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J R Lakowicz

Publications and source records attributed to J R Lakowicz.

At least 217 records · Page 12Linked to original sources
PubMed1981

Dynamic properties of the lipid-water interface of model membranes as revealed by lifetime-resolved fluorescence emission spectra.

We examined the dynamic properties of the lipid-water interface region of model membranes, on the nanosecond time scale, by using the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). In particular, we examined the steady-state emission spectra of TNS as its average lifetime was decreased by oxygen quenching. Under these quenching conditions the centers of gravity (Vcg) of the emission spectra shift ot shorter wavelengths. The lifetime dependence of these shifts reveals the time dependence of membrane relaxation around the excited-state dipole moment of TNS. The lipids investigated include dioleoyl-, dimyristoyl-, and dipalmitoyl-L-alpha-phosphatidylcholines, bilayers containing cholesterol, and an ether analogue of dipalmitoyl-L-alpha-phosphatidylcholine. For these lipids, the spectral relaxation times and the temperature dependence of the relaxations are similar in magnitude. Most relaxation times fall in the range of 0.6-6 ns, and except for the ether analogue, the inactivation energies for spectral relaxation are 10 plus or minus 2 kcal/mol. The average energy loss during spectral relaxation was 1000 cm(-1). However, for the saturated phosphatidylcholines at temperatures below their transition temperatures, smaller relaxation losses were observed (approximately 600 cm(-1)). We attribute these smaller losses to ordering of the polar head groups around the ground-state dipole moment of TNS. Overall, these results indicate that the dynamic properties of the lipid-water interface region are similar among the phosphatidylcholines and depend only slightly on the chemical composition and phase state of the acyl side chains.

Fluorescent Dyes↗
PubMed1981

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry.

The measurement of fluorescence lifetimes is known to be hindered by the wavelength-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize these effects in photomultiplier tubes used for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelengths of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatterer decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelength. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from -55 to +55 degrees C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference lifetime, to calculate the actual phase and modulation of the excitation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of NADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.

Indicators and Reagents↗
PubMed1981

Phase-sensitive fluorescence spectroscopy: a new method to resolve fluorescence lifetimes or emission spectra of components in a mixture of fluorophores.

A novel phase fluorometric method is described which permits direct recording of individual emission spectra from a mixture of two fluorescent compounds. Additionally, the lifetimes of each component may be determined by examination of the phase-sensitive fluorescence spectra. The method utilizes phase-sensitive detection of the sinusoidally modulated emission from a phase fluorometer. Resolution of the individual emission spectra in the mixture requires different fluorescence lifetimes for each component. Determination of the individual lifetimes requires knowledge of the steady-state emission spectra of the components. Use of low-frequency (approximately equal to 10(6) Hz) cross-correlated signals eliminates the need for high-frequency (approximately equal to 10(6) HZ) phase-sensitive detection. A mixture of 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS) and 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) was used to demonstrate the possibility of phase resolution of fluorophore mixture and to confirm theoretical predictions. A mixture of dibenzo[a,h]anthracene and dibenzo[c,g]carbazole was used to demonstrate that phase resolution is possible for spectra which overlap strongly and which are highly structured. In addition, the possibility of using phase-sensitive emission spectra for the resolution of excited-state reactions was demonstrated with anthracene and its diethylaniline exciplex. From a sample whose steady-state emission displayed both components we directly recorded the emission spectrum of anthracene monomer and the exciplex. For all these samples the dependence of the individual intensities on the phase angle of the detector agreed precisely with that expected on the basis of the individual fluorescence lifetimes. The detector phase angles chosen for suppression of each component in the mixture also agreed with the measured lifetimes. Thus, phase-sensitive fluorescence spectra can reveal individual spectral distributions or lifetimes. This method will be useful in the analysis of heterogeneous fluorescence emissions which frequently occur from proteins, membranes and other biological samples.

Fluorescent Dyes↗
PubMed1981

Fluorescence spectroscopic studies of Huntington fibroblast membranes.

