Biomedical subjects

E Gratton

Publications and source records attributed to E Gratton.

At least 91 records · Page 5Linked to original sources

PubMed1994

Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes.

Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered.

Binding Sites↗

PubMed1994

Cholesterol modifies water concentration and dynamics in phospholipid bilayers: a fluorescence study using Laurdan probe.

The effect of cholesterol on the gel, the liquid-crystalline, and mixed phospholipid phases has been studied using the fluorescence properties of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). Laurdan sensitivity to the polarity and to the dynamics of its environment reveals that cholesterol affects phospholipid bilayers in the gel phase by expelling water and by increasing the amount of dipolar relaxation. In the liquid-crystalline phase, the effect of cholesterol is a reduction of both water concentration and amount of dipolar relaxation. Detailed studies of Laurdan excitation and emission spectral contours as a function of cholesterol concentration show that there are some cholesterol concentrations at which Laurdan spectral properties changes discontinuously. These peculiar cholesterol concentrations are in agreement with recent observations of other workers showing the formation of local order in the liquid-crystalline phase of phospholipids upon addition of phospholipid derivatives of pyrene. A local organization of phospholipids around cholesterol molecule seems to be produced by the presence of peculiar concentrations of cholesterol itself. This local organization is stable enough to be observed during the excited state lifetime of Laurdan of approximately 5-6 ns.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.

Animals↗

PubMed1994

Evidence for an increase in water concentration in bilayers after oxidative damage of phospholipids induced by ionizing radiation.

The two membrane fluorescent probes 2-dimethyl-amino-6-lauroyl-naphthalene (Laurdan) and 2-dimethylamino-6-propionyl-naphthalene (Prodan) have been used to study the molecular basis of the damage induced in phospholipid membranes by ionizing radiation. Laurdan and Prodan display a spectral sensitivity to the polarity of their environment, showing a red shift of both excitation and emission spectra with increase of the polarity of their environment. Owing to their chemical differences, the two probes are anchored in the membrane with different strengths. In aqueous environments Laurdan is not fluorescent while Prodan shows appreciable fluorescence. Laurdan and Prodan show an opposite response to oxidative damage produced in phospholipid bilayers by ionizing radiation. The results support the model recently developed of water penetration in the bilayer as a consequence of oxidative damage.

Lipid Bilayers↗

PubMed1994

Feasibility of intracranial near-infrared optical scanning.

Light injected at a point on a surface of a scattering medium is emitted at the surface after traveling a quasisemicircular path deep into the medium. This phenomenon can be exploited to detect objects immersed in the medium from time-resolved measurements of light intensity at the surface. Our experiments on model systems demonstrate that absorbing objects, surrounded by bone and other scattering material, can be detected. The technique yields surface images of absorbing objects submerged in a scattering medium. Images of the same phantoms inside the cavity of a skull can be obtained by the same technique.

Brain↗

PubMed1993

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types.

Fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) are used to explore gel and liquid-crystalline phase domains coexistence in membranes of various cell types and in erythrocyte ghosts. Experiments and simulations were performed using liposomes composed of equimolar gel and liquid-crystalline phases in the absence and in the presence of 30 mol% cholesterol. In this model system two distinct coexisting phases can be easily recognized in the absence of cholesterol. When cholesterol is added to this phospholipid mixture, Laurdan parameters characteristic of the gel and of the liquid-crystalline phase are no longer resolvable. Coexisting domains of gel and liquid-crystalline phase were not detected in any of the examined cell membranes as judged by Laurdan excitation and emission Generalized Polarization (GP) spectra. Both in liposomes and in cell membranes, the behaviour of GP values as a function of excitation and emission wavelength corresponds to a homogeneous liquid-crystalline phase, despite the absolute GP values being relatively high, closer to the values observed in gel phase phospholipids. The presence of cholesterol appears to be the major cause for the homogeneity of phospholipids' dynamical properties in natural membranes, properties that appear close to the liquid-ordered phase state, defined to describe model systems with cholesterol concentration > or = 30 mol%.

2-Naphthylamine↗

PubMed1993

Structure and dynamics of the acidic compact state of apomyoglobin by frequency-domain fluorometry.

