Polarized light spectroscopy of photosynthetic membranes in magneto-oriented whole cells and chloroplasts. Fluorescence and dichroism

1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10–13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes.

2. The FP ratio is typically in the range of 1.2–1.9 in Chlorella, 1.20–1.25 in Scenedesmus and 1.4–1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Q_y transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented.

3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720–730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615–617).

4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01–0.06) determined by previous workers with unoriented cell suspensions.

5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered.

6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Q_y transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane.

7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena.

8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.     

The light-harvesting chlorophyll a/b protein acts as a torque aligning chloroplasts in a magnetic field

Displacement of particles from the purified light-harvesting chlorophyll a/b protein aggregate (LHC) was studied in magnetic fields of various strengths (0 to 1.6 T) by polarized fluorescence measurements. Macromolecular aggregates of LHC have a considerable magnetic susceptibility which enables the particles to rotate and align with their nematic axes parallel with H. As LHC is embedded in a transmembrane direction thylakoids should align perpendicular to H, the mode of alignment experimentally observed in thylakoids. The value of the magnetic susceptibility could be estimated by relating it to the integral susceptibility of the chlorophyll molecules in LHC. The fitting of this value with the field strength dependency of the fluorescence polarization ratio (FP) revealed a relationship between the LHC content of various photosynthetic membranes and their capacity for alignment, which suggested that LHC might be the torque ordering chloroplasts in a magnetic field.Abbreviations LHC light-harvesting chlorophyll a/b protein - FP fluorescence polarization ratio, Iz/Iy     

Effect of phospholipid:protein ratio on the state of aggregation of the (Ca2+-Mg2+)-ATPase

The organization of the (Ca2+-Mg2+)-ATPase has been studied in reconstituted systems by fluorescence polarization of the ATPase labeled with fluorescein isothiocyanate (FITC) and resonance energy transfer between ATPase labeled with FITC and with eosin isothiocyanate (EITC). The fluorescence polarization of FITC-ATPase was found to decrease with increasing labeling ratio FITC:ATPase, indicating depolarization as a result of resonance energy transfer between ATPase molecules. Fluorescence polarization was, however, independent of the molar ratio of phospholipid to protein above a molar ratio of 50:1. Resonance energy transfer between FITC-ATPase and EITC-ATPase was also found to be independent of phospholipid:protein ratio. It is suggested therefore that the ATPase is not randomly distributed in the plane of the membrane but rather forms ordered clusters (probably rows of monomers or dimers) on the fluorescence time scale (nanoseconds) even in the presence of a large excess of phospholipid. This organization within the membrane is dependent both on the chemical structure of the phospholipid and on its physical phase.     

Excitation of magnetic fields by a circularly polarized laser pulse in a plasma channel

The excitation of quasistatic magnetic fields by a circularly polarized laser pulse in a plasma channel is considered. It is shown that, to second order in the amplitude of the electric field of the laser pulse, circular rotation of the plane of polarization of the laser radiation in a radially nonuniform plasma gives rise to a nonlinear azimuthal current and leads to the excitation of the radial and axial components of the magnetic field. The dependence of the magnetic field distribution over the plasma channel on the spatial dimensions of the pulse and on the channel width is investigated for a moderate-power laser pulse. The structure of the magnetic fields excited by a relativistic laser pulse in a wide plasma channel is analyzed.     

Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure

Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3',5'-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein-dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.     

Temperature dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in phophoslipid artificial membranes.

The fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles is a function of the physical state of the lipid. Below the phase transition, the polarization approaches the theoretical maximum for total immobilization while above the phase transition the fluorescence becomes nearly completely depolarized. The discontinuity in the temperature dependence of polarization occurs within a temperature range under 5 degrees C in the case of pure phospholipids, but for mixed phospholipids occurs over a temperature range greater than 20 degrees C. From these data, phase diagrams describing the gel-sol equilibrium can be constructed; the phase diagrams correspond well with those described in the literature which were constructed using spin-label probes or from x-ray diffraction patterns. The marked change in polarization at the phase transition may be related to the packing of the probe molecule into the lipid bilayer: fluorescence measurements on oriented bilayers indicate that below the phase transition the long axis of the probe is oriented perpendicular to the plane of the membrane while above the transition the probe is oriented randomly relative to the plane of the membrane.     

