Changes In Fluorescence Polarization Research Articles

Interaction of a Multiple Antigenic Peptide of Hepatitis A Virus with Monolayers and Bilayers of Acidic, Basic, and Zwitterionic Phospholipids

The interaction of the synthetic multiple antigenic peptide construct MAP4-VP1(11-25) with lipid membranes was studied by a combination of spectroscopic and biophysical techniques. MAP4-VP1 showed an important surface activity when spread in an air–water interface, indicative of an amphipatic conformation. Since this peptide has a net anionic charge, we studied the degree of interaction with model membranes of zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic phosphatidylinositol (PI and DPPC/PI 9:1), or cationic stearylamine (SA and DPPC/SA 9:1). MAP4-VP1 was readily soluble in aqueous buffer, yet spontaneously inserted from an aqueous subphase into cationic and zwitterionic monolayers up to limiting pressures of 20–30 mN/m. The induced surface pressure increases were in the order cationic > zwitterionic > anionic monolayers. This indicates that the interaction is mainly driven by electrostatic forces, although there is an important hydrophobic component, responsible for the penetration in monolayers of neutral DPPC. Interaction of MAP4-VP1 with unilamellar vesicles of DPPC, DPPC/SA (9:1), and DPPC/PI (9:1) labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or with 1-anilino-8-naphthalene sulfonic acid (ANS) was determined by changes in fluorescence polarization as a function of temperature. Results showed that the presence of the positively charged SA in the membrane strongly enhances the incorporation of MAP4-VP1 and that the peptide increases the fluidity of the bilayer and decreases the temperature of the phase transition.

Influence of Follicular Maturation on the Susceptibility of Chicken Ovarian Granulosa Cells to Free Oxygen Radicals

The influence of ovarian follicular maturation on lipid peroxidation-induced changes in the fluidity of plasma membrane of granulosa cells was investigated using the fluorogenic polyunsaturated fatty acid cis-parinaric acid (cPNA). An increase in lipid peroxidation results in the decrease of fluorescence intensity of cPNA. A decrease in membrane fluidity results in the decrease of fluorescence polarization of cPNA. Granulosa cells isolated from the largest (F1; mature), and third largest (F3; developing) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF) of the domestic hen ovary were pre-labelled with cPNA. The cPNA-labelled cells were exposed to a free oxygen radical generating agent 2,2′azobis-(2-amidinopropane)-hydrochloride (AAPH). AAPH enhanced the rate of decrease in fluorescence intensity of cPNA-labelled cells. This effect of AAPH was greater in the more differentiated granulosa cells obtained from mature follicles than in less diferentiated cells isolated from immature follicles. Similarly, the degree of change in fluorescence polarization was greater in more differentiated F1 granulosa cells than in the less differentiated ones. The analysis of fatty acid composition of phospholipids indicated that the plasma membrane of differentiated granulosa cells contained a higher proportion of the unsaturated fatty acids, oleic and linoleic acids. The fluorescence data show that the rigidity of the plasma membrane of chicken granulosa cells decreases with advancing follicular maturation presumably due to relative increase in the unsaturated fatty acid content of plasma membrane phospholipid.

Fluorescence depolarization as an early measure of T lymphocyte stimulation

We have used the Cellscan, an apparatus capable of measuring optical properties of individual cells, to study changes in fluorescence polarization associated with T cell stimulation. We show that the fluorescence polarization of human peripheral blood lymphocytes (PBL) labeled with fluorescein diacetate (FDA) is markedly reduced upon exposure to the mitogenic lectins phytohemagglutinin (PHA), concanavalin A (ConA), or to phorbol esters. Methyl α- d-mannopyranoside ( αMM) is able to reverse the depolarizing effect induced by ConA as long as the cells are not committed to proliferate. H7 and staurosporin, both inhibitors of protein kinase C (PKC), inhibit the depolarization induced by PHA. The mitogen-induced depolarization is dependent on metabolic energy. The results support the use of fluorescence depolarization of FDA-labeled PBL, monitored by the Cellscan, as a sensitive means of measuring early lymphocyte stimulation.

Influenza A virus affects the response of human peripheral blood mononuclear cells to phytohaemagglutinin A by altering the cytoskeleton.

