Fluorescence Polarization Ratio Research Articles

Analysis of linearly polarized fluorescence of chloroplasts oriented in polyacrylamide gel

Linearly polarized fluorescence was measured in mesophyll chloroplasts of maize aligned in polyacrylamide gel. The dependence of fluorescence polarization on the squeezing parameter of the gel sample was studied at room temperature. Polarized fluorescence emission and polarization ratio spectra were recorded at low temperature (− 140°C). A theoretical model is proposed for a quantitative description of the polarized fluorescence emission and its dependence on the squeezing parameter. It is shown that chloroplasts are aligned as disc-shaped particles with no detectable deformation of their ultrastructure. The fluorescence emission and the polarization ratio spectra as well as the calculated orientation angles between the transition dipoles and the membrane plane were similar to those obtained with magnetically oriented chloroplasts.

Field concentration and temperature dependence of fluorescence polarization of magnetically oriented chloroplasts

Chloroplasts in higher magnetic fields align with their equatorial plane perpendicular to the field. Because of the nonrandom orientation of the chromophores in the membrane the fluorescence radiation will be partially polarized. The chloroplast concentration, magnetic field, and temperature dependence of the fluorescence polarization has been investigated. The results are compared with a simplified model calculation. It is shown that the concentration dependence can be related to the linear dichroism of the fluorescence radiation and self-adsorption. Taking these effects into account results in the calculation of a higher fluorescence polarization (FP) ratio and higher inclination of chlorophyll dipoles to the membrane plane. Analyzing the magnetic field dependence of the FP ratio, we conclude that in a magnetic field not only will be chloroplasts be aligned, but the thylakoid stacks as well. A decrease in the FP ratio was observed around 20 degrees C. It is suggested that this decrease reflects a phase transition in the photosynthetic membrane.

Displacement of the human apoprotein A-I by the human apoprotein A-II from complexes of (apoprotein A-I)-phosphatidylcholine-cholesterol.

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24 degrees C. 28 degrees C and 32 degrees C with either pure dimyristoyl-glycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 2, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid-cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II.

Orientation of emitting dipoles of chlorophyll A in thylakoids: considerations on the orientation factor in vivo

Orientation angles of five emitting dipoles of chlorophyll a in thylakoids were estimated from low temperature fluorescence polarization ratio spectra of magnetically oriented chloroplasts. A simple expression is given also for the evaluation of data from linear dichroism measurements. It is shown that the Qy dipoles of chlorophylls lie more in the plane of the membranes and span a larger angular interval than was previously thought. Values for the orientation factor are calculated using various models corresponding to different degrees of local order of the Qy dipoles of chlorophylls in the thylakoid. We show that the characteristic orientation pattern of the Qy dipoles of chlorophylls in the membrane, i.e., increasing dichroism toward longer wavelengths, may favour energy transfer between the antenna chlorophylls as well as funnel the excitation energy into the reaction centers.

Fluorescence polarization studies of rat liver microsomes with altered phospholipid desaturase activities.

Phospholipid desaturase activity of rat liver microsomes can be varied by dietary manipulation: rats starved and refed a "fat-free" diet have two to three times the activity of normal controls whereas starved rats have no detectable activity. These changes were accompanied by changes in fatty acid composition of the microsomal phospholipids, resulting in a double bond: saturated fatty acid mole ratio (moles double bonds per mole saturated fatty acid) of 5.7 in control and starved rats and 4.7 in starved-refed rats. Fluorescence polarization ratio P (I parallel/I perpendicular to x instrument correction factor) of cis- and trans-parinaric acid (PnA) showed no significant differences in physical state of the three microsomal preparations. However, the isolated microsomal phospholipids with trans-PnA as probe showed differences in the temperature at which onset of a change in polarization ratio occurred (starved-refed greater than normal greater than starved rats). With the cis-PnA as probe, the polarization ratio showed no change in the range 10-40 degrees C but was significantly higher (1.8) in starved-refed rats than in normal and starved rats (1.6 in both cases). These data indicate that the microsomal phospholipids of starved-refed animals were in a less fluid state than those from control and starved rats and that this decrease in fluidity was correlated with an increase in phospholipid desaturase activity.

ORIENTATION OF SHORT WAVELENGTH AND LONG WAVELENGTH PROTOCHLOROPHYLL SPECIES IN GREENING CHLOROPLASTS

Abstract— The orientation of protochlorophyll and Chi species with respect to the plane of thylakoid membranes was studied by measuring the fluorescence polarization ratio in magnetically oriented chloroplasts isolated from greening maize leaves and cucumber cotyledons. With viewing direction parallel to the plane of the photosynthetic membranes, in the spectral region of 620–660 nm, fluorescence polarization ratios of 1.0 were observed, whereas at longer wavelengths the fluorescence polarization ratios were much higher, and similar to that of fully green chloroplasts. The same result was obtained with chloroplasts isolated from leaves fed by δ‐amino levulinic acid. These data indicate that the emitting oscillators of the short and long wavelength protochlorophyll species are oriented at random with respect to the plane of thylakoid membranes. Isotropy of the protochlorophyll species is discussed in terms of isotropic structures containing Chi precursors.

Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.

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.

Polarization ratio of anthracene fluorescence

New measurements are reported of the fluorescence polarization ratios for pure anthracene crystal. The values for different crystals lie in the range b/a= 4.5–6.5 : 1. The 0—0 band group is excluded, being affected by re-absorption. The 0—1, 0—2 and 0—3 bands have PR values nearly equal to one another in any one crystal, but differing from crystal to crystal according to method of preparation and other conditions. A theoretical interpretation is discussed.

Experimental Evidence for Localized Excitons in the Spectra of Charge-Transfer Complex Molecular Crystals

The polarized absorption and emission of the anthracene—TNB complex crystal are shown to provide evidence for localization of low-energy excitation in this crystal. The absorption and fluorescence polarization ratios at 300°K are very different (5.6:1 and about 15:1, ‖c: ⊥c on 110) the former being close to an oriented gas value. The latter corresponds to a charge-transfer complex more closely aligned with the c axis of the crystal. The motion of the excited molecule is in accord with the behavior of a dipole in an electric field which is nearly along the c axis. Possible ramifications of this effect in photoconductive charge-transfer complexes are discussed. The influence of temperature on the luminescence spectrum of anthracene—TNB crystals is discussed, and apart from some artifacts due to cracking of crystals, the expected increase of emission intensity with decreasing temperature is observed.