Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter approximately 0.1 and 0.2 microm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 degrees C, this temperature corresponding closely to the heat capacity maxima (T(em)) of DNPC MLVs and LUVs (T(em) approximately 21 degrees C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T(em). This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans-->gauche isomerization.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter ∼0.1 and 0.2 μm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 °C, this temperature corresponding closely to the heat capacity maxima (T_em) of DNPC MLVs and LUVs (T_em ≈21 °C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T_em. This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans→gauche isomerization.     

Characterization of the main phase transition in 1,2-dipalmitoyl-phosphatidylcholine luvs by 1H NMR

The main phase transition (Tm) of 100 nm large unilamellar vesicles (LUVs) of 1,2-dipalmitoylphosphatidylcholine (DPPC) was investigated using 1H NMR (proton magnetic resonance) in deuterium oxide, and both DSC (differential scanning calorimetry) and IR (infrared) spectroscopy in water and deuterium oxide. The ability of 1H NMR to determine Tm was demonstrated and the values obtained were in general agreement with those observed with DSC and IR. However, the temperature range of the transition observed by NMR was significantly broader than that observed with either DSC or IR. The effect of deuterium oxide on Tm was studied by comparing results obtained in water and deuterium oxide with DSC and IR. The results showed no significant difference in Tm or temperature range of transition determined in these solvents.     

Determination of phase transition temperatures of lipids by light scattering

Various techniques have been proposed to specify the phase transition temperatures of surfactant molecules. The work reported herein deals with a new general method of T(c) determination based on the optical properties' modifications of aqueous surfactant solutions when the phase transitions occur in the phospholipid membrane. The shape alteration of supramolecular systems induced by the phase transition was correlated with the refraction and absorption coefficients of their aqueous dispersion. The mean count rate (average number of photons detected per second) measured with a Zetasizer Nano-S model ZEN1600 Dynamic Light Scattering Instrument, is representative of an emerging macroscopic phenomenon, but not directly size dependent and has been adapted to our expectations. Changes in the measured scattering intensity reflect changes in the optical properties of the material during temperature variations. Thus, this method allowed to specify the phase transition temperature of many natural or synthetic surfactants independently of their polar head or hydrophobic part.     

Dynamic fluorescence measurements on the main phase transition of dipalmytoylphosphatidylcholine vesicles

The kinetics of the main phase transition in dipalmytoylphosphatidylcholine (DPPC) vesicles have been investigated using our iodine laser-Tjump technique with fluorescence detection. A set of three fluorescent probes has been used to sense different parts of the bilayer hydrocarbon chain region. The well established membrane probes DPH and TMADPH as well as DPHPC, a labelled DPPC molecule. We report three relaxation signals in the s and ms time range, which are detected with all three probes. This result supports our model of the main phase transition in DPPC vesicles.Abbreviations DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmytoylphosphatidylcholine - DPH 1,6-Diphenylhexa-1,3,5-triene - TMADPH 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene - DPHPC Diphenylhexatriene-phosphatidylcholine - T_m Temperature of the main phase transition     

Kinetics of the main phase transition of hydrated lecithin monitored by real-time X-ray diffraction.

A method is described for observing and recording in real-time x-ray diffraction from an unoriented hydrated membrane lipid, dipalmitoylphosphatidylcholine (DPPC), through its thermotropic gel/liquid crystal phase transition. Synchrotron radiation from the Cornell High Energy Synchrotron Source (Ithaca, New York) was used as an x-ray source of extremely high brilliance and the dynamic display of the diffraction image was effected using a three-stage image intensifier tube coupled to an external fluorescent screen. The image on the output phosphor was sufficiently intense to be recorded cinematographically and to be displayed on a television monitor using a vidicon camera at 30 frames X s1. These measurements set an upper limit of 2 s on the DPPC gel----liquid crystal phase transition and indicate that the transition is a two-state process. The real-time method couples the power of x-ray diffraction as a structural probe with the ability to follow kinetics of structural changes. The method does not require an exogenous probe, is relatively nonperturbing, and can be used with membranes in a variety of physical states and with unstable samples. The method has the additional advantage over its static measurement counterpart in that it is more likely to detect transiently stable intermediates if present.     