The conformational dynamic properties of tuna apomyoglobin, a single tryptophan-containing protein, in the acidic compact state, as well as in the native and in the fully unfolded state, have been explored by frequency-domain fluorometry. Apomyoglobin at acidic pH in the presence of high salt concentration displays bimodal tryptophanyl lifetime distributions which may be related to the simultaneous presence of different populations of structural states (compact and fully unfolded states). The tryptophanyl anisotropy decay indicated that the acidic compact state displays at least two rotational correlational times, suggesting that this state possesses a complex geometrical organization. 1-Anilino-8-naphthalene sulfonate (ANS), bound both to native and compact protein forms, shows broad unimodal lifetime distributions. The small time dependence of the ANS emission spectra indicated that the solvent dipolar reorganization are either absent or they occur on a time scale much shorter than the lifetime of the excited ANS molecule bound to apomyoglobin. The anisotropy decay data relative to the extrinsic fluorophore (ANS) are consistent with the presence of a single rotational correlation time for both native (12.1 ns) and compact (6.2 ns) states.

Anilino Naphthalenesulfonates↗

PubMed1993

Effect of divalent cations on Na+,K(+)-ATPase obtained from human placenta.

Circular dichroism (CD) and acrylamide quenching studies of Na+,K(+)-ATPase from human placenta showed that its incorporation into phosphatidylcholine vesicles increased the enzymic activity by 55%. Moreover, both with the purified and the vesicle-reconstituted protein, Ca2+ and Mg2+ increased the activity, the effect being more pronounced after preincubation of the protein with Mg2+. CD data suggest that this activity increase may be linked to a change in the secondary structure of the ATPase, in particular beta-turn, beta-sheet and random coil. Acrylamide quenching studies suggest that ions could primarily interact with phospholipid head groups, but not directly with the protein.

Calcium↗

PubMed1993

Molten globule monomers in human superoxide dismutase.

The time-resolved fluorescence decay and anisotropy of Cu/Zn human superoxide dismutase (HSOD) were studied as a function of temperature and denaturant concentration. In addition, circular dichroism (CD) measurements were performed on HSOD as a function of denaturant concentration in the amide and aromatic regions. The time-resolved fluorescence decay results reveal the existence of structural microheterogeneity in HSOD. Furthermore, CD measurements and a global analysis decomposition of the time-resolved fluorescence decay over denaturant concentration shows the presence of an intermediate in the unfolding of HSOD by guanidinium hydrochloride. Considering our previous measurements of partially denatured HSOD as a function of protein concentration (Mei et al., Biochemistry 31 (1992) 7224-7230), our results strongly suggest that the unfolding intermediate is a monomer that displays a molten globule state.

Circular Dichroism↗

PubMed1993

Designing matrix models for fluorescence energy transfer between moving donors and acceptors.

A recipe is given for designing theoretical models for donor-acceptor systems in which fluorescence energy transfer and motion takes place simultaneously. This recipe is based on the idea that a system exhibiting both motion and fluorescence energy transfer can be modeled by specifying a number of "states" and the rates of transitions between them. A state in this context is a set of specific coordinates and conditions that describe the system at a certain moment in time. As time goes on, the coordinates and conditions for the system change, and this evolution can be described as a series of transitions from one state to the next. The recipe is applied to a number of example systems in which the donors and/or acceptors undergo either rotational or translational motion. In each example, fluorescence intensities and anisotropies for the donor and acceptor are calculated from solutions of eigensystems. The proposed method allows for analyzing time-resolved fluorescence energy transfer data without restrictive assumptions for motional averaging regimes and the orientation factor. It is shown that the fluorescence quantities depend on the size of the motional step (i.e., on the number of states), only if fluorescence energy transfer occurs. This finding indicates that fluorescence energy transfer studies may reveal whether the dynamics of a system (e.g., a protein) is better described in terms of transitions between a relatively small number of discrete states (jumping) or a large number of dense states (diffusion).

Biophysical Phenomena↗

PubMed1993

Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence.