In the present study, we demonstrate that the infection of human peripheral blood mononuclear cells (PBMC) with influenza A virus caused changes in intracellular fluorescein fluorescence polarization (IFFP) which, as previously described, reflect alterations in the polymerization of the cytoskeleton. Kinetic measurements revealed two cycles of an approximate 10% decrease in IFFP within 3.5 and 5 h after infection. Infection win influenza A virus also altered the response of PBMC to phytohaemagglutinin (PHA), which was manifested as changes of 5.3 and 4% in IFFP at 1 and 2 h after infection, respectively. the changes in IFFP correlated with DNA synthesis measured 72 h after exposure to PHA. These results show the ability of IFFP measurements to identify early intracellular metabolic events induced in virus-infected cells.

Purinergically induced membrane fluidization in ciliary cells: characterization and control by calcium and membrane potential

To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways.

Inhibition of mitogen-induced changes in intracellular fluorescein fluorescence polarization of human peripheral blood lymphocytes by colchicine, vinblastine and cytochalasin B.

We have previously reported that the exposure of human peripheral blood lymphocytes (PBL) to a variety of stimulants caused rapid changes in intracellular fluorescein fluorescence polarization (IFFP) in the activated cells. In the present study we further analyzed possible mechanisms responsible for the changes in IFFP in PBL exposed to phytohaemagglutinin (PHA) and anti-CD3 antibody. By employing several agents which are known to affect the polymerization of the cytoskeleton we showed that both cytochalasin B, which regulates the microfilaments structure, and vinblastine and colchicine, which affect the microtubules, completely abolished the changes induced in IFFP of human PBL by both PHA and anti-CD3. This effect was dose dependent and was noted at concentrations ranging from 10 to 100 microM of cytochalasin B and 10 microM of vinblastine and colchicine. The effect of these cytoskeleton modulators occurred within 20 minutes after the initiation of activation with PHA. Our results indicate that activation with PHA and anti-CD3 causes early changes in the microtubules and microfilaments components of the cytoskeleton. The possible application of IFFP measurement in analyzing early changes in the cytoskeleton following cell activation is discussed.

Binding of biologically important molecules to DNA, probed using electro-fluorescence polarization spectroscopy

When small fluorescent tag molecules are bound with a single geometry to larger carrier molecules the fluorescence excited using polarized light may itself be partially polarized. If this is the case then alignment of the carrier molecules changes the fluorescence polarization. Electro-fluorescence polarization spectroscopy (EFPS) makes use of the fluorescence polarization changes which occur when the carrier molecules are aligned by an electric field. EFPS allows the determination of the binding geometry of the small tag molecules to the larger carrier molecules. In this study, EFPS has been used to probe the interactions between DNA and a series of chromosomal stains and anti-neoplastic (anti-cancer) agents. Results are presented for calf thymus DNA, treated with the dyes acridine orange, ethidium bromide, proflavine, Hoechst 33258, Hoechst 33342, 4′,6-diamidino-2-phenylindole and the anti-tumour agents, doxorubicin, daunomycin, actinomycin C and actinomycin V.

Fluorescence polarization changes in the lymphocytic cytoplasm in the various stages of breast cancer

Post-surgical pathological staging and ancillary tests determine the initial treatment of breast cancer. Because change in the structuredness of the cytoplasmic matrix (SCM) of peripheral lymphocytes (as assessed by measurement of fluorescein fluorescence polarization, FFP) has already emerged as diagnostic for breast cancer and an aid to staging in other cancers, testing was carried out in 113 pre-surgical patients to see whether such change could contribute to accurate staging of breast cancer. The FFP test was able to distinguish grouped stages of locoregional disease from metastatic disease but was unable to distinguish between any single locoregional stage and the metastatic stage. Technological improvements now underway may create a role for assessment of SCM changes in breast cancer staging.

Decrease of intracellular fluorescein fluorescence polarization (IFFP) in human peripheral blood lymphocytes undergoing stimulation with phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody

In the present study we describe the induction of changes in intracellular fluorescein fluorescence polarization (IFFP) in lymphocytes undergoing activation with a variety of stimulants. These stimulants included the lectins phytohaemagglutinin (PHA), concanavalin (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody. Changes in IFFP were detected in individual cells using the Cellscan apparatus. Our results show that by employing mitogenic concentrations of PHA, as revealed in a [ 3H]-thymidine incorporation assay, a decrease in the IFFP in human peripheral blood lymphocytes (PBL) occurred within 40 min. ConA and anti-CD3 affected similarly IFFP, whereas PWM, a B lymphocyte lectin, had no effect on IFFP at the concentrations employed. Kinetic analysis revealed that changes in IFFP occurred within 20–40 min after exposure to the stimulants and lasted for 24 h. Our results show that stimulants which activate CD3 + lymphocytes caused immediate changes in IFFP, in an enriched population of human PBL. The possible mechanisms involved in IFFP modulation following exposure to selected stimulants are discussed.

