Degree Of Fluorescence Polarization Research Articles

Determination of Fluorescence Polarization and Absorption Anisotropy in Molecular Complexes Having Threefold Rotational Symmetry

Abstract— The current work concerns investigation of the polarization properties of complex molecular ensembles exhibiting threefold (C3) rotational symmetry, particularly with regard to the interplay between their structure and dynamics of internal energy transfer. We assume that the molecules or chromophores in such complexes possess strongly overlapped spectra both for absorption and fluorescence. Such trimeric structures are widely found in biological preparations, as for example the trimer of C‐phycocyanin (C‐PC). Higher order aggregates, e.g. hex‐amers and three‐hexamer rods, are also investigated and compared with the trimer case. The theory addresses both steady‐state and 8‐pulse excitation and establishes some links between them. Monochromophoric, bichro‐mophoric and trichromophoric molecular complexes are individually examined.For steady‐state excitation, analytical formulas are reported for the degree of fluorescence polarization and absorption anisotropy. It is shown that the polarization is dependent on the chromophore inclination relative to the symmetry axis, the relative efficiencies of absorption and fluorescence by chromophores of different spectral types, and the rates of energy equilibration. To assess the validity of the theory, it has been applied to C‐PC aggregates. Here it was found that different C‐PC aggregates provide practically identical polarization response. For S‐pulse excitation we give analytical formulas for determination of the fluorescence depolarization, and also the depolarization associated with absorption recovery, both for a monochromophoric trimer and some particular cases of bichromophoric trimer. More complicated systems are analyzed by computer modeling. Thus it transpires that the initial polarization anisotropy r(t = 0) takes the value 0.4 for all considered aggregates; the long‐time limit r(t →∞) has about the same value as is associated with steady‐state excitation. We also show that with steady‐state excitation the degree of fluorescence polarization is practically equal for various C3 aggregates of C‐PC, and that the major factor determining the polarization is the chromophore orientation relative to the symmetry axis.

Influence of an n-6 polyunsaturated fatty acid-enriched diet on the development of tolerance during chronic ethanol administration in rats.

This study investigates the effects of n-6 polyunsaturated fatty acids (PUFAs), in the form of dietary Evening Primrose Oil (EPO) and safflower oil, on the development of tolerance to ethanol. The degree of fluorescence polarization of the fluoroprobes DPH, PROP-DPH, and TMA-DPH in isolated cortical synaptosomal membranes was measured. In addition, the development of tolerance, as shown by changes in synaptosomal membrane fluidity after an acute in vitro ethanol challenge, was also determined after 20 weeks of ethanol administration, either alone or together with a PUFA-enriched diet. Although the administration of EPO-enriched diet did not significantly render the inner core of the cortical synaptosomal membrane tolerant to the acute ethanol challenge, concomitant administration of ethanol and EPO was found to increase further the rigidity and tolerance to the acute ethanol challenge in the inner core. Chronic administration of safflower oil, which lacks gamma-linolenic acid (18:3, n-6) but like EPO contains linoleic acid, either alone or together with chronic ethanol had no effect on synaptosomal membrane fluidity after an acute ethanol challenge. The results suggest that gamma-linolenic acid or its metabolites may have an important role to play in the development of tolerance to chronic ethanol.

Cytosolic Ca2+ content and membrane fluidity of platelets and polymorphonuclear leucocytes in diabetes mellitus.

Considering the role played by platelets and leucocytes in diabetic disease and keeping in mind the strong correlation between functional and metabolic aspects that characterizes this clinical condition, we evaluated, in two groups of diabetics, respectively the platelet and polymorphonuclear (PMN) cytosolic Ca2+ content (employing the fluorescent probe Fura 2-AM) and membrane fluidity (using the fluorescent probe TMA-DPH and considering the fluorescence polarization degree, inversely related to the membrane fluidity). From the obtained results, it is evident that the platelet cytosolic Ca2+ content does not distinguish normals from diabetics of type 1 and 2; the platelet membrane fluidity instead does not discriminate normals from diabetics, but differentiates diabetics of type 1 and 2 (type 1 = 0.284 +/- 0.015; type 2 = 0.314 +/- 0.018; p < 0.001). PMN cytosolic Ca2+ content and membrane fluidity do not discriminate normals from diabetics. In the two groups of diabetics none of the platelet and PMN parameters (cytosolic Ca2+ content and membrane fluidity) are related to the glycometabolic pattern.