Effect of weak combined low frequency constant and alternative magnetic fields on intrinsic fluorescence of proteins in aqueous solutions]

It was shown that weak combined static (42 microT) and low-frequency variable (40 nT; 3-5 Hz) magnetic fields change the intensity of intrinsic fluorescence of some proteins (cytochrome c, bovine serum albumin, horseradish peroxidase, alkaline phosphatase). The effect can be interpreted as a change in the conformational state of the protein in water environment by the action of weak magnetic fields. The dynamics of the process, the concentration dependence, the binding of proteins to the fluorescence probe 1,8-ANS after treatment with magnetic fields, the frequency dependence of these reactions, and the dependence of the effect on the presence of the static constituent of the magnetic field were studied. It was shown that the changes in the intrinsic fluorescence of some enzymes (horseradish peroxidase, alkaline phosphatase) are related to changes in their functional activity. It was found that the effect is partially transferred via a solvent (water, 0.01 M NaCl) preliminarily treated with magnetic field. In the solvent, changes in its intrinsic fluorescence by the action of weak magnetic fields were also registered.     

Fluorescence anisotropy and the structure of myelin. I. The theory of the method

Theoretical aspects of the experiment dealing with measurements of fluorescence polarization degrees in stained myelinated nerve fibres are considered. Fluorescence polarization (FP) largely depends on the excitation azimuth, i.e. on the angle between the electrical vector of the polarized exciting light and the nerve fibre axis (geometrically it is a figure with the cylinder symmetry). The dependence of FP on the excitation azimuth is shown to be related to the degree of molecular orientation of dyes adsorbed on anisotropic layers of myelin membranes. This permits to associate FP with the structural changes of myelin membranes.     

Influence of a stationary magnetic field on water relations in lettuce seeds. Part I: theoretical considerations.

The theoretical calculation about the dependence of the ionic current density across the cellular membrane on the intensity of the magnetic field applied to cellular tissue is presented. This interaction induces changes in the magnitude of the ionic current density across the cellular membrane and in the ionic concentration, and it also causes alterations in the osmotic pressure and in the capacity of the cellular tissues to absorb water. The magnetic field dependence of the ionic current densities J(p) (B) (positive ions) and J(n) (B) (negative ions), the membrane conductivity sigma (B), the ionic concentration in both membrane sides c(B), the osmotic pressure pi (B), and the water uptake rate by seeds k(w) (B) are presented. The increase in water uptake rate due to the applied magnetic field may be the explanation of the recently reported increase in the germination speed of the seeds treated with stationary magnetic fields.     

Spatial and temporal electroselection patterns in electric field stimulation of polarized luminescence from photosynthetic membrane vesicles

Electroselection processes of charge recombination are manifested in the study of electric field induced polarized emission from photosynthetic membrane vesicles. The study explores the coupled spatial-temporal characteristics of electric field induced charge recombination by examining the dependence of the integrated polarized emission and the time dependent polarization on electric field strength. The experimental results were fitted to theoretical models by computer simulations employing empirical parameters. Simulation of the dependence of the integrated polarized components of emission on electric field strength, suggests field-dependent increased ratio between radiative and nonradiative rates of charge recombination. The observation that the initial polarization values are independent of electric field strength supports the assumption that electric field induced emission originates from the pole area and then spreads away from it towards the equator. The propagation rate of this electric field induced charge recombination from the pole area towards the equator is reflected by the decay of polarization which increases upon raising the electric field strength. Simulation of the polarization's decay, based on a calculated angle of 26.3 ± 0.4° between the transition moment of emission and the plane of the membrane, establishes coupled temporal spatial patterns of electroselection in intramembrane electron transfer invoked by exposing preilluminated photosynthetic vesicles to a homogeneous electric field.     

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes. A fluorescence probe study

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles (out greater than in) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47 degrees C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.     

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. II. Stabilization of the membranes by polyamines

The effects of polyamines, spermine, spermidine and putrescine on the stabilization of the membrane organization of Escherichia coli cells were studied using measurements of fluorescence polarization change of extrinsic fluorescence probes in membrane specimens as a function of temperature. The effects of the polyamines on the restoration of the cell viability after freeze-thawing were also investigated. In logarithmic-phase membrane specimens, polyamines depressed the polarization ratio increase below the transition temperatures in a dose-dependent manner. The physiologically relevant concentration of polyamines repressed the ratios to the same levels as are obtained with the stationary-phase specimens. In the stationary-phase specimens, no effect of polyamines on repression of the polarization increase was observed. A preliminary exposure of logarithmic-phase cells to polyamines protected the cells from the reduction of viability in freeze-thawing. However, a considerably high concentration and a certain length of preincubation time were required in order to an effect to be exerted. These results indicate that the intracellular polyamines could stabilize the membrane organization of logarithmic-phase cells to the same extent as in the stationary-phase cell membranes. It is conjectured that the membrane stability which is mediated by the polyamines results in cellular resistance to freeze-thawing, as it is attained by increasing the growth phase of the cells.     

