Determination of Fluorescence Polarization of Membrane Probes in Intact Erythrocytes

Kutchai, Howard; Huxley, Virginia H.; Chandler, Laura H.

doi:10.1016/s0006-3495(82)84512-8

Cited by 17 publications

(3 citation statements)

References 10 publications

(5 reference statements)

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“…In the early stage of the experiments, part of the unsealed ghosts were labeled with 1 ,6-diphenyl-1 ,3,5-hexatriene (DPH) and the fluorescence anisotropy, r, was measured. At room temperature the calculated r value of 0.238 ± 0.012 (mean ± SD) lies well between the values reported by Eisinger et al (6) and Kutchai et al (7). Fig.…”

Section: Resultssupporting

confidence: 90%

Water permeability through biological membranes by isotopic effects of fluorescence and light scattering

Lawaczeck¹

1984

Biophysical Journal

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A light-scattering technique used to measure the water permeability across closed biomembranes is described, which is based on the different indices of refraction of D2O and H2O. This transient technique is compared with a similar method using D2O-sensitive fluorophores in the intravesicular space. The results of both techniques are equivalent although the signal-to-noise ratio favors the light-scattering or turbidity experiment. The light-scattering method is only applicable to larger particles (no point-scatterers) and is easily extended to biological objects. Data on the H2O/D2O exchange across membranes of ghosts from human erythrocytes suggest two mechanisms: the D2O and H2O permeation through the membrane and a slower D2O-induced conformational change of membraneous proteins.

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Section: Resultssupporting

confidence: 90%

Water permeability through biological membranes by isotopic effects of fluorescence and light scattering

Lawaczeck¹

1984

Biophysical Journal

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“…Our results are in agreement with the work of Williamson et al (1982) using a dye binding method but not with the fluorescence polarization data of Schachter and collaborators (Cogan & Schachter, 1981;Schachter et al, 1982). While we have no precise explanation for such discrepancy, it may be remarked that other authors have stressed that fluorescence polarization data on erythrocytes and ghosts may be hampered by artifacts due to light scattering as well as to the ghosting procedure (Aloni et al, 1974;Kutchai et al, 1982). Finally, our results are at variance with those of Rimon et al (1980) and Henis et al (1982).…”

Section: Discussionsupporting

confidence: 84%

Asymmetric lipid fluidity in human erythrocyte membrane: new spin-label evidence

Seigneuret¹,

Zachowski²,

Hermann³

et al. 1984

Biochemistry

141

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We have synthesized spin-labeled analogues of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine with a short beta chain (C5) bearing a doxyl group at the fourth position. When added to an erythrocyte suspension, the labels immediately incorporate in the membrane. The orientation of the spin-labels was assessed in the bilayer (i) by addition in the medium of a nonpermeant reducer (ascorbate at 5 degrees C) or (ii) by following spontaneous reduction at 37 degrees C due to the endogenous reducing agents present in the cytosol. Both techniques prove that the spin-labels are originally incorporated in the outer leaflet and redistribute differently after incubation. After a 5-h incubation at 5 degrees C, the phosphatidylcholine derivative remained in the outer layer, while the phosphatidylethanolamine and phosphatidylserine derivatives were found principally in the inner leaflet. During the incubation, a small fraction of the spin-labels is hydrolyzed, particularly the phosphatidylserine derivative, presumably by an endogenous phospholipase A2. Because the hydrolyzed spin-labeled fatty acids are rejected in the aqueous phase, the spectra of the intact membrane-bound phospholipids can be obtained by an adequate spectral subtraction. The ESR spectrum corresponding to a probe in the outer leaflet indicates a more restricted motion than that associated with probes in the inner leaflet. Additional experiments have been carried out to prove that the difference in viscosity, which is likely to be due to anisotropic cholesterol distribution, is not attributable to modification of the cell morphology.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Similar difficulties attend the measurement of the polarization of light emitted by fluorophores in turbid samples, since both the excitation and emission light is depolarized in passing through such samples. The depolarization of fluorescence resulting from dipole (i.e., molecular) scattering is less severe and was analyzed in detail by Teale (1), while the importance of correcting fluorescence polarization for scattering by large assemblies like erythrocytes was recently discussed by Kutchai et al (2). Lentz and his collaborators (3) have proposed an approximate correction factor for the emission anisotropy, which is linear with the sample's turbidity, as measured by a spectrophotometer.…”

Section: Introductionmentioning

confidence: 99%

Fluorometry of turbid and absorbant samples and the membrane fluidity of intact erythrocytes

Eisinger¹,

Flores²

1985

Biophysical Journal

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In employing intrinsic or extrinsic fluorophores in the study of whole cells, or other strongly absorbant and/or scattering samples, the measured fluorescence intensity and polarization is seriously affected by absorption and scattering within the sample cuvet. These artifacts are analyzed and simple protocols are provided for overcoming them. An expression relating attenuation of the observed emission anisotropy to sample turbidity is derived. The validity of the method is confirmed by experiments in which the emission anisotropies and fluorescence yields of membrane probes in intact erythrocytes was measured with precision. It is also shown that the rotational mobility of the membrane probe 1-phenyl-3-(2-naphthyl)-2-pyrazoline is the same for intact erythrocytes and ghosts. These protocols are particularly useful in measuring the intrinsic fluorescence yield ratio for excimeric and monomeric emission of pyrene-containing membrane probes. This provides a method for determining the local lateral mobility of excimeric probes in intact erythrocytes.

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Determination of Fluorescence Polarization of Membrane Probes in Intact Erythrocytes

Cited by 17 publications

References 10 publications

Water permeability through biological membranes by isotopic effects of fluorescence and light scattering

Water permeability through biological membranes by isotopic effects of fluorescence and light scattering

Asymmetric lipid fluidity in human erythrocyte membrane: new spin-label evidence

Fluorometry of turbid and absorbant samples and the membrane fluidity of intact erythrocytes

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