A fluorescence study on the gel-to-sol transition of kappa-carrageenan

The fluorescence technique was employed to study gel-to-sol transitions in kappa-carrageenan systems with various carrageenan contents. Pyranine was used as a fluorescence probe for monitoring these transitions. Fluorescence, Ip and scattered light, Isc intensities were measured against temperature to determine critical phase transition temperatures and exponents. It was observed that gel-to-sol transition temperatures, Tgs are found to be dependent to the carrageenan content. The measured critical exponents gamma' and beta' were found to be very close to the weight average degree of polymerization, DPw and gel fraction G, exponents (gamma and beta) of the classical Percolation Model.     

Dynamic light scattering studies of irradiated kappa carrageenan

Investigation of the dynamic behavior of irradiated kappa carrageenan (in KCl) as a function of irradiation dose and temperature was done by dynamic light scattering (DLS). The intensity correlation function (ICF) shifted towards shorter relaxation times with increasing radiation dose as a result of radiolysis. The characteristic decay time distribution function, G(gamma), indicates the presence of fast and slow mode peaks respectively at around 0.1-10 ms and 100-1000 ms. A peak broadening of the fast mode peak in G(gamma) appeared with decreasing temperature, indicating that coil-to-helical conformational transition took place. The conformation transition temperature (CTT) decreased with increasing radiation dose. No transition was observed for kappa-carrageenan irradiated at 200 kGy. A new faster relaxation mode appeared at around 0.1-1 ms at temperatures below the CTT. This peak is found in kappa-carrageenan irradiated at doses exclusively between 75 and 175 kGy. The peak height of this mode is largest at 100 kGy which corresponds to the optimum biologic activity of kappa-carrageenan reported previously.     

Phosphatidylinositol 3-phosphate is found in microdomains of early endosomes

Phosphatidylinositol 3-phosphate [PI(3)P] is a phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P₂, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.David J. Gillooly and Camilla Raiborg contributed equally     

Conformational states of yeast tRNA Phe in the complex with cognate and non cognate synthetases.

The influence of phenylalanyl-tRNA synthetase and seryl-tRNA synthetase on the conformation and structural kinetics of yeast tRNA Phe was investigated. Ethidium substituted for dihydrouracil at position 16 or 17 was used as a structural probe, showing the existence of three conformational states in tRNA. The distribution of states (T1, T2, T3) is changed only by the cognate synthetase towards T3 which probably is related to the X-ray structure. The binding of phenylalanyl-tRNA synthetase leads to an about 10-fold increase in the fast transition T1 in equilibrium or formed from T2 which has been assigned to changes in the anticodon loop conformation and to a 2-3 fold increase in the slow transition which probably extends to other parts of the tRNA molecule. The observed rates for the transition T2 in equilibrium or formed from T3 are close to that observed for the transfer of the activated phenylalanine to tRNA Phe. This raises the possibility that the conformational transition in tRNA is the rate limiting step in the charging reaction.     

Microviscosity in dilute aqueous solutions of SDS and non-ionic cellulose derivatives of different hydrophobicity: fluorescence probe investigations

The microviscosity in mixed micelles formed in dilute aqueous solutions of sodium dodecyl sulphate (SDS) and a set of non-ionic cellulose ethers of different hydrophobicity has been determined by means of steady-state fluorescence probe techniques. Two hydrophobic probes have been applied in this investigation: 1,3-di(1-pyrenyl)propane (P3P) and perylene. Reference measurements of microviscosity have also been performed on SDS solutions including the uncharged polymers poly(ethyleneoxide) (PEO) or poly(vinylpyrrolidone) (PVP). All compositions investigated showed qualitatively the same general behaviour with an abrupt increase in microviscosity at the critical surfactant concentration where the polymer-surfactant interaction starts (c₁) followed by a maximum and an asymptotically declining region as the surfactant concentration was increased further. Comparison with a recent investigation of a specific ethyl(hydroxyethyl)cellulose (EHEC fraction CST-103)/ SDS/water system (Evertsson & Nilsson (1997) Macromolecules, 30, 2377) revealed that the maximum in microviscosity generally corresponds to a low degree of SDS adsorption (≈ 0.5 mmol of SDS per gram of polymer) and consequently to a high polymer content of the mixed micelles formed in the type of systems studied herein. The hydrophobicity of the cellulose derivatives was found to correlate to the amplitude of the overall microviscosity pattern for the mixed micelles, i.e. an increased polymer hydrophobicity gave an increased rigidity of the polymersurfactant aggregates. An approximately exponential relation was demonstrated between the maxima in microviscosity of the different mixed micelles and the surface activities of the corresponding cellulose derivatives. All polymer/surfactant combinations investigated gave aggregates with a higher rigidity than ordinary SDS micelles. The microviscosity of the mixed micelles of the cellulose derivatives and SDS formed close to c₁ increased as the temperature rose from 20 to 50 °C. This effect was attributed to an increased hydrophobicity of the cellulose ethers upon temperature elevation, hence giving rise to further close-packing of the aggregate structures.     