Effect of phospholipids on the reconstitution and thermal stability of delipidated rat ovarian luteinizing hormone/human chorionic gonadotropin receptors in proteoliposomes

The role of lipids and a possible structure-functional alteration of delipidated LH/hCG receptor reconstituted into proteoliposomes was analyzed by thermal perturbation techniques. Delipidated receptor lost to a great extent its binding activity and thermal stability. The LH/hCG receptor was almost fully reactivated by the reconstitution into proteoliposomes with phosphatidylcholine (PC) and sphingomyelin (SpM) and partly with phosphatidylethanolamine (PE) and phosphatidic acid (PA). The heat inactivation profile of delipidated LH/hCG binding sites was shifted to a lower temperature by about 4°C ( T 50 values). Thermal inactivation of the receptor by delipidation was entirely inverted by treatment with soybean PC, dioleoyl PC and dipalmitoyl PC and partially with SpM. The presence of negatively charged phospholipids, phosphatidylserine (PS), phosphatidylglycerol (PGl) and PA, did not change the heat-inactivation profile of the LH/hCG binding sites of delipidated proteoliposomes. On the other hand, the next decline of thermal stability of the receptor was found in proteoliposomes reconstituted in the presence of lyso-derivatives of PC, as well as of cholesteryl hemisuccinate. Delipidation of the LH/hCG receptor modified the differential scanning calorimetric profile and the quenching of protein fluorescence characteristic for control proteoliposomes. Delipidation increased membrane lipid rigidity. Reconstitution of delipidated proteoliposomes with soybean PC, dioleoyl PC, PGl, PS and PA decreased, and that of dipalmitoyl PC, lysoPC, SpM and cholesterol increased the degree of fluorescence polarization of DPH of proteoliposomes. The different action of phospholipids on the reconstitution, thermal inactivation of the receptor and membrane lipid fluidity in proteoliposomes suggests that lipid fluidity is not related to the stabilizing action of phospholipids on the LH/hCG receptor.

Theory of polarized fluorescence and absorption in molecular complexes comprising two chromophores with non-parallel absorption and emission transition dipole moments

This work concerns theoretical determination of the degree of fluorescence polarization and absorption anisotropy in double-chromophore complexes with non-parallel absorption and emission transition dipole moments. Specially, the theory addresses the case in which energy may incoherently transfer within each complex between chromophores with broad and overlapped spectra. Inside each complex the chromophores are rigidly positioned and oriented relative to each other, whilst the complexes themselves are randomly distributed and oriented in space. The cases of steady-state and of δ-pulse excitation are both considered. In general the results obtained apply not only to the specifically considered case of energy migration but more widely, for example to systems exhibiting fast trans-cis isomerization, electron or proton transfer, etc., either alone or in conjunction with conventional energy transfer.

Fluorescence polarization of triple-chromophore complexes with energy transfer

The formula for the determination of a fluorescence polarization degree is derived for triple-chromophore complexes with energy transfer: P = (3B - 1 + 2A)/(3 + B + 4A). In this formula B = q(12) cos(2)(?(12)) + q(13) cos(2)(?(13)) + q(23) cos(2)(?(23)) and parameters A and q(ij) are dependent on the kinetic parameters of energy exchange and the chromophore spectroscopic parameters. The angle ?ij is the angle between the transition dipole moments of the i and j chromophores. The formula was tested for the particular case of the C-phycocyanin (C-PC) of the blue-green algae Agmenellum quadruplicatum, which contain three chromophores, α-84, β-84, and β155. Polarized spectra were calculated for both the emission and the excitation spectra of C-PC β subunits and monomers.