Effect of reductive alkylation on transferrin conformation and physicochemical properties

Lauryl moieties were linked to the lysine ε-amino groups of native transferrin using lauryl aldehyde and sodium cyanoborohydride. The level of derivatization, determined by amino acid analysis, was 50 and 100%, according to the decrease in lysine content. The protein was titrated with 8-anilino-1-naphthylsulphonic acid and changes in fluorescence intensity, λ max and polarization were determined. The intrinsic fluorescence of the protein was determined as a function of the phospholipids' content. The ability of derivatized transferrin to destabilize bilayers was checked using carboxyfluorescein loaded liposomes. The surface activity and the interaction with monolayers were determined for both proteins and compared with the same values before derivatization. The binding of native and derivatized transferrin to bilayers was determined at different phospholipid to protein ratios and found to be highly dependent on the hydrophobicity of protein. The results show that derivatization induces a new structure, the alkyl chains being located inside the protein core. No strong differences were found between 50 and 100% derivatized transferrin.

Fluorescence Evidence for Formation of a Hyaluronate Ion Dodecyltrimethylammonium Bromide Complex

Structural changes of the complex formed by the polysaccharide hyaluronic acid, as its salt, partially modified by a covalently bound fluorescence probe, 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, and dodecyltrimethylammonium bromide, were observed by measuring fluorescence depolarization with 0.175 M NaBr as a function of surfactant concentration. Values of depolarization change significantly between 2.0 and 10.0 mM surfactant. The critical micelle concentrations of the surfactant in 0.175 M NaBr in the absence and presence of the polymer are in the surfactant concentration range at which fluorescence polarization changes. Formation of the surfactant-polymer complex at high salt concentration is consistent with appearance of free micelles, which associate with the anionic polymer.

Erythrocyte membrane lateral sterol domains: A dehydroergosterol fluorescence polarization study

Structural domains of cholesterol and their regulation in the erythrocyte membrane are poorly understood. Dehydroergosterol fluorescence polarization change was used to continuously monitor the kinetics of sterol exchange and sterol domain size in erythrocyte ghost membranes. Direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements was obtained without separation of donor and acceptor membranes. Three important observations were made. First, sterol exchange between small unilamellar vesicles (SUV) with the same cholesterol/phospholipid ratio as the erythrocyte membrane (1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol = 1:1) was resolved into three kinetic cholesterol domains: 23 +/- 9% of total sterol was rapidly exchangeable, with t1/2 = 23 +/- 6 min; 59 +/- 9% of total sterol was slowly exchangeable, with t1/2 = 135 +/- 3 min; and 19 +/- 9% of total sterol was essentially nonexchangeable, with a t1/2 of days. Second, the substitution of erythrocyte ghosts for SUV as an acceptor significantly altered the kinetic parameters of sterol exchange from donor SUV, graphically showing that both the properties of the acceptor and spontaneous desorption of cholesterol from the donor SUV influenced spontaneous cholesterol transfer. Third, studies of exchange between erythrocyte ghosts revealed multiple kinetic pools of sterol differing from those in the SUV: 4 +/- 2% of total sterol was rapidly exchangeable, with t1/2 = 32 +/- 9 min; 29 +/- 3% of total sterol was very slowly exchangeable, with t1/2 = 23 +/- 7 h; and a surprisingly large 67 +/- 2% of total sterol was nonexchangeable, with a t1/2 of days.

Binding of IAA and ABA to phospholipid bilayers

The plant hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) are shown to bind differently to phospholipid bilayer membranes. As followed by fluorescence polarization of membrane interior (1,6 diphenyl-1,3,5-hexatriene) and aqueous interface (1-(4-trimethylammoninum-phenyl)-6-phenyl-1,3,5-hexatriene and octadecyl-anthracene-9-carboxylate) probes, non-dissociated IAA binds to liquid crystalline state membranes composed of either saturated or unsaturated phospholipids. Weak binding ( K d = 3.4 mM) is also measured by a novel method following a change in fluorescence polarization of IAA as it binds. Particularly altered by IAA are the aqueous interface probes indicating that IAA acts on the membrane surface. In sharp contrast, ABA does not appreciably affect polarization of the same probes on the same series of phospholipid bilayers that are affected by IAA. From these observations and prior reports of the hormones' effects on phospholipid vesicle permeability, aggregation and fusion, we propose that IAA perturbs the membrane surface while ABA acts at regions of membrane packing defects.

