Quantitation of Hindered Rotations of Diphenylhexatriene in Lipid Bilayers by Differential Polarized Phase Fluorometry

Lakowicz,; Prendergast, F. G.

doi:10.1126/science.663620

Cited by 106 publications

(32 citation statements)

References 14 publications

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“…Anisotropy decay experiments were performed by differential phase fluorimetry using vertically and horizontally polarized excitation light modulated at 30 MHz (Lakowicz & Prendergast, 1978;Lakowicz et al, 1979). The differential phase angle A is defined by,…”

Section: Methodsmentioning

confidence: 99%

Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

Cabrini

Verkman

1986

J. Membrain Biol.

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The equilibrium binding mechanism and kinetics of binding of diS-C3-(5) (3,3'-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS-C3-(5) exists as a monomer at concentrations less than 5 microM with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropy r = 0.102, and lifetime tau = 1.2 nsec (23 degrees C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K+/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS-C3-(5) rotation is unhindered with a rotational rate R = 0.57 nsec-1 while membrane monomer is hindered with steady-state anisotropy r = 0.190, lifetime tau = 2.1 nsec, R = 0.58 nsec-1 and limiting anisotropy r infinity = 0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant, Kd = [M]2/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS-C3-(5). There is rapid binding of diS-C3-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.

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Section: Methodsmentioning

confidence: 99%

Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

Cabrini

Verkman

1986

J. Membrain Biol.

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“…Fluorescence lifetimes were always measured with a polarizer in the emission path oriented to 54.7O to eliminate effects due to Brownian rotation, and invariably a solution of POPOP in ethanol was used as a reference lifetime standard ( T~~~ = 1.35 ns in ethanol; F. G. Prendergast and P. J. Callahan, unpublished results) to minimize possible artifacts due to wavelength-dependent response of the photomultiplier tubes. Differential phase lifetimes were determined by the method originally described by Mantulin & Weber (1977) as used by Lakowicz & Prendergast (1978). Values for r, were determined by the method of Weber & Bablouzian (1966); ro values for TMA-DPH were determined by measurement of rss for the fluorophore dissolved in propylene glycol, maintained at -65 OC, and found to be 0.390.…”

Section: Methodsmentioning

confidence: 99%

1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties and use as a fluorescence probe of lipid bilayers

Prendergast¹,

Haugland²,

1981

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1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a cationic analogue of diphenylhexatriene (DPH), has photophysical properties that are generally similar to those of DPH. In solution the fluorescence lifetime (tau) of TMA-DPH is short (less than 1.5 ns), but tau increases to approximately 7 ns when the probe is embedded in lipid bilayers at temperatures less than the thermal transition temperature (Tc) of the lipid. The cationic charge ensures that the probe is anchored at the lipid-water interface, most likely with the DPH moiety intercalated between the upper portions of the fatty acyl chains. The profiles of changes in steady-state anisotropies (rss) and limiting hindered anisotropies (r infinity) are similar for both TMA-DPH and DPH embedded in lipid bilayers, but r infinity values for TMA-DPH even at T much greater than Tc are generally greater than 0.14, e.g., at 35 degrees C in 1,2-dimyristoylglycero-3-phosphocholine (DMPC) (cf. 0.03 for DPH in DMPC at 35 degrees C). Electrostatic interactions of the cationic probe with head groups of phospholipids do not appear to significantly influence the apparent dynamics of the probe. TMA-DPH should prove useful in the study of the dynamics of phospholipid monolayers, e.g., in native or reconstituted lipoproteins.

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“…Apparent rotation rates were calculated from steady-state anisotropy and fluorescence lifetime measurements (phase, [5,19,20,22,23,241.…”

Section: Fluorescence Spectroscopymentioning

confidence: 99%

Platelet membrane abnormality in Alzheimer's disease

Zubenko

Cohen

Boller

et al. 1987

Annals of Neurology

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Fluorescence studies of membrane fluidity were conducted double-blind using platelet and red cell membranes prepared from 24 demented patients with probable Alzheimer's disease and 36 neurologically healthy subjects. The fluidity of the hydrocarbon and lipid-aqueous interface regions of cell membranes was determined at 37 degrees C by fluorescence spectroscopy using the lipid probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), respectively. The rotation rate of TMA-DPH in labeled platelet membranes did not differ between the groups. In contrast, the rotation rate of DPH in labeled platelet membranes from the demented patients (2.15 +/- 0.24 X 10(8)/sec, SD) was greater than that for the normal controls (1.93 +/- 0.13 X 10(8)/sec, SD, p = 3.8 X 10(-5)). This difference was reflected by a reduction in the steady-state anisotropy of DPH in labeled platelets from the demented group (0.1887 +/- 0.0085, SD) when compared to the respective mean for the controls (0.2000 +/- 0.0060, SD; p = 1.3 X 10(-7)). Abnormal membrane fluidity was significantly correlated with severity of dementia, but not with duration of illness or apparent age of onset. The findings do not support the hypothesis that Alzheimer's disease results from a pathological acceleration of the normal aging process, since normal aging is associated with decreased fluidity of cell membranes from platelets, as well as from lymphocytes, hepatocytes, and neurons.

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Quantitation of Hindered Rotations of Diphenylhexatriene in Lipid Bilayers by Differential Polarized Phase Fluorometry

Cited by 106 publications

References 14 publications

Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties and use as a fluorescence probe of lipid bilayers

Platelet membrane abnormality in Alzheimer's disease

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