Mechanism and kinetics of merocyanine 540 binding to phospholipid membranes

The physicochemical mechanism for merocyanine 540 (M540) binding to unilamellar phosphatidylcholine (PC) vesicles was examined by steady-state and dynamic fluorescence and fluorescence stopped-flow methods. At 530-nm excitation, aqueous M540 has an emission peak at 565 nm, which red shifts to 580 nm with formation of membrane-bound monomers (M); bound dimers (D) are nonfluorescent. Equilibrium fluorescence titrations show that 50% of total M540 partitions into the membrane to form D at [M540]/[PC] (Rm/p)_approximately 0.6. M and D concentrations are equal at Rm/p approximately 0.05. For Rm/p less than 0.1, M540 has a single fluorescence lifetime (tau), which decreases with Rm/p [tau-1 (ns-1) = 0.48 + 3.3Rm/p], indicating a rapid collisional rate between M to form D. Dynamic depolarization studies show that hindered rotation of M (r infinity = 0.13 at Rm/p = 0.006) becomes more rapid (rotational rate 0.2-1.9 ns-1) with increasing Rm/p (0.006-0.075). The efficiencies of energy transfer between n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12, 16) and bound M540 suggest that M is oriented parallel to the phospholipids near the membrane surface; studies of efficiencies of n-AF quenching by D are consistent with an orientation of D perpendicular to the phospholipids. In stopped-flow fluorescence measurements in which M540 is mixed with PC vesicles, there is a rapid (1 ms) followed by a slower (10-50 ms) concentration-dependent fluorescence increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol-free phospholipid domains may be the membrane feature selected by N epsilon-dansyl-L-lysine and merocyanine 540

We have used N epsilon-dansyl-L-lysine as a fluorescent membrane probe, to study cells taken from tissues concerned with immune function. There is a striking similarity between the staining selectivity of this compound and that reported by others for merocyanine 540. Both compounds stain leukemic, human, peripheral leukocytes, an erythroleukemia line, and some mouse bone marrow cells, suggesting common selectivity for a membrane feature of hemopoietic cells. Both compounds fail to stain red blood cells, normal human leukocytes, mouse spleen and thymus cells. We have recently reported that dansyl-lysine apparently selects for cholesterol-free phospholipid domains in liposomes and now report similar selectivity for merocyanine 540 staining of liposomes.     

Abnormal membrane protein methylation and merocyanine 540 fluorescence in sickle erythrocyte membranes

Sickle cell erythrocytes exhibit reduced carboxyl methylation of membrane proteins compared to normal erythrocytes. This altered methylation in sickle membrane proteins is also observable when extracted membranes, both intact and alkali treated, were used as substrates for the homologous protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24). However, when glycophorin A, one of the major methyl acceptors in both membranes, was extracted by lithium diiodosalicylate and used as the methyl acceptor, the proteins from both membranes were methylated equally, suggesting an involvement of membrane structure in membrane-bound protein methylation. Merocyanine 540 (MC-540), a fluorescent probe, was used to determine if the membranes differed in organization. Incubation of both normal and sickle erythrocytes membranes with MC-540 produced a marked increase in extrinsic fluorescence, reflecting a relatively nonpolar environment for the dye bound to the membranes. The fluorescence from sickle cell ghosts was only 87% as intense as that from normal ghosts, while the actual amount of MC-540 associated with sickle cell membranes was only 62% of normal. These data suggest that differences exist in the distribution of surface charges on these plasma membranes. These results are consistent with the hypothesis that abnormal levels of membrane protein methylation observed in sickle erythrocytes may be a result of abnormal membrane organization characteristic to sickle cell anemia.     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Laurdan is a fluorescent probe that detects changes in membrane phase properties through its sensitivity to the polarity of its environment in the bilayer. Variations in membrane water content cause shifts in the laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). We tested whether laurdan fluorescence could be used to distinguish differences in phospholipid order from changes in membrane fluidity by examining the temperature dependence of laurdan GP and fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) vesicles. The phase transition from the solid ordered phase to the liquid disordered phase was observed as a decrease in laurdan GP values from 0.7 to −0.14 and a reduction in anisotropy from 0.25 to 0.12. Inclusion of various amounts of cholesterol in the membranes to generate a liquid ordered phase caused an increase in the apparent melting temperature detected by laurdan GP. In contrast, cholesterol decreased the apparent melting temperature estimated from anisotropy measurements. Based on these results, it appeared that laurdan anisotropy detected changes in membrane fluidity while laurdan GP sensed changes in phospholipid order. Thus, the same fluorescent probe can be used to distinguish effects of perturbations on membrane order and fluidity by comparing the results of fluorescence emission and anisotropy measurements.     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Sialyltransferase activity has been determined in membrane preparations containing the Golgi apparatus that were isolated from atherosclerotic and normal human aortic intima as well as in plasma of patients with documented atherosclerosis and healthy donors by measuring the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to asialofetuin. The asialofetuin sialyltransferase activity was found to be 2 times higher in the atherosclerotic intima as compared to the normal intima and 2-fold higher in patients' plasma than in that from healthy donors. The mean values of the apparent Michaelis constant (K(m)) for the sialylating enzyme for both tissues did not differ and were close for the intima and plasma. In contrast, the maximal velocity (V(max)) was 2 times higher for the atherosclerotic intima than for the normal intima and 3 times higher for patients' plasma than for that of the donors. These results suggest that the activity of asialofetuin sialyltransferases of aortal intima is enhanced in atherosclerosis as is the secretion of their soluble forms into patients' plasma.     