Steady-state and dynamic fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) have been used to ascertain the coexistence of separate phase domains and their dynamic properties in phospholipid vesicles composed of different mole ratios of dilauroyl- and dipalmitoyl-phosphatidylcholine (DLPC and DPPC, respectively). The recently introduced generalized polarization together with time-resolved emission spectra have been utilized for detecting changes. The results indicate the coexistence of phospholipid phase domains in vesicle compositions in the range between 30 mol% and 70 mol% DPPC in DLPC. Below and above these concentrations a homogeneous phase is observed, with averaged properties. In the case of coexisting phase domains, the properties of each individual phase are largely influenced by the presence of the other phase. Implications on fluctuations between the coexisting phases and on the size and shape of domains are discussed.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1993

Lipid phases in renal brush border membranes revealed by Laurdan fluorescence.

Steady-state fluorescence properties of 6-dodecanoyl-2-dimethylaminonapththalene (Laurdan) have been used to determine the coexistence of separate lipid-phase domains in apical brush border membrane vesicles isolated from the rat renal cortex. The temperature dependence of generalized polarization has been utilized to quantitate the lipid phases. Finally, the effect of cholesterol enrichment on these parameters has been studied. The results indicate the coexistence of lipid-phase domains in brush border membranes based on the values of the generalized polarization and assuming that the membrane state can be described by coexistence of gel and liquid crystalline state. At 37 degrees C, approximately 19-32% of the brush border membrane lipids are in the gel phase, and cholesterol enrichment causes a further concentration-dependent increase in the gel phase of brush border membrane lipids.

2-Naphthylamine↗

PubMed1993

Use of Laurdan fluorescence in studying plasma membrane organization of polymorphonuclear leukocytes during the respiratory burst.

The changes in plasma membrane polarity of polymorphonuclear leukocytes (PMN) during the activation of the respiratory burst were investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-lauroyl) naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surroundings. Laurdan shows a marked steady-state emission blue shift in nonpolar solvents, with respect to polar solvents. Our results show a blue shift of the fluorescence emission spectra of Laurdan during activation of PMN with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine. These results suggest that the activation of the respiratory burst of PMN is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.

2-Naphthylamine↗

PubMed1993

Multifrequency cross-correlation phase fluorometry of chlorophyll a fluorescence in thylakoid and PSII-enriched membranes.

We present here a comparative study on the decay of chlorophyll (Chl) a fluorescence yield in thylakoid membranes and photosystem II (PSII)-enriched samples, measured with multifrequency cross-correlation phase fluorometry. These measurements confirm the general conclusions of Van Mieghem et al. (Biochim. Biophys. Acta 1100, 198-206, 1992), obtained with a flash method, on the effects of reduction of the primary quinone acceptor (QA) on Chl a fluorescence yield of PSII. Different states of the reaction centers of PSII were produced by: (1) pretreatment with sodium dithionite and methyl viologen followed by laser illumination: the doubly reduced QA (QAH2) centers; (2) with laser illumination or pretreatment with diuron: QA- centers; and (3) the addition of micromolar concentration of dichlorobenzoquinone (DCBQ): oxidized QA centers. The data were analyzed with Lorentzian distribution as well as with multiexponential fluorescence decay functions. The analysis with Lorentzian distribution function showed that upon formation of QA-, the major lifetime distribution peak shifted to longer lifetimes: from 0.25 ns to 1.66 ns (pea thylakoid membranes) and from 0.24 ns to 1.31 ns (core PSII). However, when QAH2 was formed, the lifetime distribution peaks shifted back to shorter lifetimes (0.57-0.77 ns) both in thylakoids and PSII membranes. Multiexponential analysis showed three lifetime components: fast (40-400 ps), middle (300-1500 ps) and slow (5-25 ns). When QA- was formed in PSII centers, the amplitude of the fast component decreased, but both the amplitude and the lifetime of the middle component increased severalfold.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorophyll↗

PubMed1993

Propagation of photon-density waves in strongly scattering media containing an absorbing semi-infinite plane bounded by a straight edge.

Light propagation in strongly scattering media can be described by the diffusion approximation to the Boltzmann transport equation. We have derived analytical expressions based on the diffusion approximation that describe the photon density in a uniform, infinite, strongly scattering medium that contains a sinusoidally intensity-modulated point source of light. These expressions predict that the photon density will propagate outward from the light source as a spherical wave of constant phase velocity with an amplitude that attenuates with distance r from the source as exp(-alpha r)/r. The properties of the photon-density wave are given in terms of the spectral properties of the scattering medium. We have used the Green's function obtained from the diffusion approximation to the Boltzmann transport equation with a sinusoidally modulated point source to derive analytic expressions describing the diffraction and the reflection of photon-density waves from an absorbing and/or reflecting semi-infinite plane bounded by a straight edge immersed in a strongly scattering medium. The analytic expressions given are in agreement with the results of frequency-domain experiments performed in skim-milk media and with Monte Carlo simulations. These studies provide a basis for the understanding of photon diffusion in strongly scattering media in the presence of absorbing and reflecting objects and allow for a determination of the conditions for obtaining maximum resolution and penetration for applications to optical tomography.