Structure of aggregating kappa-carrageenan fractions studied by light scattering

We have studied kappa-carrageenan fractions with varying molar mass, obtained by sonication, using static and dynamic light scattering and polarimetry. The samples were characterised in 0.1 M NaCl and 0.1 M NaI, i.e. in the coil and helix conformation, respectively. We find that the molar mass and size of the untreated sample are the same in the coil and helix conformation. For the sonicated samples, we find larger average molar masses and sizes in the helix conformation. The critical temperature, T(c), below which the coil-helix transition sets in, decreases with decreasing molar mass. Aggregation is induced by lowering the temperature in the presence of 0.01 M KCl, which leads to the formation of locally rigid bundles of kappa-carrageenan chains. The thickness of the bundles increases slowly with time and we have not observed stabilisation, even after 24 h at 10 degrees C below T(c). The local structure of the aggregates is the same for all fractions, but at a given temperature, the rate of aggregation decreases with decreasing molar mass.     

The fluorescence studies of the sol-gel transition by styrylpyridine derivative

The gelation process of tetraethylorthosilanes in acid environment was monitored with the trans-4-(p-N,N-dimethylaminostyryl)-N-vinylbenzylopyridinium chloride (vbDMASP) fluorescent probe. The fluorescence steady-state and anisotropy measurements of material during sol-gel transition are reported. The results are compared with fluorescence studies of the probe in a modeled viscous system of water-glycerol mixtures. A strong increase of anisotropy, from 0.1 to 0.9, with gelation time as well with wavelength, was observed. Although the increase of anisotropy with wavelength is due to specificity of the compounds exhibiting charge transfer properties, the increase of the anisotropy with gelation time is due to an increase of microviscosity. On this basis, suitability of the applied fluorophore in recording of viscosity changes during sol-gel transition is discussed. The molecular structure of vbDMASP in the excited states in dependence on environmental polarity was optimized using the HyperChem and Amsol program. The dynamics of torsional angle C35-C34-N31-C28 of the multichromophore dye in correlation with micropolarity and microviscosity of the network formation during the sol-gel transition is discussed.     

Stage-specific changes in membrane microviscosity in <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> embryos

Natural and probe fluorescence as well as membrane microviscosity was studied in eggs and embryos of Misgurnus fossilis by fluorescence microscopy. The lateral mobility of the probe (pyrene) increased in loach embryos from early to late blastula, which indicates a decrease in plasma membrane microviscosity. At the later stage of mid-gastrula, the microviscosity remained largely invariant. Considering that the embryo exposure to different temperatures changes the quantum yield of fluorescence and the degree of pyrene excimerization, one can gain information about both the temperature-induced structural changes and changes in membrane microviscosity in the embryos. Natural and probe fluorescence of embryonic membranes is proposed as at tool to study morphogenetic mechanisms.     