Quinidine is a strong perturber of acidic phospholipid bilayer order and fluidity

The effect of an antiarrhythmic drug, quinidine, on the organization of model phospholipid membranes was studied by the spin-labeling technique. Quinidine strongly perturbs the molecular organization of lipid bilayers prepared from acidic phospholipids (phosphatidylserine, phosphatidic acid) and has only a slight effect on neutral phosphatidylcholine membranes. The interaction of the drug with acidic phospholipids manifests itself in a pronounced increase in the order parameter of the region close to the polar surface of the bilayer and in some decrease in its inner hydrocarbon core fluidity. It is suggested that the perturbation in the organization of membrane lipids may contribute to the mechanisms by which quinidine exerts its pharmacological effects.     

Perturbations of membrane structure by optical probes: I. Location and structural sensitivity of merocyanine 540 bound to phospholipid membranes

Summary The maximum monomer absorption wavelength of a frequently used external membrane probe, Merocyanine 540, can be related to the location of the binding site for the dye within lipid membranes. Solvent studies indicate the occurrence of very specific and mutual perturbances between the probe and its microenvironment, that are of relevance, when investigating structural and functional events in biomembranes with the aid of this dye. Merocyanine 540 (MC 540) is an excellent probe for structural altions in the lipids including phase transitions. The extinction coefficient and _max place the location of the dye-chromophore slightly above the domain of the glycerol of backbone of neutral and charged phospholipids. This explains the sensitivity of MC 540 to structural variations in the head-group region of several synthetic dipalmitoyl-lecithin analogues. The major physical parameters involved in variations of the optical signals associated with changes in the membrane structure are the dye/lipid partition coefficient and the monomer-dimer dissociation constant of the dye bound to the lipids. A temperature dependent transition from the liquid-crystalline to the crystalline state leads mainly to an exclusion of the dye from the lipid phase with a concomitant dimerization of the dye molecules still in contact with the polarhead group region of the lipid. The relevance of this finding for the mechanism of transient optical signals in connection with the occurrence of action potentials in excitable membranes is discussed. Our findings underline the necessary caution when applying external optical probes and analyzing membrane features from the spectral data, because of inevitable perturbances in the microenvironment of every probe molecule.     

A chemometric approach to the estimation of the absorption spectra of dye probe merocyanine 540 in aqueous and phospholipid environments

