The components of merocyanine‐540 absorption spectra in aqueous, micellar and bilayer environments

Abstract

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...

Folding graft copolymer with pendant drug segments for co-delivery of anticancer drugs

“…The folding process of graft copolymers can be monitored by the dye micellization (Figure 3B) [43]. Dye merocyanine 540 is known to show a shift of the maximum wavelength (λ max ) during the formation of micelle [57–58]. As shown in Figure 3B, the absorbance intensity of the micelled dye peak at 575 nm, associated with a micellar environment, steadily increased as the concentration of the graft copolymer increased, clearly indicating the formation of a micelle-like folded nanostructure.…”

Spectral Imaging of MC540 During Murine and Human Colon Carcinoma Cell Differentiation

“…It is well established that the fluorescence intensity of fluorophores is affected by the polarity of their microenvironment (Kaschny and Goni 1992;Malik et al 1997) and that their absorbance depends on the ratio between lipophilic and hydrophilic media (Ehrenberg and Pevzner 1993). Spectral measurements revealed maximal light absorbance of MC540 at 480 nm in aqueous environments and at 570 nm in lipophilic media and membranes (Figure 1).…”

“…In some previous studies, MC540 has been used as a probe for measuring changes in the membrane potential of liposomes (Williamson et al 1983;Ehrenberg and Pevzner 1993). These changes depend on two major components of the electric field, the cross membrane potential and the surface potential, and were expressed by a slight alteration in the absorption and fluorescence spectra (Sieber 1987;Singth et al 1991;Kaschny and Goni 1992;Ehrenberg and Pevzner 1993). The first component, also known as the Nernst potential, arises from differences in ion concentrations between the two sides of the membrane, and the second component arises from charged groups at the membrane-water interface.…”

Photobehavior of merocyanine 540 bound to human serum albumin

“…On the other hand, in phosphate buffer there is a high dimer contribution, in agreement with previous reports. 36 Accordingly, the addition of HSA or Triton X-100 to MC aqueous solution leads to a decrease in the A 500 /A 550 ratio from 1 to 0.8 and 0.2, for 40 mM HSA and 10 mM Triton, respectively. From this result, it is possible to infer that the presence of HSA or Triton decreases the MC aggregation degree.…”