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.     

Mg~(2+)加强嵌有H~+-ATP酶脂酶体脂质分子的堆积(packing)

用亲脂性的灵敏的荧光MC 540标记在有Mg~(2+)(1mM)与无Mg~(2+)条件下重建的线粒体H~+-ATP酶脂酶体,后者的荧光强度较前者增加30%左右.这提示,含Mg~(2+)的脂酶体的脂质分子间的堆积紧密度增加.在N-AF系列(n=27和16)探剂与MC 540之间的能量转移效率,又以反应靠近脂双层表面变化的2-AP与MC 54O之间最高.这进一步表明,含Mg~(2+)的脂酶体具有较适合流动性是与Mg~(2+)通过调节靠近脂双层表面的脂质分子具有适度的堆积相关的.这对阐明我们已提出的Mg~(2+)促进线粒体H~+-ATP酶重建作用模型进一步提供了较直接的证据.     

Abnormal erythrocyte membrane phospholipid organisation in chronic myeloid leukaemia

The membrane phospholipid organisation in the red cells of humans suffering from chronic myeloid leukaemia has been analysed using the amino-group labelling reagent trinitrobenzenesulphonic acid and the fluid-sensing fluorophore, Merocyanine 540. Unlike the normal human erythrocytes, trinitrobenzenesulphonic acid in intact chronic myeloid leukaemia erythrocytes modified about 30% phosphatidylserine, under controlled conditions. Also, the chronic myeloid laukaemia red cells, but not the normal cells, were found to bind the fluorescent dye Merocyanine 540. These results demonstrate that loss of the transmembrane phospholipid asymmetry in chronic myeloid leukaemia erythrocytes is accompanied by an enhancement in the outer surface fluidity and, therefore, suggest that the red cells membrane phase-state asymmetry originates probably from the asymmetric arrangements of phospholipids across the membrane bilayer.     

Increased adherence of oxidant-treated human and bovine erythrocytes to cultured endothelial cells

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H2O2 or diamide (1-3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30-33%) in the external leaflet of the bilayer and a concomitant increased (four- to five-fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H2O2 at low concentration (0.5 mM) resulted in cross-linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15-20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant-induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant-treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC-540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H2O2 (0.5 mM)-treated erythrocytes compared to 30% of untreated RBCs exhibited MC-540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant-treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross-linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte-endothelial cell interaction.     

Temperature dependence of the lipid packing in thylakoid membranes studied by time- and spectrally resolved fluorescence of Merocyanine 540

The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above -30 degrees C and that a phase transition occurs only above 45 degrees C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases. It is demonstrated that the degree and way of incorporation differs for most lipid phases--upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 degrees C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 degrees C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane.     

Merocyanine 540 recognizes membrane abnormalities of erythrocytes in chronic myelogenous leukemia

Merocyanine 540 is a fluorescent dye which stains erythrocytes that have lost their normal membrane phospholipid asymmetry. Because erythrocytes from patients with chronic myelogenous leukemia have been reported to display this abnormal phenotype, peripheral blood erythrocytes from such patients were examined for their ability to stain with the dye. Erythrocytes from all patients with active disease states stained, whereas neither erythrocytes from normal, healthy individuals nor from a patient whose disease symptoms were eliminated by chemotherapy stained. These results suggest that merocyanine 540 may have utility in the clinical evaluation of chronic myelogenous leukemia.     

Differential Sensitivity of Mouse Hematopoietic Stem Cells to Merocyanine 540

In vivo and in vitro clonal assays of immature mouse blood cells showed that diffferent populations of hematopoietic progenitor cells differ considerably with respect to their sensitivity to photodynamic damages caused by the fluorescent dye Merocyanine 540. Late erythroid progenitors were the most sensitive cells followed in order of decreasing sensitivity by pluripotent stem cells, early erythroid progenitors, and granulocyte/macrophage progenitors. Only about 2%–4% of all nucleated marrow cells were stained with Merocyanine 540 which correlated well with current frequency estimates of progenitor cells in mouse bone marrow. Our findings indicate that the expression of Merocyanine binding sites is developmentally regulated and might, therefore, provide a useful molecular marker for blood cell differentiation and a basis for an effective purification of hematopoietic progenitor cells.     

Effect of vanadium compounds on the lipid organization of liposomes and cell membranes

The influence of vanadate on the adsorption properties of Merocyanine 540 (MC540) to UMR cells was studied by means of specrofluorometry. An increment in the fluorescence was observed in the osteoblasts incubated with 0.1 mM vanadate. This effect could be interpreted in terms of vanadate inhibitory effects on aminotraslocase activity. However, vanadate promotes a similar behavior to that found in UMR 106 cells when it was added to lipid vesicles composed of phosphatidylcholine. The effect of vanadium in different oxidation states, such as vanadate(V) and vanadyl(IV) on lipid membrane properties was examined in large unilamellar vesicles by means of spectrofluorometry employing different probes. Merocyanine 540 and 1,6-diphenylhexatriene were used in order to sense the changes at interfacial and hydrophobic core of membranes, respectively. In contrast to vanadate, vanadyl decreased the fluorescence of MC540. Both vanadium compounds slightly perturbed the hydrocarbon core. The results can be interpreted by the specific adsorption of both compounds on the polar head groups of phospholipid and suggest a possible influence of vanadium compounds on the lipid organization of cell membranes.     