Merocyanine 540 (MC540) is a widely used dye probe for membranous environments. However, fundamental knowledge of the spectral features of this dye in aqueous and hydrophobic environments is still lacking. Such knowledge is important because biomembranes involve a hydrophobic environment surrounded by a hydrophilic environment. Because many investigations so far have been performed based on indistinct spectral estimations, the interpretation of the data obtained using this dye as a fluorescent transmembrane probe remains controversial. In order to determine the exact spectra in both aqueous and hydrophobic environments, we adopted principal factor analysis (PFA), a method of multivariate analysis. The PFA method can also determine the number of molecular species present in the reaction mixture, which is three in pure water and two in phospholipid suspension. Two of the species in both water and phospholipid suspension were the monomer and dimer. The third species in water was the trimer, but its amount was so small at 10 microM MC540 solution that the spectral data in water can be approximated neglecting this molecular species. The monomer spectrum changed its form markedly with a bathochromic shift when transferred from the water to phospholipid environment, whereas the dimer remained similar in its shape except for a remarkable red shift. In water, the dissociation constants, K(1) and K(2), for the assumed stacking-model reactions, M+M <--> M(2) and M+M(2) <--> M(3), were 3.1 x 10(-4) M and 5.7 x 10(-4) M, respectively. In the phospholipid environment, the dissociation constant K* for the assumed stacking-model reaction, M(*)+M(*) <--> *M(2), was 1.9x10(-5)M. The fluorescent intensities of MC540 were also measured in both water and phospholipid environments. A comparison based on the absorption and fluorescence spectra suggested that the temporal increase in the amount of the monomer on the excitable membrane contributes to the fluorescent intensity change observed in the transmembrane potential change.     

Substrate-supported phospholipid membranes studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates.     

Verteporfin, photofrin II, and merocyanine 540 as PDT photosensitizers against melanoma cells

The efficiency of photodynamic effect (PDE) for Photofrin II (PfII), Verteporfin, and Merocyanine 540 (MC540) was compared against neoplastic cells. Triplet state lifetimes and singlet molecular oxygen quantum yields were correlated with biological effect. PfII triplet lifetime was two times longer than that of Verteporfin, however, its singlet molecular oxygen quantum yield was two times lower in comparison with Verteporfin. High singlet molecular oxygen quantum yield of Verteporfin resulted in high biological efficacy. To achieve 50% mortality of cells four times lower light dose and five times lower concentration of Verteporfin were applied in comparison with PfII. The same level of cell damage was reached using 10 times higher light dose and two times higher concentration of MC540 in comparison with PfII. Our results confirm that singlet molecular oxygen based mechanism, prevalent for Verteporfin and PfII, was highly effective against melanoma cells. Verteporfin can be used at small doses with high cellular damage efficiency.     

Perturbation of binary phospholipid mixtures by melittin: a fluorescence and raman spectroscopy study

The effect of melittin on different binary mixtures of phospholipids has been studied by polarization of DPH fluorescence in order to determine if melittin can induce phase separation. Since the interaction between lipids and melittin is sensitive to both electrostatic and hydrophobic forces, we have studied the effect of the acyl chain length and of the polar head group of the lipids. In spite of the difference of the chain length between dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), no phase separation occurs in an equimolar mixture of these lipids in presence of melittin. However, when the charged lipid dipalmitoylphosphatidylglycerol (DPPG) is mixed with either DPPC or DSPC, the addition of melittin leads to phase separation. The DSPC/DPPG/melittin system, which shows a very complex thermotropism, has also been studied by Raman spectroscopy using DPPG with deuteriated chains in order to monitor each lipid independently. The results suggest that the higher affinity of melittin for DPPG leads to a partial phase separation. We propose the formation of DPPG-rich domains perturbed by melittin and peptide-free regions enriched in DSPC triggered by the head group charge and chain-length differences.     

Singlet oxygen production and fluorescence yields of merocyanine 540: a comparative study in solution and model membrane systems.

Singlet oxygen and fluorescence quantum yields of merocyanine 540 were measured in solution (methanol, ethanol, n-heptanol) and in model membrane systems (cationic micelles, unilamellar dimyristoyl- and dipalmitoylphosphatidylcholine vesicles). Both singlet oxygen quantum yields and fluorescence quantum yields increase with increasing viscosity/rigidity of the surrounding medium: the yield of singlet oxygen production (24 degrees C) goes from 0.002 in methanol to 0.04 in dipalmitoylphosphatidylcholine vesicles, and fluorescence yields (25 degrees C) change from 0.14 to 0.61 in the same media. The data are consistent with previous findings that photoisomerization is in direct competition with intersystem crossing and radiative relaxation. Therefore, a singlet oxygen yield close to the maximum value of 0.11 can only be achieved after both photoisomerization and internal conversion are prevented by a highly viscous environment.     

Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Perturbations of membrane structure by optical probes: II. Differential scanning calorimetry of dipalmitoyllecithin and its analogs interacting with merocyanine 540

Summary Differential scanning calorimetry of multilamellar liposomes, interacting with the optical probe Merocyanine 540, yields quantitative information about perturbances of the bilayer structure induced by this dye. At low dye: lipid ratios, the dye perturbs primarily its own microenvironment, which is laterally separated from the unmodified lipid domain and exhibits modified thermotropic properties. A further increase in the dye concentration results in a perturbance of the whole lipid bilayer. The degree of perturbance is sensitive to structural modifications in the head-group region of the lipids. It is concluded that Merocyanine 540 reports in every case, even at infinite dilution, on localized events originating from a perturbed microenvironment.An abstract of this paper was presented at the XI Congress of Biochemistry, Toronto, in July 1979.     

EPR studies of phospholipid bilayers after lipoperoxidation. 1. Inner molecular order and fluidity gradient

The molecular order of fatty acyl chains in oriented lipid bilayers on solid support (SPB), made of either natural or synthetic phospholipids oxidized by Fenton reagent and probed with spin labeled lecithin (5-DSPC) was studied by means of EPR spectrometry. Phospholipids (ASPC, EYPC, mitochondrial extract) were oxidized as either aqueous buffer/methanol dispersions or reverse-phase evaporation vesicles (REV) suspensions. Oxidation was preliminarily revealed both by assaying MDA and by detecting conjugated dienes. Oxidized phospholipid species was quantified by preparative TLC. The degree of order in oriented lipid bilayers of samples containing oxidized phospholipids was estimated by the loss of EPR spectral anisotropy, and an empirical index of the related bilayer disorder was calculated from the second derivative spectra. Bilayers made of each non-oxidized phospholipid species from either ethanol solutions or REV suspensions showed the highest anisotropy, while the increasing presence of oxidized lipids in the samples resulted in progressive loss of EPR spectral anisotropy. In contrast, vesicles containing 40% of the oxidized species maintained an unaltered fluidity gradient, while REV formation was hindered by oxidized phospholipid percentages higher than 45% for ASPC and EYPC, and 35% for Mitochondrial lipids (MtL). It is concluded that the early stages of lipoperoxidation bring about disordering of the phospholipid bilayer interior rather than fluidity alterations, and that prolonged oxidation may result in loss of structural and chemical properties of the bilayer until the structure no longer holds.     

Interaction of propranolol with model phospholipid membranes Monolayer,spin label and fluorescence spectroscopy studies

The interaction of propranolol with model phospholipid membranes was studied using various experimental techniques. The partition coefficient of propranolol in the negatively charged membranes of vesicles prepared from phosphatidylserine and phosphatidic acid was found to be more than 20-times higher than in neutral phosphatidylcholine membranes. Preferential interaction of propranolol with acidic phospholipid membranes was confirmed using the monolayer compression isotherm technique and the spin-labeling method. Phosphatidylserine monolayers were markedly expanded even at a relatively low drug concentration ($\text{5 · 10}{}^{\mathrm{?6}}$ M). In contrast, the effect of propranolol on phosphatidylcholine monolayers was much smaller, being detectable only at a higher concentration of the drug ($\text{1 · 10}{}^{\mathrm{?4}}$ M). Spin-labeling experiments show that propranolol exerts marked ordering effect on bilayers prepared from acidic phospholipids and does not change the order parameter of phosphatidylcholine membranes. The dependence of the propranolol fluorescence spectrum on the polarity of the solvent allowed us to identify the intercalation region of the drug in the membrane. The fluorophore moiety of propranolol was found to be localized in the lipid polar head groups region of the bilayer. The role of electrostatic and hydrophobic effects in propranolol-membrane interaction is discussed and the effect of propranolol on the ordering of phospholipid bilayers is compared with the effects of other anesthetic-like molecules.     

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.     