Properties of chloroplasts isolated by phase partition

Summary Chloroplasts from spinach can be separated into at least three different populations by countercurrent distribution using polymer two-phase systems. The chloroplast particles of the three populations differ in protein/chlorophyll ratio, ultrastructure and metabolism. One population, peak I, consists of intact chloroplasts surrounded by the chloroplast envelope; the second population, peak II, consists of chloroplasts, which have lost their envelopes and much of their stromal material; the third population, peak III, consists of particles containing intact chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and peroxisomes.Rapid batch procedures of peak I chloroplasts incorporated¹⁴C almost entirely into glycolate and intermediates of the Calvin cycle and starch synthesis. Only small amounts were found in sucrose and amino acids. On the other hand preparations of peak III chloroplasts gave a much broader spectrum of¹⁴C-labelled products. Sucrose, malate and some amino acids contained about 40% of the¹⁴C incorporated. It is concluded from these experiments that sucrose is formed not within the chloroplast but in the cytoplasm from intermediates exported by the chloroplast.The origin of peak III particles and their use for studying the cooperation between the chloroplast and the surrounding cytoplasm including mitochondria and peroxisomes is discussed.An invited article.     

Interaction of liposome with immobilized chitosan during main phase transition

It has been recently demonstrated that chitosan in aqueous solution alters the phase behavior and structure of a phospholipid bilayer (Fang, N.; et al. Biomacromolecules 2001, 2, 1161-1168). Until now, the physical driving forces between chitosan and the phospholipid bilayer upon their initial encounter remains unknown. In this study, confocal reflectance interference contrast microscopy (C-RICM), phase contrast microscopy and bioadhesion modeling are concurrently applied to probe the interaction of phospholipid vesicle with immobilized chitosan at various temperatures, pH, and osmotic stress. First, the successful immobilization of chitosan on amino-silanized glass is indicated by the increases in both the degree of vesicle deformation and adhesion energy of vesicles adhering on chitosan modified substrate in comparison with those on amino-silanized glass. Second, the phase transition of a phospholipid bilayer does not modulate the adhesion strength at the chitosan-biomembrane interface at pH 7.4. With increase of the degree of protonation on the chitsoan backbone at pH 4, the adhesion energy is increased by 5-fold for vesicles of all sizes compared to that in pH 7.4. Furthermore, pH reduction amplifies the thermal-induced response of larger vesicles on the immobilized chitosan layer. Interestingly, a moderate increase of osmotic stress maximizes the degree of vesicle deformation and adhesion energy at 23 degrees C and dampens the effect of phase transition on vesicle adhesion. Overall, this study demonstrates the quantitation of chitosan-biomembrane interactions that will be critical for future applications of chitosan in biological systems.     

Substrate-induced deformation and adhesion of phospholipid vesicles at the main phase transition

The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T(m). In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T(m) and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 microm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.     

Application of the change in partition coefficient with pH to the structure determination of alkyl substituted guanosines.

A versatile method for the determination of the ionization of guanosine is described. Suitably derivatized alkyl-guanosines are partitioned between organic solvents and aqueous buffer solutions at various pH values. Ionization is revealed by a change in partition coefficient with pH. The method is ideally suited for application to micro samples since any quantitative method can be used to determine the partition coefficient. A procedure for distinguishing N¹ or O⁶, N², and C⁸ alkylation of guanosine is described.     

The kinetics of the main phase transition of aqueous dispersions of phospholipids induced by pressure jump and monitored by raman spectroscopy

The sensitivity of the melting transition temperature of aqueous dispersions of dipalmitoyl- and distearoylphosphatidylcholine to hydrostatic pressure is used to allow measurement of the rates of isothermal freezing and melting of the lipids by rapidly changing the pressure. The degree of order of the lipids is measured by monitoring a ratio of two points in the Raman spectrum of the lipids which changes sharply at the melting temperature. Use of this Raman order ratio allows correlation between the order of the sample and the rates of transition in a manner which is impossible by monitoring only turbidity. Our longest relaxation times range upwards from a few seconds for both compounds. The freezing rates are slowest when the samples are initially fully melted, and the melting rates are slowest when the samples are initially frozen. These results imply that nucleation of the growing phase dominates the kinetics of both freezing and melting.     