Effect of Escherichia coli lipopolysaccharide on the microviscosity of liver plasma membranes and hepatocyte suspensions and monolayers

The fluorescence probe 1,6-diphenylhexa-1,3,5-triene (DPH) was used for monitoring structural perturbations induced by lipopolysaccharide (LPS) of Escherichia coli (0111:B4) in plasma membranes of rat liver. Changes in microviscosity were observed in plasma membrane preparations from control rats after treatment with LPS and in plasma membrane preparations from liver perfused with LPS. In both systems fluorescence polarization was measured from which microviscosity was calculated. This parameter increases with LPS treatment. From temperature dependence studies was inferred that LPS interaction with plasma membrane preparations induces an increase of both the polarization term (r0/r-1)-1 and flow activation energy (delta E). Addition of LPS to hepatocyte suspensions also induces an increase on microviscosity and a delay in the fall of microviscosity induced by a temperature rise in hepatocyte monolayers grown on microcover slides. These data suggest that LPS interaction can be attributed to its binding to membrane hydrophobic regions in a non-specific manner.     

Microviscosity of mucosal cellular membranes in toad urinary bladder: Relation to antidiuretic hormone action on water permeability

Summary The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad,Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23°C, 5mm dibutyryl cyclic 3,5-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P<0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.     

Modeling the kinetics of the pectin methylesterase catalyzed de-esterfication of pectin in frozen systems

The applicability of the William, Landel, and Ferry (WLF) equation with a modification to take into account the effect of melt-dilution and an empirical log-logistic equation were evaluated to model the kinetics of diffusion-controlled reactions in frozen systems. Kinetic data for the pectin methylesterase catalyzed hydrolysis of pectin in four model systems with different glass transition temperatures: sucrose, maltodextrin (DE = 16.5-19.5), carboxymethylcellulose (CMC) and fructose in a temperature range of -24 to 0 degrees C were used. The modified WLF equation was evaluated with a concentration-dependent glass transition temperature (T(g)) as well as the glass transition temperature of the maximally freeze-concentrated matrix (T(g)') as reference temperatures. The equation with temperature-dependent T(g) described the reaction kinetics reasonably well in all the model systems studied. However the kinetics was better described by a linear relationship between log(V(0)/V(0ref)) and (T - T(ref)) in all cases except CMC. The log-logistic equation also described the kinetics reasonably well. The effect of melt-dilution on reactant concentration was found to be minimal in all cases.     

Nonsymmetrical polymethine dyes as fluorescent probes for the study of microviscosity of membrane phospholipid bilayers

Nonsymmetrical polymethine dyes are shown to be applied as a new approach in the studies of phospholipid membrane microviscosity. The method requires determination of the intensity ratio for the long-wave length (Ig) and short-wave length (Ik) bands of fluorescence spectra in the region of 730-770 nm at exitation 600 nm. It allows determination of microviscosity by comparative measurements of the fluorescence parameter Ig/Ik in the model medium of the known viscosity (glycerol) and the object under study. Microviscosity in egg phosphatidylcholine vesicules (liposomes) is found to be 0.6-1.2 P. It depends on the surface curvature (size of vesicle), cholesterol content and temperature. It the studies of dimiristoylphosphatidyl choline liposomes the changes in microviscosity at the phase transition temperature are shown to be from 4.5 to 1.1 P. The transition temperature is 24.5 degrees C, the transition range being 2.2 degrees C. The results of this work demonstrate the advantages of the suggested approach to study mobility in phospholipid membranes and confirm it to be promising to study natural membranes and whole cells.     

Viscous solutions, networks and the glass transition in high sugar galactomannan and kappa-carrageenan mixtures

Small-deformation rotational oscillation was used to examine the effect of small additions of galactomannan and kappa-carrageenan on the vitrification of glucose syrup at a total level of solids of 83%. The method of reduced variables allowed construction of composite curves covering the glass transition and glassy state (from 10(5) to 10(9.5) Pa) over a wide frequency range (up to 15 orders of magnitude). The combined WLF/free volume framework was employed to determine the rheological glass transition temperature (T(g)), fractional free volume and thermal expansion coefficient of the samples. It was found that the WLF-predicted glass transition temperature matched the cross over of experimental modulus traces in the passage from the glass transition (GG') to the glassy state (GG"). This coincides with the mechanistic transformation from free volume effects to the Arrhenius-type phenomena, thus ascribing physical significance to the rheological T(g). The T(g) value of 83% glucose syrup at a scan rate of 2 degrees C min(-1) was -25.3 degrees C. Replacing, for example, 1% glucose syrup with guar gum shifted the T(g) of the mixture to -19.7 degrees C. Network formation via the K(+)-supported junction zones of the kappa-carrageenan chains further increased the T(g) to about -1 degrees C. It appears that the low rates of relaxation processes and diffusion mobility in the presence of a polysaccharide network accelerate the collapse of the free volume thus inducing vitrification of the high sugar/polysaccharide mixture at high temperatures.     