Effects of partial delipidation of rat ovarian membranes on thermal stability of LH/hCG receptors

The role of lipids and of possible structure-functional properties of the LH/hCG receptor were analyzed by thermal perturbation techniques in its native membrane environment. A method for the reversible removal of lipids from membranes with a mild detergent Tween 20 was developed. The receptor was reactivated with phosphatidylcholine (PC) by its reconstitution into proteoliposomes. The heat inactivation profile of LH/hCG binding sites in delipidated membranes was shifted to a temperature lower by approx. 8 C° ( T 50 values). Thermal inactivation of the delipidated LH/hCG receptor was found to be a quick process. Occupation of receptor binding sites by the agonist before thermal perturbation induced stabilization of the receptor. Thermal inactivation of the receptor by delipidation was fully reversed by treatment with soybean PC, diolcoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC), partly with phosphatidylethanolamine (PE) and sphingomyelin (SpM), but not with phosphatidylserine (PS), phosphatidylglycerol (PGI) or cholesterol. Delipidation modified the differential scanning calorimetric profile characteristic of control membranes. Delipidation of ovarian membranes also increased membrane lipid rigidity. The addition of PC, DOPC and PS to delipidated membranes decreased, that of DPPC and SpM increased, while PGI did not change the degree of fluorescence polarization of DPH, suggesting that membrane lipid fluidity was not involved in the stabilizing action of specific phospholipids against thermal inactivation of the ovarian LH/hCG receptor.

Fluorescence study of polyelectrolyte complex formation III: Mobility of probes in microdomains

AbstractThe mobility of fluorescent probe molecules in the microdomains formed by polyelectrolyte complexes (PEC) in aqueous solution was investigated by the fluorescence polarization method. The degree of fluorescence polarization (p) for the anthracene (Anth) chromophores attached to anionic polyelectrolytes increased with complexation, indicating the restricted mobility of probe molecules in the PEC microdomains. These p values depended on the properties of added polycations, which were not in parallel with the fluorescence intensities of the Anth chromophores.

Calcium and magnesium induced changes in the relative fluidity of phosphatidylcholine liposomes

The effect of Ca2+ and Mg2+ on relative fluidity of phosphatidylcholine liposomes was studied by measuring the degree of chlorophyll fluorescence polarization. An increase in the degree of fluorescence polarization was observed on incubation of liposomes with different concentrations of Ca2+ or Mg2+. The results have been interpreted on the basis of increase in the size of liposomes which could be brought about by calcium or magnesium induced fusion of small unilamellar liposomes to form larger vesicles. Fusion of liposomes has also been confirmed by the experiments on efficiency of energy transfer from chlorophyll b to chlorophyll a, and transmission electron microscopy of liposomes before and after incubation with Ca2+ and Mg2+.

Time-resolved polarization measurements of porphyrin fluorescence in solution and in single cells

Porphyrin monomers, dimers and aggregates, which can be differentiated on the basis of their fluorescence lifetimes, are shown to possess different degrees of fluorescence polarization. This opens up new possibilities for microscopic imaging of these individual components in photosensitization and tumour detection. A rough estimate of the size of the porphyrin aggregates is obtained from the data of time-resolved fluorescence anisotropy.

Coherence effects in the polarization of photofragments

The general quantum-mechanical treatment of coherence effects in the polarization of the fluorescence, as well as in the orientation of photofragments, is reviewed. They are the result of simultaneous excitation of dissociative electronic states with different symmetry correlating to the same limit. The coherent excitation produces interferences which depend both on the photoabsorption probability amplitudes as well as on the relative phases of the continuum vibrational wavefunctions at large distances. Oscillations in the polarization degree of photofragments fluorescence as a function of the photon energy are predicted. Examples are presented in the case of direct dissociation of diatomic molecules. The case of slow predissociation is also analysed. Finally, possible extensions to the photodissociation of polyatomic molecules are considered.