Using fluorescence spectroscopic methods, we compared the membrane properties of intact fibroblasts from both normal subjects and patients with Huntington disease (HD). Cells were stained with various fluorophores, including 1-anilino-8-naphthalene sulfonic acid (ANS), 2-toluidinyl-6-naphthalene sulfonic acid (TNS), 1,6-diphenyl-1,3,5-hexatriene (DPH), and 6-lauroyl-2-(dimethylamino)-naphthalene (LAURDAN). Using these labeled cells, we measured fluorescence yields and emission maxima (ANS, TNS, and LAURDAN), polarizations (TNS, DPH, and LAURDAN), lifetimes (TNS), and differential polarized lifetimes (DPH). In each instance, comparisons were made between cells from normal and from HD individuals. These cultures were controlled for passage number in culture and for age of donor. We found no significant differences between the HD and the control fibroblasts in experiments using the above-mentioned probes and spectroscopic parameters.

Cell Membrane↗
PubMed1980

Transport of a carcinogen, benzo[a]pyrene, from particulates to lipid bilayers: a model for the fate of particle-adsorbed polynuclear aromatic hydrocarbons which are retained in the lungs.

Fluorescence spectroscopic methods were used to investigate the effects of adsorption of benzo[a]pyrene to particulate matter on its rate of uptake into model membranes composed of dipalmitoyl L-alpha-phosphatidylcholine. From these experiments we conclude the following: 1. Adsorption of benzo[a]pyrene to four types of asbestos (anthophyllite, crocidolite, chrysotile, and amosite) and a variety of non-fibrous particles (hematite, silica, titanium dioxide, porous glass and talc) results in increased rates of membrane uptake when compared with aqueous suspensions of benzo[a]pyrene microcrystals. Benzo[a]pyrene was not released from carbon black. 2. Asbestos-adsorbed benzo[a]pyrene was transferred to the membranes most rapidly. 3. Adsorption of benzo[a]pyrene to the surface of the particles is necessary for its enhanced transport into membranes. That is, simple mixtures of benzo[a]pyrene microcrystals and particulates do not show enhanced transport. 4. Particle-enhanced transport of benzo[a]pyrene is not correlated with the effects of the particles on vesicle integrity, binding of vesicles to the particles, or the concentrations of either particles or vesicles. The rate limiting step for transport of benzo[a]pyrene into vesicles appears to be its rate of desorption from the surface of the particle. Following desorption, membrane uptake of benzo[a]pyrene is rapid.

Adsorption↗
PubMed1980

Hindered depolarizing rotations of perylene in lipid bilayers. Detection by lifetime-resolved fluorescence anisotropy measurements.

Oxygen quenching of perylene fluorescence was used to vary its fluorescence lifetime. Steady-state fluorescence anisotropy measurements under these quenching conditions were used to investigate the diffusive motions of perylene in the isotropic solvent propylene glycol and in lipid bilayers. These lifetime-resolved anisotropy measurements indicate that the anisotropy of perylene in propylene glycol decays to zero at times long compared to its fluorescence lifetime. In contrast, the asymptotic or "limiting" anistropy values at these long times (r infinity) are nonzero in vesicles of dimyristoyl-phosphatidylcholine (DMPC). r infinity values are largest at temperatures below the DMPC phase transition temperature of 23 degrees C. Representative values of r infinity for perylene in DMPC vesicles are 0.16 and 0.02 at 5 and 47 degrees C, respectively. Thus, in contrast to the free rotations observed for perylene in propylene glycol, perylene rotations are hindered in lipid bilayers. Less marked, yet significant, rotational hindrance was observed in dioleoylphosphatidylcholine (DOPC) vesicles. Representative values for r infinity in this unsaturated lipid are 0.05 and 0.01 at 2 and 45 degrees C, respectively. Steady-state anisotropy measurements with short-wavelength excitations were used to investigate whether the in-plane or out-of-plane rotations of perylene were responsible for the observed r infinity values. In DMPC vesicles we conclude that both rotations are partially hindered. In DOPC vesicles we can only conclude that one or both of these rotations are partially hindered, but both are not free. Most importantly, the existence of fundamentally different diffusive behavior for perylene in solvents and in lipids calls into question the meaning of membrane microviscosities which are derived via such comparisons.

Benz(a)Anthracenes↗
PubMed1980

Dipolar relaxation in proteins on the nanosecond timescale observed by wavelength-resolved phase fluorometry of tryptophan fluorescence.

