, A. M., Jr & Pownall, H. J. (1981) J. Biol. Chem. 256, 10815-10819]. The relationship of the intensity of the excimer at 475 nm to that of the monomer at 396 nm was measured before and after lipolysis of VLDL by milk lipoprotein lipase. These fluorescent changes parallel the release of free fatty acids from VLDL and their uptake by albumin. The rate of increase of monomer to excimer fluorescence was dependent upon the enzyme, substrate and albumin concentration. The lipolysis reaction, as monitored by fluorescence changes, followed Michaelis-Menten kinetics with a Km of 1.7 M for milk lipoprotein lipase. The use of the fluorescent triacylglycerol probe increases the sensitivity of the technique by a factor 50-80 compared to a technique previously reported using a fluorescent phospholipid. The present method is applicable to 2-10 micrograms triacylglycerol corresponding to about 50-100 microliters of newborn plasma or 30-50 microliters normal adult plasma. The use of an Airfuge ultracentrifuge for VLDL isolation, in conjunction with that of DPBG as a fluorescent probe enables a rapid study of VLDL lipolysis on minimal sample amounts. It can therefore be easily applied to normal and dyslipoproteinemic samples and to the newborns.     

Phase transition characteristics of diphosphatidyl-glycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers. Mono- and divalent metal ion effects

Synthesis and phase transition chaaracteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature (Tm) and the enthalpy of transition (delta H) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; Tm for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric sn-3,3- and sn-1,1-phosphatidyldiacylglycerol and for the diastereomeric, meso-sn-1,3-phosphatidyldiacylglycerol are approximately the same. The molar enthalpy for the transition of cardiolipin-NH+4 bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C16-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e., the highest Tm is observed for Ca2+-cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca2+. The lowest Tm is observed for Rb+-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.