Photodynamic action of merocyanine 540 on erythrocyte membranes: structural perturbation of lipid and protein constituents.

erocyanine 540 (MC540) is a membrane-directed photosensitizing dye with antileukemic and antiviral properties. In this study, biophysical and biochemical techniques have been used to examine MC540-sensitized photooxidative damage in the lipid and protein compartments of a test membrane, the human erythrocyte ghost. Irradiation of MC540-sensitized ghosts with white light resulted in oxidative damage to proteins, as manifested by (i) loss of sulfhydryl groups; (ii) intermolecular cross-linking of major polypeptides; and (iii) loss of Mg(2+)-ATPase and Na+,K(+)-ATPase activities. Photooxidation also produced a rapid and progressive increase in general protein motion, as measured by electron paramagnetic resonance spectrometry (EPR) with the sulfhydryl spin label MAL-6. In addition to these effects, ghosts exposed to MC540 and light underwent lipid peroxidation. EPR with two lipophilic spin probes, 5-doxylstearate and 16-doxylstearate, showed that lipid peroxidation is accompanied by a progressive decrease in bilayer fluidity (motional freedom). At a given dye concentration, structural perturbations of proteins were detected at much lower light fluences than those of lipids. When photoreactions were carried out in the presence of ascorbate and iron, there was a strong stimulation of lipid peroxidation (attributed to free radical chain reactions), with a concomitant greater decrease in lipid mobility. Thus, the deleterious effects of photoperoxidation on lipid structure and motional freedom were greatly exacerbated by ascorbate and iron. Membrane damage similar to that described here may play a role in the phototherapeutic activity of MC540.     

Light-induced permeabilization and merocyanine 540 staining of mouse spleen cells

Merocyanine 540 (M540) is a potential-sensitive, hydrophobic dye that preferentially incorporates into the 'fluid' domains of cellular membranes, distinguishing between hemopoietic cells according to their differentiation state. A bright staining with M540 is usually achieved by UV illumination of the cells during staining. We show by flow cytometric analysis that: (1) staining is greatly enhanced by UV illumination of mouse spleen cells before addition of the dye; (2) UV treatment causes an increased permeability toward propidium iodide and intracellular fluorescein as well; (3) the increment in M540 fluorescence precedes permeabilization to propidium iodide, while the latter precedes leakage of fluorescein. We also describe an overshoot and accelerated recovery of M540 fluorescence after photobleaching by a 514 nm laser beam. It is suggested that penetration of M540 to the more fluid inner membrane structures explains the fluorescence increment in both experiments.     

Echinocytosis and microvesiculation of human erythrocytes induced by insertion of merocyanine 540 into the outer membrane leaflet

Echinocytosis and release of microvesicles from human erythrocytes treated with the impermeant fluorescent dye merocyanine 540 (MC540) has been correlated with the extent of dye binding to intact cells and ghosts. At 20 degrees C binding appeared to saturate at about 9.3.10(6) molecules per cell (3.6 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 2.7%. Stage 3 echinocytes were formed upon binding of (3-4).10(6) molecules of MC540/cell (about 1.3 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 1.0%. Negligible release of microvesicles was observed with MC540 at 20 degrees C. Binding of MC540 to permeable ghosts was approximately twice that to cells suggesting that there was no selective binding to the unsaturated (more fluid) phospholipids which are concentrated in the inner lipid leaflet of the membrane. At 37 degrees C apparent maximal binding of MC540 was about 3.2 mol/100 mol phospholipid and correlated with the maximal release of microvesicles from the cells as measured by release of phospholipid and acetylcholinesterase. These results are discussed in relation to the bilayer couple hypothesis of Sheetz and Singer (Proc. Natl. Acad. Sci. USA 71 (1974) 4457-4461).     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A₅₄₉ and resistant A₅₄₉/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A₅₄₉ cell and 0.194 for the resistant A₅₄₉/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A₅₄₉/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A₅₄₉ cell membranes was looser than that of the A₅₄₉/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A₅₄₉/DDP cells was also lower than that of A₅₄₉ cells. Taken together, it was proposed that the alteration of membrane lipid biophysical state may be involved in the resistance of A₅₄₉/DDP cells to cisplatin.     

Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A₅₄₉ and resistant A₅₄₉/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A₅₄₉ cell and 0.194 for the resistant A₅₄₉/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A₅₄₉/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A₅₄₉ cell membranes was looser than that of the A₅₄₉/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A₅₄₉/DDP cells was also lower than that of A₅₄₉ cells. Taken together, it was proposed that the alteration of membrane lipid biophysical state may be involved in the resistance of A₅₄₉/DDP cells to cisplatin.     

Interaction of low-molecular-weight chitosan with mimic membrane studied by electrochemical methods and surface plasmon resonance

Chitosan has shown its potential as a non-viral gene carrier and an adsorption enhancer for subsequent drug delivery to cells. These results showed that chitosan acted as a membrane perturbant. However, there is currently a lack of direct experimental evidence of this membrane perturbance effect, especially for chitosans with low molecular weight (LMW). In this report, the interaction between a lipid (didodecyl dimethylammonium bromide; DDAB) bilayer and chitosan with molecular weight (MW) of 4200 Da was studied with cyclic voltammetry (CV), electrochemical impedance spectroscopy and surface plasmon resonance (SPR). A lipid bilayer was formed by fusion of oppositely charged lipid vesicles on a mercaptopropionic acid (MPA)-modified gold surface to mimic a cell membrane. The results showed that the LMW chitosan could disrupt the lipid bilayer, and the effect seemed to be in a concentration-dependent manner.     

Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type—WT) occur at 4–7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures.     

The parasitophorous vacuole membrane of Plasmodium falciparum: demonstration of vesicle formation using an immunoprobe

We have applied several immunolabeling techniques using a monoclonal antibody to a Plasmodium falciparum antigen to differentiate morphologically dissimilar membranous structures present in infected erythrocytes. Evidence is presented that cytoplasmic clefts, multimembranous structures and vesicles within the infected cell originate from the parasitophorous vacuole membrane by a process described as budding off. The parasitophorous vacuole membrane and related structures in infected, parasitized erythrocytes reacted with the cyanine dye Merocyanine 540, demonstrating that they are accessible to molecules from the extracellular environment. Immunogold labeling of freeze-fractured preparations and of thin sections of parasitized cells using pre- and post-embedding techniques revealed that each of the membranous structures carried a common parasite antigen, QF 116, which was identified by monoclonal antibody 8E7/55.     

Susceptibility to merocyanine 540-mediated photosensitization: a differentiation marker on murine hematopoietic progenitor cells

Merocyanine 540 (MC 540) is an impermeant fluorescent dye that binds preferentially to fluidlike domains of the cell membrane. Photoexcitation of membrane-bound dye causes a breakdown of the normal permeability properties of the membrane and, eventually, cell death. We have used in vitro and in vivo clonal assays to determine the relative sensitivities of different classes of normal murine hematopoietic progenitor cells to MC 540-mediated photosensitization. Late erythroid progenitors (CFU-E) were the most sensitive cells, followed in order of decreasing sensitivity by early erythroid progenitors (BFU-E), megakaryocyte progenitors (CFU-Meg), day 7-spleen colony forming cells (day 7-CFU-S), granulocyte/macrophage progenitors (CFU-GM), and day 11-spleen colony forming cells (day 11-CFU-S). Bipotent progenitors of the granulocyte/macrophage lineage were more sensitive than unipotent macrophage progenitors but less sensitive than unipotent granulocyte progenitors. Progenitors giving rise to large granulocyte/macrophage colonies were more sensitive than progenitors giving rise to small colonies ("clusters"). We conclude that sensitivity to MC 540-mediated photosensitization is develop-mentally regulated and that differences occur even between the most closely related classes of progenitor cells. Our findings indicate the usefulness of MC 540 as a plasma membrane probe. They also support the contention that early and late-appearing spleen colonies are the progeny of two distinct classes of progenitor cells.     

Interactions of voltage-sensing dyes with membranes. III. Electrical properties induced by merocyanine 540

The effects of merocyanine 540 on the electrical properties of lipid bilayer membranes have been investigated. The alterations this dye was found to produce in the intrinsic conductances of these membranes were minimal, but it profoundly altered the conductances produced by extrinsic permeant species. These alterations were much larger for neutral membranes than for negatively charged ones. The dye increased the conductances mediated by positively charged permeant species and decreased those by negatively charged permeant species, suggesting that it produces a negative electrostatic potential on the membrane; it also altered the kinetics and the voltage dependencies of permeation by these charge carriers. The magnitudes of dye-mediated conductance changes were much larger for positively charged permeants than for negatively charged ones; also, changes in ionic strength altered these dye effects in opposite directions from those predicted by the Stern equation, and the dependence of the conductance alteration on dye concentration was steeper than that predicted by this equation. Finally, only very small changes in liposome zeta potentials were induced by the dye. Calculations show that a large fraction of these effects can be accounted for by the dipole potential produced by merocyanine at the membrane surface, but that additional effects of the dye must be postulated as well.     

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.