The effect of pressure on the lipid microviscosity and phase transition of lung surfactant

The effect of pressure on the lipid dynamics of the rat lung surfactant was studied in liposomes made of the natural lung surfactant of the rat and of model phospholipid mixture. The determined parameter was the lipid microviscosity, monitored by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Osmotic pressure of up to 47 atm, as well as hydrostatic pressure of up to 1.4 kbar, were applied at a constant temperature. The effect of pressure was monitored by the change in the lipid microviscosity of the system. The maximal change achieved with osmotic pressure at a constant temperature was only 30%. This suggests that the conversion of melted lipid to its solid phase above the lipid critical temperature requires several hundred atmospheres. Similarly, measurements of lipid microviscosity under increased hydrostatic pressure revealed transitions which occurred at above 400 atm. Since such pressures are far beyond the physiological scale, it excludes the possibility that pressure alone can be responsible for a full phase transition of the lung surfactant during respiration. Upon decompression, microviscosity of the examined lipid system was found to return to its original values, confirming the reversibility of the process.     

Rheology of kappa-carrageenan in mixtures of sodium and cesium iodide: two types of gels

Recent studies on dilute solutions (Borgström et al. (1996), Int. J. Biol. Macromol. 18, 223) have shown that kappa-carrageenan helices associate into superhelical rigid rods in mixed 0.1 M aqueous solutions of NaI and CsI above a critical mole fraction (x_Cs = 0.4) of Cs. This work concerns the temperature-dependent rheology of more concentrated systems in mixed and pure solutions of the same salts. Gels with low moduli were even found in NaI alone, although this salt is known to impede the gelation of kappa-carrageenan, but only above 0.9% (w/w) of carrageenan. These gels were reminiscent of iota-carrageenan gels in two respects: the (low) magnitude of the shear storage modulus (G′), and the absence of hysteresis in the sol-gel transition. On the other hand, both the threshold concentration for gelation and the ratio between the loss and storage moduli were substantially higher for the kappa-carrageenan gels in NaI. In mixed solutions of CsI and NaI, two types of kappa-carrageenan gels could be distinguished, depending on the cesium content. The transition occurred at x_Cs = 0.4, as in the previous studies on dilute solutions. Below x_Cs = 0.4, the gels were similar to those in NaI alone. Above x_Cs = 0.4, the gels were similar to ‘conventional’ kappa-carrageenan gels, formed in salts such as KC1: a pronounced thermal hysteresis appeared in the sol-gel transition, the gels showed tendencies for syneresis, and G′ increased dramatically with increasing cesium content.     

天花粉蛋白小结构域N端同源片段的溶液构象：T18肽的圆二色性和荧光研究

应用圆二色性,内源荧光和疏水荧光探针法进一步研究Ｔｌ８肽的溶液构象及其相互转化。发现Ｔ１８肽在水溶液中为β折叠结构,且在高浓度（＞１ｍｇ／ｍｌ）时形成疏水聚合物;Ｌｙｓ１５和Ｉｌｅ３－Ｉｌｅ４是形成β折叠疏水簇的关键因素。并讨论了蛋白质肽链和溶剂环境对肽段二级结构的调制作用。     

Fluidity of the lipid phase of bovine serum high density lipoprotein from fluorescence polarization measurements.