7-Diethylamino-3-((4′-iodoacetylamino)phenyl)-4-methylcoumarin, a fluorescent probe of the hydrophobic cleft in the tropomyosin coiled coil

A sulfhydryl-specific fluorescent reagent, 7-diethylamino-3-((4′-iodoacetylamino)phenyl)-4-methylcoumarin (DCIA) was used to label cysteine residues on tropomyosin (TM) from rabbit cardiac and rabbit skeletal muscles. The emission maximum at 486 nm, the high degree of fluorescence polarization, and the limited accessibility of the bound probe to quenching by iodide suggest that the probe is bound in the hydrophobic cleft between polypeptide chains of the TM coiled coil, as well as being bound covalently at a cysteine residue. The labelled TMs retain their abilities to bind F-actin and are able to interact with deoxyribonuclease I. They, however, show a reduced tendency to aggregate into filaments in low ionic strength solutions.

Intramolecular charge-transfer fluorescence as a mobility probe in poly(methylmethacrylate)

The (time-resolved) fluorescence spectra of 1-phenyl-4-(4-cyano-1-naphthylmethylene)piperidine (1) have been measured during the polymerization of methylmethacrylate (MMA). It was found that the fluorescence maximum of 1 shows a large hypsochromic shift as polymerization of the MMA medium progresses. While the emission maximum is located at 565 nm in the monomer, it is found at 450 ± 5 nm in the polymer (PMMA). The polarization of the fluorescence increases strongly during the polymerization, indicating that the rotational motion of 1 ceases on the timescale of the fluorescence lifetime (about 10 ns). Fluorescence measurements performed with a detection time delay with respect to the exciting laser pulse showed that within the time range 0–100 ns the molecule 1 undergoes reorientation and/or internal relaxation even in a PMMA matrix; a red shift of the emission with time occurs, together with a decrease of the degree of fluorescence polarization.

Fluorescence studies of thermal stability of staphylococcal exfoliative toxins A and B

The quantum yields of intrinsic fluorescence of staphylococcal exfoliative toxins ETA and ETB were determined after excitation at 295 nm and 275 nm respectively. The variations in the intrinsic fluorescence and degree of fluorescence polarization showed conformational modifications of ETA around 57–59°C and of ETB around 52–54°C. Above these transition temperatures, ETB precipitated out and its biological activity was lost, whereas ETA showed almost no precipitation and no change in its biological activity, despite the structural changes in the molecule.

Fluorescence studies of thermal stability of staphylococcal exfoliative toxins A and B

The quantum yields of intrinsic fluorescence of staphylococcal exfoliative toxins ETA and ETB were determined after excitation at 295 nm and 275 nm respectively. The variations in the intrinsic fluorescence and degree of fluorescence polarization showed conformational modifications of ETA around 57–59°C and of ETB around 52–54°C. Above these transition temperatures, ETB precipitated out and its biological activity was lost, whereas ETA showed almost no precipitation and no change in its biological activity, despite the structural changes in the molecule.

Investigation of the polarized fluorescence of prolate-shaped molecules by subnanosecond laser spectroscopy

The degree of fluorescence polarization ( p) was investigated for prolate molecules in solvents of different viscosity (η) over the temperature ( T) range 283 to 313 K. The decay times (τ) for these solvents were also measured. The linear dependence of 1/ p on τ T η was confirmed for oligophenyl compounds.

Impairment of human polymorphonuclear leukocyte chemotaxis by 2,5-hexanedione

The effect of n-hexane metabolites on human polymorphonuclear leukocyte chemotaxis and luminol-dependent chemiluminescence was investigated. No effect was detected when 2-hexanol, 2-hexanone and gamma-valerolactone were used; 2,5-hexanedione at 75 micrograms/ml inhibited chemotaxis and a direct correlation between increasing the xenobiotic concentration and the degree of inhibition was found. Chemotactic peptide-induced chemiluminescence was not affected by 2,5-hexanedione. In order to clarify the phenomenon, plasma membrane fluidity was investigated by fluorescence polarization of the fluorescent probe trimethylammonium diphenylhexatriene. 2,5-hexanedione increased the membrane fluidity, while the other n-hexane metabolites did not change the degree of fluorescence polarization. Results suggest that the cellular functions modulated by membrane-cytoskeletal organization are affected by 2,5-hexanedione also at the low concentrations.

Fluorescence study of brevin, the Mr 92 000 actin-capping and -fragmenting protein isolated from serum. Effect of Ca2+ on protein conformation.