Molecular order and fluidity of the plasma membrane of human platelets from time-resolved fluorescence depolarization

The ability of seven fluorescence polarization probes (1,6-diphenyl-1,3,5-hexatriene, 1-[(4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene, 16(9-anthroyloxy)palmitic acid, CIS-parinaric acid, trans-parinaric acid and perylene) to report changes induced by temperature and Ca²⁺ in the plasma membrane of human platelets has been examined. The steady-state fluorescence anisotropy of the probes was compared after being incorporated into whole resting platelets, fragments of platelet plasma membrane and multilayers of lipids extracted from these membranes. In addition, we have investigated the molecular order and dynamics of the three preparations by time-resolved fluorescence depolarization of DPH and CE-DPH as a function of temperature and Ca²⁺ concentration. The high values of the order parameters found in intact platelets (S_{DPH, 36.c}=0.70) were almost identical to those in membrane fragments and lipid vesicles, suggesting that lipid-lipid interactions and, therefore, the lipid composition are the main factors influencing the probe order parameter. Other lipid interactions such as those with membrane proteins and intracellular components have little effect on the SDP, in platelets. These measurements also showed that the stationary fluorescence anisotropy of DPH and CE-DPH in platelets is largely determined (80%) by the structural order of the lipid bilayer. Therefore, the previous microviscosity values based on stationary anisotropy data reflect the alignment and packing rather than the mobility of the bilayer components. The dynamic component of the anisotropy decay of these probes was analyzed in terms of the wobbling-in-cone model, allowing an estimation of the apparent viscosity of platelet plasma membrane (DPH, 36°C =–0–5 P) that is similar to that of the erythrocyte membrane. This value decreased substantially in multilayers of native lipids, indicating a large effect of the lipidprotein interactions on the probe dynamics within the bilayer. When the temperature was raised from 25° to 36°C a pronounced decrease was observed in the order parameter and apparent viscosity, followed by a tendency to level-off in the 36°-40°C interval. This may be related to the end-point of the lipid phase separation reported by Gordon et al. (1983). Finally, the rigidifying (lipid ordering) effect of Ca²⁺ on the platelet plasma membrane could also be observed by the fluorescence anisotropy measurements, in the form of an increase (2%) of the order parameter of CE-DPH for Ca²⁺ concentrations in the millimolar range.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - TMA-DPH 1-[(4-trimethyl-amino)phenyl]-6-phenyl-1,3,5-hexatriene - CE-DPH (2-carboxyethyl)-1,6-diphenyl-1,3,5-hexatriene - 16AP 16-(9-anthroyloxy)-palmitic acid; c-PnA, CIS-parinaric acid; t-PnA, trans-parinaric acid - PER perylene - POPOP p-bis[2(5-phenyl-oxazolyl)benzene] - ESR electron spin resonance Offprint requests to: A. U. Acuña     

Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy.

We have utilized both fluorescent and nitroxide derivatives of stearic acid as probes of membrane structural heterogeneity in phospholipid vesicles under physiological conditions, as well as conditions of varying ionic strengths and temperatures where spectral heterogeneity has been previously observed and attributed to multiple ionization states of the probes. To identify the source of this spectral heterogeneity, we have utilized complimentary measurements of the relaxation properties (lifetimes) and motion of both (a) spin labeled and anthroyloxy derivatives of stearic acid (i.e., SASL and AS) and (b) a diphenylhexatriene derivative of phosphatidylcholine (DPH-PC) in single component membranes containing dimyristoylphosphatidylcholine (DMPC). We use an 15N stearic-acid spin label for optimal sensitivity to membrane heterogeneity. The lifetime and dynamics of the fluorescent phospholipid analogue DPH-PC (with no ionizable groups over this pH range) were compared with those of AS, allowing us to discriminate between changes in membrane structure and the ionization of the label. The quantum yield and rotational dynamics of DPH-PC are independent of pH, indicating that changes in pH do not affect the conformation of the host phospholipids. However, both EPR spectra of SASL and the lifetime or dynamics of AS are affected profoundly by changes in solution pH. The apparent pKa's of these two probes in DMPC membranes were determined to be near pH 6.3, implying that at physiological pH and ionic strength these stearic-acid labels exist predominantly as a single ionized population in membranes. Therefore, the observed temperature- and ionic-strength-dependent alterations in the spectra of SASL as well as the lifetime or dynamics of AS in DMPC membranes at neutral pH are due to changes in membrane structure rather than the ionization of the probes. The possibility that ionic gradients across biological membranes induce alterations in phospholipid structures, thereby modulating lipid-protein interactions is discussed.