The microviscosity of the lipid phase of bovine serum high density lipoprotein was determined by fluorescence polarization measurements on a lipophilic probe (1,6-diphenyl-1,3,5-hexatriene) dissolved in the lipoprotein. At 25°C the average microviscosity was 6.1 ± 0.5 poise, and the activation energy calculated from a plot of log η versus $\text{1}\text{T}$ was 13±3Kcal/mole. A constant slope for the Arrhenius plot from 0 to 46°C indicated no apparent phase transitions in this temperature range.Comparison of the present results with reported microviscosity values for rat lymphocyte membranes and liposomes [Shinitzky and Inbar (1974) $\text{J. Mol. Biol. 85}$, 603] indicates a more rigid environment of the probe in the high density lipoprotein system fluidity of the lipid appears to be considerably decreased in the lipoprotein relative to organic solvent or oil solutions of lipids, probably as a result of the anisotropic environment of the probe, high total cholesterol, and presence of protein in these particles.     

Calorimetric and chiroptical evidence of aggregate-driven helix formation in carrageenan systems

Thermally induced, order-disorder transitions of iota- and kappa-carrageenan have been monitored by optical rotation and differential-scanning calorimetry in various ionic environments. Conformational ordering in kappa-carrageenan is observed only in the presence of cations that have been shown previously to promote helix-helix aggregation, and shows marked hysteresis between heating and cooling. Iota-carrageenan, by contrast, shows an order-disorder transition in the non-aggregating, tetramethylammonium salt form, at substantially lower temperature than for kappa-carrageenan, and without hysteresis. In the presence of potassium ions, which are known to promote aggregation, iota-carrageenan shows two distinct thermal-transitions, one without hysteresis at the same temperature as observed under non-aggregating conditions, and one with significant hysteresis close to the temperature of the kappa-carrageenan transition. We interpret these transitions as helix-to-coil and aggregated helix-to-coil, respectively. This interpretation is supported by measurements of the enthalpy changes of the transitions; ΔH values show a systematic increase with increasing aggregation and hysteresis. We conclude that the double helix of iota-carrageenan can exist as a stable entity in isolation, but may be further stabilised by aggregation, whereas the kappa-carrageenan helix is stable only when aggregated.     

Effect of the cluster size on the micro phase separation in mixtures of beta-lactoglobulin clusters and kappa-carrageenan

The phase separation of globular protein clusters formed by heat-denatured beta-lactoglobulin (beta-lg) in mixtures with the polysaccharide kappa-carrageenan (kappa-car) has been studied at pH 7 and 20 degrees C. The effect of the protein cluster size on the phase separation was investigated by preparing clusters with radii between 20 nm and 1 mum. The formation of protein rich microdomains led to an increase of the turbidity starting at a minimum kappa-car concentration that decreased with increasing cluster size, but was only weakly dependent on the protein concentration. The size and number of microdomains do not depend much on the cluster size, but their density decreases with increasing cluster size leading to a lower turbidity.     

Macromolecular crowding and size effects on probe microviscosity

Development of biologically relevant crowding solutions necessitates improved understanding of how the relative size and density of mobile obstacles affect probe diffusion. Both the crowding density and relative size of each co-solute in a mixture will contribute to the measured microviscosity as assessed by altered translational mobility. Using multiphoton fluorescent correlation spectroscopy, this study addresses how excluded volume of dextran polymers from 10 to 500 kDa affect microviscosity quantified by measurements of calmodulin labeled with green fluorescent protein as the diffusing probe. Autocorrelation functions were fit using both a multiple-component model with maximum entropy method (MEMFCS) and an anomalous model. Anomalous diffusion was not detected, but fits of the data with the multiple-component model revealed separable modes of diffusion. When the dominant mode of diffusion from the MEMFCS analysis was used, we observed that increased excluded volume slows probe mobility as a simple exponential with crowder concentration. This behavior can be modeled with a single parameter, β, which depends on the dextran size composition. Two additional modes of diffusion were observed using MEMFCS and were interpreted as unique microviscosities. The fast mode corresponded to unhindered free diffusion as in buffer, whereas the slower agreed well with the bulk viscosity. At 10% crowder concentration, one finds a microviscosity approximately three times that of water, which mimics that reported for intracellular viscosity.