We used phase fluorometry to investigate the wavelength dependence of the fluorescence lifetimes of N-acetyl-L-tryptophanamide (NATA) in solvents of varying viscosity and the lifetimes of tryptophan in human serum albumin, melittin, and liver alcohol dehydrogenase. In highly fluid solvents, and in completely vitrified solvents, the lifetime of NATA was constant across its emission spectrum. In viscous solvents, such as propylene glycol at -9 degrees C, the lifetimes of NATA increased across its emission spectrum, with the values being 3.3, 5.5, and 8.1 ns at 317, 344, and 400 nm, respectively. These wavelength-dependent lifetimes appear to be a result of reorientations of solvent dipoles around the excited state dipole moment of the indole moiety. For the three proteins investigated, the fluorescence lifetimes of tryptophan increased with increasing wavelength in a manner comparable to that observed for NATA in propylene glycol. These observations indicate that these protein matrices can reorientation around their tryptophan residues on the nanosecond timescale, and illustrate the potential of phase fluorometry for quantifying the details of these dipolar relaxation processes.

Alcohol Oxidoreductases↗
PubMed1980

Benzo[a]pyrene uptake into rat liver microsomes: effects of adsorption of benzo[a]pyrene to asbestos and non-fibrous mineral particulates.

The fluorescence yield of benzo[a]pyrene (BP) increases dramatically upon its transfer from the surface of particulates to rat liver microsomes. Adsorption of BP to Canadian chrysotile, anthophyllite, hematite and silica results in greatly enhanced uptake rates into microsomes when compared to uptake from a microcrystalline dispersion of BP. The fibrous minerals chrysotile and anthophyllite were more effective than silica and hematite in enhancing BP uptake. Simple mixtures of BP microcrystals and particles did not display enhanced transport, indicating that adsorption of BP to the particulate surface is necessary for enhanced microsomal uptake. BP was not released into microsomes from carbon black. We suggest that particulate-enhanced availability of BP may be of significance in the co-carcinogenesis between particulates and polynuclear aromatic hydrocarbons. However, other mechanisms are also possible, and are not excluded by our experiments. The fluorescence methodology, described in this paper provides a novel and convenient means to quantify microsomal uptake of BP and thereby investigate further the mechanisms of cocarcinogenesis.

Adsorption↗
PubMed1980

Permeability of lipid bilayers to methylmercuric chloride: quantification by fluorescence quenching of a carbazole-labeled phospholipid.

We investigated the permeabilities of lipid bilayers to the neurotoxin methylmercuric chloride (MMC). This mercurial is an efficient collisional quencher of the fluorescence of N-alkyl carbazole derivatives. Quenching of the fluorescence of beta-(3-(9-carbazole)-propionyl-L-alpha-phosphatidylcholine (CPA-PC) in vesicles of dimyristoyl phosphatidylcholine and of dioleoyl phosphatidylcholine reveal rapid diffusion of MMC in the alkyl side chain regions of these bilayers. By a combination of (1) the lipid concentration dependence of the apparent quenching constants, (2) the solubility of MMC in concentrated lipid dispersions and (3) the 270 MHz proton magnetic resonance of methylmercury in the presence of lipid bilayers we conclude that the lipid-water partition coefficient of this mercurial is less than or equal to two. Using the fluorescence quenching and the partitioning data we estimate the diffusion coefficient of MMC in these bilayers to range from 0.13 to 0.31 X 10(-5) cm2/sec, or 20--47% of its diffusion coefficient in ethanol. These data indicate that lipid bilayers do not pose a significant permeability barrier to the diffusional transport of MMC.

Diffusion↗
PubMed1980

Chlorinated hydrocarbon-cell membrane interactions studied by the fluorescence quenching of carbazole-labeled phospholipids: probe synthesis and characterization of the quenching methodology.