Effects of hyperthermia and membrane-active compounds or low pH on the membrane fluidity of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells heated in the presence of the membrane-active agents procaine, ethanol, butylated hydroxytoluene (BHT) and glycerol were analysed for changes in fluorescence polarization of the lipid probe (1-[4(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, an indicator of plasma membrane fluidity). Cells were heated in normal and acid (pH 6.6) medium. Procaine, ethanol, BHT and low pH sensitized cells to heat-killing. Procaine, ethanol and BHT decreased fluorescence polarization (increased plasma membrane fluidity) significantly. Polarization distributions for cells heated with these sensitizers were broadened substantially and were skewed toward lower polarization values. Glycerol, a heat-protector, inhibited changes in fluorescence polarization due to heating at temperatures up to 45.0 degrees C. Heating cells at either 42 or 45 degrees C in pH 6.6 medium had no significant effect on the fluorescence polarization compared with controls, while survival was reduced substantially. Thus, heat-sensitization of low pH cannot be ascribed to changes in membrane fluidity. The changes in membrane fluidity caused by other sensitizers and protector indicate that membrane fluidity changes may be a contributing cause of cell killing by hyperthermia.

Sterol domains in phospholipid membranes: dehydroergosterol polarization measures molecular sterol transfer

The domain structure of cholesterol in membranes and factors affecting it are not well understood. A method, based on kinetics of Δ5,7,9,(11),22-ergostatetraen-3β-ol (dehydroergosterol) fluorescence polarization change and not requiring separation of donor and acceptor membranes, was used to examine sterol domains in three-component cholesterol: dehydroergosterol: phospholipid small unilamellar vesicles (SUV). A new mathematical data treatment was developed to provide a direct correlation between molecular sterol exchnage and steady-state dehydroergosterol fluorescence polarization measurements. The method identified multiple kinetic pools of sterol in SUV: a small but rapidly exchanging pool, a predominant slowly exchanging pool, and a very slowly exchangeable (nonexchangeable) pool. The relative sizes of the pools and half-times of exchange were highly dependent on the presence of acidic phospholipids and on cytosolic proteins involved in sterol transfer. Thus, the method provides a direct measure of molecular sterol transfer between membranes without separating donor and acceptor membranes.

Lipid hydrogenation induces elevated 18:1-CoA desaturase activity in Candida lipolytica microsomes

Microsomal membranes prepared from the mesophilic yeast Candida lipolytica grown at 10 degrees C were hydrogenated by the homogeneous Pd-catalyst, palladium di (sodium alizarine sulfonate) (Pd(QS)2). After hydrogenation to various levels, the microsomes were washed free of the Pd-complex and transferred to a reaction mixture (containing NADH, MgCl2, ATP, CoA and [14C]18:1-CoA) for assay of 18:1-CoA desaturase activity. Microviscosity alterations were also followed by measuring changes in DPH fluorescence polarization. Rapid catalytic hydrogenation of unsaturated fatty acids of the lipids occurred within 20-120 s, resulting in large increases in 16:0, 18:0 and 18:1 acids and decreases in 18:2 acid. In the range 7-20% 18:0 content, a pronounced increase in desaturase activity was observed, with a maximum of greater than 2-fold at a 18:0 content of 12%, followed by a decrease to the initial activity at 33% 18:0 content. These changes were well-correlated with changes in microviscosity, maximal desaturase activity occurring in the DPH fluorescence anisotropy range of 0.23-0.24; above and below this range, desaturase activities were close to the initial control values. It is suggested that the hydrogenation-induced increase in the formation of 18:2 from 18:1-CoA (proceeding partly through direct desaturation of PC) may be due to changes in conformation of the membrane-bound desaturase enzyme complex as a result of controlled rigidification of the surrounding lipids. The operation of such a self-regulating control mechanism would be consistent with a previously proposed model for microsomal desaturase action.