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.     

The orientation of the primary donor in bacterial photosynthesis.

The triplet state EPR spectra of magnetically aligned whole cells of Rhodopseudomonas viridis and Rhodopseudomonas palustris display a marked dependence on the orientation of the static EPR field with respect to the alignment field direction. This observation implies that the primary donor species on which the triplets are localized are ordered within the membranes. We have developed a theoretical model for the system to enable calculation of the orientation of the magnetic axes of the primary donor species with respect to the membranes in which they reside. The triplet state spectra are generated by an ensemble of partially ordered magnetic systems and a computer simulation of the experimental results. The triplet orientation is very similar for the two organisms studied, where one axis lies predominantly in the plane of the membrane and the other two axes have approximately equal projections onto the normal to the membrane.     

What causes the variation of polarization degree across the emission spectrum of proteins?

A gradual decrease in fluorescence polarization across the emission spectrum on increase in wavelength has been recorded for a number of proteins and also for tryptophan, N-acetyltryptophan and glycyltryptophan. Various factors responsible for this dependence have been analyzed. It is shown that if the emission originates from both the 1La and 1Lb states, the position and form of the fluorescence spectrum polarization components as well as the slope of the dependence of the degree of polarization upon emission wavelength must always vary with the excitation wavelength. However, this condition, although necessary, is not enough to prove the participation of 1Lb in emission. The dependence of the form of the emission polarization spectrum upon excitation wavelength obtained for some proteins is explained by tyrosine residues contributing to the emission. Consequently, there are no reasons for assuming that the 1Lb oscillator participates in emission. It has been observed that for individual emitting centres, the slope of the dependence of the degree of polarization upon emission wavelength is determined by alteration of the vibrational substates, between which the transition with radiation takes place. The heterogeneity in the microenvironment properties of separate tryptophan residues in multitryptophan proteins and the existence, under certain conditions, of a correlation between the radiative lifetime of the emitting centre (determining the degree of the emission polarization) and the completeness of the microenvironment orientational relaxation (determining the emitted quantum of energy) can also affect the slope of this dependence.     

Polarization of electromagnetic radiation at the second harmonic of the electron gyrofrequency in a turbulent plasma

A study is made of the polarization of electromagnetic radiation at the second harmonic of the electron gyrofrequency. The radiation is emitted by a highly ionized collisionless plasma in which the turbulence is excited at electron gyrofrequencies in a strong magnetic field. The mechanism for the generation of electromagnetic waves during mergings of the gyrofrequency plasmons is analyzed. It is shown that, even in a strong magnetic field, the degree of circular polarization of electromagnetic radiation at the second harmonic of the electron gyrofrequency may be moderate or weak.     

Cell membrane orientation visualized by polarized total internal reflection fluorescence.

In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C(18)-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane.     

Theoretical calculation of the dielectric constant of a bilayer membrane.

The dielectric constant (epsilon) and refractive index (n) of a bilayer lipid membrane is determined from the known values of the polarizabilities of the carbon-carbon and carbon-hydrogen bonds. It is assumed that the hydrocarbon chains are hexagonally arranged in an all-trans conformation perpendicular to the plane of the membrane. The only variable in the calculation is the average separation between the chains and the theory relates epsilon to this separation. The calculation and results differ significantly from those presented in a 1968 publication by Ohki. It is shown that a thin membrane is not homogeneously polarized by the applied field. This effect is analysed and the dependence of epsilon on the membrane thickness is determined. The theoretical results are in good quantitative agreement with experimental measurements on bulk paraffins and on oriented multilayers of saturated fatty acids. The most important conclusion is that the dielectric constant for an applied field perpendicular to the membrane (which is the appropriate value for capacitance measurements) differs by only a few percent from the value for the macroscopic (bulk) liquid hydrocarbon. Thus the dielectric constant of a bilayer membrane can be approximated by the value for the appropriate bulk hydrocarbon.     

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes: A fluorescence probe study

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles ($\text{out > in}$) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47°C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.