Spectral-data-processing and curve-fitting techniques have been applied to the decomposition of merocyanine-540 absorption spectra in aqueous, micellar and bilayer environments. The various resolved component bands have been assigned to dye monomers, dimers, or larger aggregates, either in polar or non-polar environments. The analysis of spectral parameters (A, , , and integrated intensity) of the overall spectra and of each component has revealed that merocyanine 540 is a useful probe in studies of membrane structure and dynamics using visible-absorption spectroscopy. In particular, the monomer A, ,, and the integrated intensity, i.e. area, of the dimer population are very useful in this respect. The monomer A, , , is especially sensitive to polarity changes and is thus useful, e.g. in the precise determination of critical micellar concentrations. The fractional area of the dimer increases with the packing density of the phospholipid-hydrocarbon region near the interface and is thus very sensitive to changes in vesicle curvature and to the presence of sterols or intrinsic polypeptides in the bilayer.Cyanine dyes have been frequently used as optical probes in membrane studies. In particular, the anionic dye merocyanine 540 (MCS40) has found widespread application [l]. Due to its amphipathic nature, MC540 ( Fig. 1) is soluble in solvents of a wide polarity range, including water and, to a limited extent, chloroform. The dye can easily be incorporated into the bilayer of phospholipid model membranes and partitions into the lipid phase when added externally to an aqueous liposome suspension [2,3]. MC540 exhibits an equilibrium between fluorescent monomers and non-fluorescing dimers which depends on the dispersing medium. In phospholipid bilayers the monomer chromophore is thought to be located near the membrane/water interface [3].The A, , , of the dye's visible spectrum correlates very well with the dielectric constant of the solvent [4]. This supports the use of MC540 as a solvatochromic dye; polarity changes in the microenvironment of the chromophore are monitored as I,,, shifts. Most important, however, is its ability to show variations in surface potential by changes in its absorbance and fluorescence spectra [ 5 , 61. The electrochromic properties of the dye are of particular interest when the object of study is too small to allow the application of microelectrodes. Furthermore, MC540 selectively stains leukemic and immature hemopoietic cells, also acting as a photosensitizing agent [7, 81. Correspondence to F. M. Go@ Department of Biochemistry, Faculty of Science, University of the Basque Country, Apartado 644, E-48080 Bilbao, SpainAbbreviations. CMC, critical micellar concentration; LUV, large unilamellar vesicles; MC540, merocyanine 540; MLV, multilamellar vesicles; PtdCho, phosphatidylcholine; SUV, small unilamellar vesicles.The versatility of the dye as an optical membrane probe explains the importance of a precise analysis of its spectrometric properties. This is particularly valid for the evaluation...

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The cyanine dye merocyanine 540 (MC540) was incorporated as an optical sensor into small and large unilamellar phospholipid vesicles. The absorption spectrum of the amphipathic dye was used to monitor the sublytic and lytic actions of the nonionic detergent Triton X-100 on model membrane systems. Curve-fitting techniques were applied to separate the monomer and dimer components of the MC540 visible spectrum.The maximum absorption wavelength ( A , , ) of the monomer band is sensitive to micropolarity changes, while dimer formation is related to environmental polarity as well as to vesicle curvature. Triton X-100 incorporation into the bilayer leads to a more polar environment in the hydrocarbon region near the interface, so that solubilization is accompanied by a blue-shift of A, , and a decrease in the monomeddimer area ratio.A surfactant-induced transient increase in size of small unilamellar vesicles, which occurs within a given range of sublytic surfactant concentrations, is also detected by MC540 as an increase in the proportion of probe monomers. Effective detergenaipid ratios ReoN and Reom have been computed, corresponding to the real detergent-to-lipid molar ratio in the bilayer (or mixed micelle) at which a spectral feature of MC540 is first or last detected. ReoN for the decrease in monomeddimer ratio is 0.13, well ahead of the earliest signs of solubilization, detected by light scattering at Re = 0.71. At the other end of the process, light scattering informs that solubilization is complete at Re = 3.0, while Reom for the blue-shift of Am= is 4.5, this parameter reporting on changes in the mixed micelles apparently not detected by light scattering. It is concluded that MC540 is a sensitive and informative optical probe, supplying information along an unusually broad range of detergenaipid ratios.

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