Increased Membrane Order of Erythrocytes in Alcoholics as Measured by Fluorescence Polarization:

The erythrocyte membranes from 9 healthy non-alcoholic controls and 8 alcoholic patients (fulfilled DSM-III-R criteria for alcohol dependence) were compared for changes in membrane order as measured by fluorescence polarization. The mean amount of change in fluorescence polarization in response to in vitro exposure to ethanol was significantly less in the alcoholics than controls. This increased membrane order of the peripheral blood erythrocytes suggests the presence of pharmacodynamic tolerance to ethanol in the alcoholic patients.

Hemolysis of erythrocytes and fluorescence polarization changes elicited by peptide toxins, aliphatic alcohols, related glycols and benzylidene derivatives

Hemolysis rates of human erythrocytes induced by C 2 and C 8–C 14 straight chain 1-alkanols, 1,2-alkanediols and the corresponding benzylidene derivatives (benzaldehyde acetals) have been studied and compared with hemolysis rates obtained by three peptide toxins. The peak of activity occurs at C 12 for the alkanols and glycols and at C 10 for the benzylidene derivatives. The most active compound is 1-dodecanol, followed by 1,2-dodecanediol and the C 10 benzylidene acetal, which show 50% hemolysis at 15, 99 and 151 μM, respectively, at 37°C. A few lysolecithins and longer chain cis-unsaturated alcohols were studied for comparison purposes, and were found to be more active than 1-dodecanol. The most active were the 16:0 lysolecithin and cis-9-tetradecene-1-ol, which gave 50% hemolysis at concentrations of 2.8 and 5.6 μM respectively. The hemolytic activities of 1-dodecanol, 1,2-dodecanediol and the C 10 benzylidene acetal were compared to activities of Pyrularia thionin and melittin with cow, horse, sheep, pig and human erythrocytes. Whereas the peptide toxins showed clear specificity for human erythrocytes, no selectivity was shown by any of the other compounds tested. Addition of the thionin or Naja naja kaouthia cardiotoxin to erythrocyte ghosts caused a slight but reproducible increase in the order of the phospholipid bilayer, as measured with the fluorescent probe NBD-PC. Cardiotoxin gave a greater response than did the P thionin, and extensively iodinated P thionin gave a smaller change than did P thionin. Similar results were obtained with melittin, but this peptide gave a markedly greater response than all other peptides. Addition of dodecanol or the C 10 benzylidene acetal caused a marked increase in membrane fluidity. All of these data indicate that the organic compounds interact directly with and are incorporated nonspecifically into the membrane lipid bilayer, but the peptide toxins interact specifically with some component on the surface of the membrane, either a protein or specific phospholipid domain, followed by insertion into the membrane and decreasing phospholipid movement.

Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.

A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe. Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label. Formation of protein-DNA complex was monitored as a function of cAMP concentration. Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP-(cAMP)2 to DNA. These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA. These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions. This technique also has the potential of providing information regarding the structure of protein-DNA complexes.

Fluorescence Polarization Changes with Gestational Age in Amniotic Fluid of Rabbit and Guinea Pig

The variation of amniotic fluid microviscosity with gestational age was measured in rat, rabbit, and guinea pig. In rat, the changes followed the same pattern as known for women, microviscosity being high during early and mid-gestation and markedly lowering 12 h before delivery. Surprisingly, an opposite trend was observed in rabbit and guinea pig amniotic fluid. Moreover, the lecithin to sphingomyelin ratio markedly rose in late gestation in all species considered. Qualitative and quantitative analysis of lipids and phospholipids were performed in woman and rabbit amniotic fluids at early and late gestational ages. Among all the parameters measured, the most important differences that can influence the amniotic fluid microviscosity are the presence of very high levels of lysophosphatidylcholine both in early and late gestation in rabbit (much higher than in woman) and the cholesterol to total phospholipid ratio which decreased with gestational age in woman but remained stable in rabbit. The Arrhenius plot of the logarithm of microviscosity against the reciprocal of absolute temperature of mature and immature amniotic fluids from woman and rabbit was also determined. The temperature profiles confirmed the differences in lipid profile between woman and rabbit in early and late gestation which could be quantified on a physicochemical basis by determining the activation energy (delta E) at 25 degrees and 37 degrees C for each curve. This confirmed the opposite patterns in woman and rabbit and showed that amniotic fluid from the immature rabbit was the most fluid.