In recognition of the need to understand better the interactions of the chlorinated hydrocarbon insecticides with cell membranes we investigated the use of fluorescence quenching of membrane-bound fluorophores by these chlorinated hydrocarbons. An extensive survey of potential fluorophores identified the N-alkyl derivatives of carbazole as being especially suitable fluorophores. The fluorescence emission of these derivatives is quenched by a wide variety of commonly-used chlorinated hydrocarbons. This quenching is collisional and does not result in significant photodecomposition. Four structurally distinct carbazole-labeled phospholipids were synthesized, and their structures were confirmed by 270 MHz proton NMR and by chromatographic and chemical means. The carbazole moiety of each labeled phospholipid should be localized at a different depth in lipid bilayer. However, water soluble quenchers indicate that the fluorophores are inaccessible to the aqueous phase, irrespective of their point of attachment to the phospholipids. When incorporated into lipid bilayers, the fluorescence lifetime of these carbazole-labeled phospholipids reveals the collisional frequency between the fluorophore and the chlorinated hydrocarbon. As a result quenching of membrane-bound fluorophores may be used to measure: (1) the diffusional rate of the chlorinated hydrocarbon in the bilayer; (2) the lipid-water partition coefficient; (3) the maximum binding capacity of the membrane for the chlorinated hydrocarbon. Examples of all these measurements are given, and the fluorometric results are confirmed by direct chemical analysis.

Carbazoles↗
PubMed1980

Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies.

Steady-state and lifetime-resolved fluorescence anisotropy measurements of protein fluorescence were used to investigate the depolarizing motions of tryptophan residues in proteins. Lifetime resolution was achieved by oxygen quenching. The proteins investigated were carbonic anhydrase, carboxypeptidase A, alpha-chymotrypsin, trypsin, pepsin, and bovine and human serum albumin. When corrected for overall protein rotation, the steady state anisotropies indicate that, on the average, the tryptophan residues in these proteins rotate 29 degrees +/- 6 degrees during the unquenched excited state lifetimes of these proteins, which range from 1.7 to 6.1 ns. The lifetime-resolved anisotropies reveal correlation times for these displacements ranging from 1 to 12 ns. On the average these correlation times are tenfold shorter than that expected for overall protein rotation. We conclude that the tryptophan residues in these proteins display remarkable freedom of motion within the protein matrix, which implies that these matrices are highly flexible on the nanosecond time scale.

Animals↗
PubMed1979

Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations.

Differential polarized phase fluorometry has been used to investigate the depolarizing rotations of 1,6-diphenyl-1,3,5-hexatriene (DPH) in isotropic solvents and in lipid bilayers. For DPH dissolved in isotropic solvents, there is a precise agreement between the observed and predicted values for maximum differential tangents, indicating that in these media DPH is a free isotropic rotator. In lipid bilayers the tangent defects (i.e., the differences between the calculated and the observed maximum differential tangents) are too large to be explained by anisotropy in the depolarizing rotations but are accounted for by hindered isotropic torsional motions for the fluorophore [Weber, G (1978) Acta Phys. Pol A 54, 173]. This theory describes the depolarizing rotations of the fluorophore by its rotational rate R (in radians/second) and the limiting fluorescence anisotropy (r) at times long compared with the fluorescence lifetime. Through the combined use of both steady-state anisotropy measurements and differential phase measurements, we have demonstrated that one may obtain unique solutions for both R and r. For DPH embedded in vesicles prepared from dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholines, the depolarizing motions are highly hindered at temperatures below the transition temperature (Tc) but are unhindered above Tc. The apparent rotational rates of the probe do not change significantly at Tc. These data suggest that the changes observed in the steady-state anisotropy near Tc derive primarily from changes in the degree to which the probe's rotations are hindered, and only to a small extent from changes in rotational rate. For DPH embedded in bilayers that contained 25 mol % cholesterol, no clear transition occurred and the rotations appeared to be hindered at all temperatures. The rotational motions of DPH embedded in dioleolyphosphatidylcholine were found to be far less hindered, but the rotational rates were similar to those obtained in the saturated phosphatidylcholines. Finally, the data show that in an anisotropic environment, such as that of a lipid bilayer, steady-state fluorescence anisotropy measurements alone cannot yield quantitatively meaningful rotational rates. Extrapolation of steady-state aniosotropy data to the quantitation of membrane viscosity is therefore difficult, if not invalid; however, qualitative comparisons can be useful.

Fluorescence Polarization↗