The fluorescence characteristics of brevin and the effects of Ca2+ on the protein conformation were fully investigated. Brevin contains 18 tryptophans and 27 tyrosines. Analysis of the fluorescence spectra and the accessibility to quenching molecules indicate that the emitting tryptophans are located in a hydrophobic environment (lambda max = 324 nm) close to the protein surface. In native brevin, tyrosyl residues do not contribute to the fluorescence emission. Partial quenching of these chromophores has to be attributed to tyrosine----tryptophan resonance energy transfer which is highly efficient. The effect of brevin on actin polymerization has been shown to be Ca2+ sensitive [Harris, D. A., & Schwartz, J. H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6798-6802; Thorstensson, R., Utter, G., & Norberg, R. (1982) Eur. J. Biochem. 126, 11-16; Wilkins, J. A., Schwartz, J. H. & Harris, D. A. (1983) Cell Biol. Int. Rep. 7, 1097-1104; Harris, H. E., & Weeds, A. G. (1983) Biochemistry 22, 2728-2741] and brevin binding to hydrophobic matrices to be Ca2+ dependent (Z. Soua, personal communication). Ca2+ binding to brevin decreases the tryptophan fluorescence polarization degree (without affecting the excited-state lifetime), which suggests a higher chromophore mobility. This effect may be partly related to the slight unshielding of the tryptophan residues observed in fluorescence quenching experiments. Moreover, the reactivity of brevin sulfhydryl groups toward 5,5'-dithiobis(2-nitrobenzoic acid) increases in the presence of Ca2+. On the other hand, fluorescence spectra, quantum yields, excited-state lifetimes, and thermostability remain unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometry is a new method for the characterization of intestinal plasma membrane.

The highly differentiated plasma membrane of rabbit enterocytes constitutes an interesting model for membrane studies. Flow cytometry allows combined measurements of the size of membrane vesicles by light-scatter and fluorescence polarization at a single-particle level. The degree of fluorescence polarization of 1,6-diphenylhexa-1,3,5-triene was determined at 4, 18 and 37 degrees C in the brush-border and basolateral plasma membranes. The fluorescence polarization was considerably higher in brush-border than in basolateral membranes. After incubation with dimethyl sulphoxide, the membrane fluidity decreased in both types of membranes. Moreover, a time-effect study of dimethyl sulphoxide showed changes in fluorescence polarization. Only in brush-border membrane a temporary fluid phase was observed. The different properties of the two membrane domains in relation to the lipid-protein dynamics of enterocytes are discussed.

Study of polysaccharides by the fluorescence method. III. Chain length dependence of the micro‐Brownian motion of amylose in an aqueous solution

AbstractThe fluorescence polarization method was applied to the investigation of the micro‐Brownian motion of amylose chains having a wide range of degree of polymerization (DP). We prepared two types of fluorescent conjugates of amylose: amylose conjugated with fluorescein randomly throughout the chain (F‐amylose) and amylose conjugated locally on a terminal segment (t‐F‐amylose). The degree of fluorescence polarization of these conjugates was measured by changing the solvent viscosity at a constant temperature (25°C). The data obtained were analyzed by a Perrin‐type equation to calculate the mean rotational relaxation time, 〈ρ〉. By examination of the plots of 〈ρ〉 vs DP, and by comparison of 〈ρ〉 with the theoretical rotational relaxation time of the whole molecule at a given DP, it was found that 〈ρ〉 mainly reflects the segmental motion of the amylose chain in the high‐DP range. Thus, the fact that 〈ρ〉 for t‐F‐amylose is much smaller than that for F‐amylose at a sufficiently high DP shows that a terminal segment undergoes a more rapid micro‐Brownian motion than interior segments. In the low‐DP range, we felt that the rotational diffusion of the whole molecule contributes significantly to the relaxation process. We also examined, for comparison, the segmental motion of dextran and pullulan in a similar manner and found that these segmental motions are more rapid than those of amylose. Based on the results obtained, the segmental mobility and conformation of the amylose molecule are discussed in relation to its chain length.