gramicidin-a and merocyanine-dye

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 (lambda max 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 lambda max and the integrated intensity, i.e. area, of the dimer population are very useful in this respect. The monomer lambda max 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.

Topics: Cholesterol; Detergents; Fluorescent Dyes; Gramicidin; Lipid Bilayers; Micelles; Octoxynol; Phospholipids; Polyethylene Glycols; Pyrimidinones; Spectrometry, Fluorescence

Changes in the fluorescence intensity of merocyanine-540 were measured in suspensions of synaptic plasma membrane ghosts isolated from rat brain cortex. With preincubation of the membrane ghosts in isotonic KCl or NaCl solution, K- and Na-enriched ghosts samples were prepared. In suspensions of both sort of synaptic membrane ghosts, merocyanine-540 showed a fluorescnece emission peak at a wavelength of 590 nm. Under a fixed total concentration of NaCl and KCl, high external K+ induced an increase in the fluorescence intensity, such an increase being proportional to logarithm of K+ concentration. Replacing K+ by Rb+, NH4+ or Cs+, a similar effect was observed. Rb+ was about as effective as K+ ; NH4 is about 2/3 and Cs+ 1/4 as effective. But the changes in fluorescence with increasing K+ concentration were larger in K-ghosts than in Na-ghosts. K+-induced fluorescence changes were very small when gramicidin D was added to the suspension. K+-induced fluorescence changes were not observed in ultrasonicated ghost suspensions. Such findings seem to indicates that the K+-ions induced fluorescence increase reflect the depolarization in the isolated synaptic plasma membrane ghosts. Furthermore, the permeability ratio PNa/PK (PNa, PK: the permeability constant for Na+ and K+) was estimated to be smaller than 0.03.

Topics: Animals; Benzoxazoles; Cell Fractionation; Cerebral Cortex; Fluorescence; Fluorescent Dyes; Gramicidin; In Vitro Techniques; Membrane Potentials; Potassium; Pyrimidinones; Rats; Synaptic Membranes