Animals↗

PubMed1992

Denaturation of human Cu/Zn superoxide dismutase by guanidine hydrochloride: a dynamic fluorescence study.

The unfolding of holo and apo forms of human Cu/Zn superoxide dismutase by guanidine hydrochloride has been investigated by steady-state and dynamic fluorescence. In agreement with previous observations, a stabilizing effect of the metal ions on the protein tertiary structure was apparent from comparison of apo- and holoproteins, which both showed a sharp sigmoidal transition though at different denaturant concentrations. The transition was also followed by circular dichroism to check the extent of secondary structure present at each denaturant concentration. The results are incompatible with a simple two-state mechanism for denaturation. The occurrence of a more complicated process is supported by the emission decay properties of the single tryptophanyl residue at different denaturant concentrations. A complex decay function, namely, two discrete exponentials or a continuous distribution of lifetimes, was always required to fit the data. In particular, the width of the lifetime distribution, which is maximum at the transition midpoint, reflects heterogeneity of the tryptophan microenvironment and thus the presence of different species along the denaturation pathway. In the unfolded state, the width of the lifetime distribution is broader than in the folded state probably because the tryptophan residue is affected by a larger number of local conformations. The dissociation of the dimer was also studied by varying the protein concentration at different denaturant concentrations. This process affects primarly the surface of the protein rather than its secondary structure as shown by a comparison between the tryptophan emission decay and circular dichroism data under the same conditions. Another consequence of dissociation is a greater instability in the structure of the monomers, which are more easily unfolded.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoenzymes↗

PubMed1992

Membrane aging during cell growth ascertained by Laurdan generalized polarization.

The sensitivity of the fluorescent probe Laurdan to the phase state of lipids has been utilized to detect modifications in the composition and physical state of cell membranes during cell growth. In phospholipid vesicles, the Laurdan emission spectrum shows a 50-nm red shift by passing from the gel to the liquid-crystalline phase. The Generalized Polarization (GP) value has been used for the data treatment instead of the ratiometric method common in investigations utilizing other fluorescent probes that display spectral sensitivity to medium properties. The GP value can be measured easily and quickly and possesses all the properties of "classical" polarization, including the additivity rule. Once Laurdan limiting GP values have been established for the gel and the liquid-crystalline phase of lipids, the quantitative determination of coexisting phases in natural samples is possible. In the present work the observation of a relevant decrease in the fractional intensity of the liquid-crystalline phase in K562 cell membranes during 5 days of asynchronous growth is reported. A decrease in the "fluidity" of cell membranes in K562 cells kept in culture for several months is also reported. The procedure developed for labeling cell membranes with Laurdan is reported and the influence of cell metabolism on fluorescence parameters is discussed. Also discussed is the influence of cholesterol on Laurdan GP.

2-Naphthylamine↗

PubMed1992

Membrane oxidative damage induced by ionizing radiation detected by diphenylhexatriene fluorescence lifetime distributions.

The sensitivity of the fluorescence lifetime of 1,6-diphenyl- 1,3,5-hexatriene (DPH) to the dielectric constant of its environment has been used to detect oxidative damage to phospholipid membranes induced by ionizing radiation. The DPH fluorescence decay in phospholipid vesicles is described well by a continuous distribution of lifetime values, reflecting the various DPH depths in the bilayer and related to the gradient of the dielectric constant. Ionizing radiation oxidizes unsaturated acyl residues of phospholipids, altering the dielectric constant across the bilayer, sharpening the distribution of DPH lifetimes and increasing the centre of the distribution. Ionizing radiation doses between 22 and 110 Gy were used, and were effective only in the presence of oxygen. A model based on the formation of packing defects in the bilayer describes the phenomenon.

Diphenylhexatriene↗