Determination of the overall volumetric mass transfer coefficient kLa,by a temperature dependent change of gas solubility

Summary The solubility of oxygen in the liquid phase of a bioreactor was changed by a ramp change of temperature, and k_La was determined from the resulting return to equilibrium of dissolved oxygen activity. The maximum k_La that can be measured by this method in a standard laboratory scale bioreactor is 145 h^–1 corresponding to a temperature change rate of 320°C h^–1.Nomenclature p Difference between p_G and p_L (% saturation) - T Ramp change of temperature (°C) - E Temperature-compensated output from the oxygen electrode (A) - E_u Uncompensated output from the oxygen electrode (A) - k_La Overall volumetric mass transfer coefficient (h^–1) - k_La_Tm Overall volumetric mass transfer coefficient at temperature T_m (h^–1) - P_G Dissolved oxygen activity in equilibrium with the gas phase (% saturation) - p_L Dissolved oxygen activity (% saturation) - p_Lm Dissolved oxygen activity at time t_m (% saturation) - t Time (h) - t_m Time of maximum p (h) - T Temperature (°C) - T_cal Calibration temperature of the oxygen electrode (°C) - T_m Final temperature after a temperature shift (°C) - T_n Temperature at time t_n     

Determination of membrane lipid phase transition temperature from C-NMR intensities

A new and simple approach for the determination of the temperature of gel-to-liquid crystalline phase transitions (T_C) of biological (chloroplast) membrane lipids from ¹³C-NMR resonance intensities is proposed. The variation of intensity of a temperature-sensitive NMR resonance is monitored by recording the spectra of the sample at a range of temperatures. From such a series of spectra recorded at different temperatures, a temperature-insensitive resonance is located. Then the ratio of the intensity of the temperature-sensitive to the intensity of the temperature-insensitive resonance is calculated from each spectrum to even out the procedural error, if any. The values of this ratio at different temperatures, when plotted against sample temperature, shows a break at T_C as confirmed by spin label ESR studies.     

Determination of lipid loss during aqueous and phase partition fixation using formalin and glutaraldehyde

Phase partition fixation permits fixation of tissue in a nonaqueous environment, thus eliminating osmotic effects. It was shown in an earlier investigation that retention of protein in liver blocks can be improved by phase partition fixation. By using radioisotopic labeling techniques, the effects of phase partition fixation on lipid retention during fixation, dehydration, and clearing have been determined and compared with those of standard aqueous fixation techniques. In this article we show that retention of total lipid in liver blocks following phase partition fixation using formalin was comparable to or better than that with aqueous formalin fixation and processing. Fixation with glutaraldehyde using phase partition fixation resulted in somewhat greater loss of total lipid than that observed for aqueous buffered glutaraldehyde-fixed blocks.     

Characterization of the main transition of dinervonoylphosphocholine liposomes by fluorescence spectroscopy

The structural dynamics of the main phase transition of large unilamellar dinervonoylphosphocholine (DNPC) vesicles was investigated by steady state and time-resolved fluorescence spectroscopy of the membrane incorporated fluorescent lipid analog, 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC). These data were supplemented by differential scanning calorimetry (DSC) and fluorescence anisotropy measured for 1-palmitoyl-2-(3-(diphenylhexatrienyl) propanoyl)-sn-glycero-3-phosphocholine (DPHPC). The collected data displayed several discontinuities in the course of the main transition and the pretransition. The discontinuities seen in the fluorescence properties may require modification of the existing models for phospholipid main transition as a first order process. From our previous study on dipalmitoylphosphocholine (DPPC), we concluded the transition to involve a first-order process resulting in the formation of an intermediate phase, which then converts into the liquid crystalline state by a second order process. Changes in the physical properties of the DNPC matrix influencing probe behavior were similar to those reported previously for PPDPC in DPPC. In gel state DNPC [(T-T(m))<-10] the high values for excimer/monomer emission ratio (I(e)/I(m)) suggest enrichment of the probe in clusters. In this temperature range, excimer fluorescence for PPDPC (mole fraction X(PPDPC)=0.02) is described by two formation times up to (T-T(m)) approximately -10, with a gradual disappearance of the fractional intensity (I(R1)) of the shorter formation time (tau(R1)) with increasing temperature up to (T-T(m)) approximately -10. This would be consistent with the initiation of the bilayer melting at the PPDPC clusters and the subsequent dispersion of the one population of PPDPC domains. A pronounced decrement in I(e) starts at (T-T(m))=-10, continuing until T(m) is reached. No decrease was observed in fluorescence quantum yield in contrast to our previous study on DPPC/PPDPC large unilamellar vesicles (LUVs) [J. Phys. Chem., B 107 (2003) 1251], suggesting that a lack of proper hydrophobic mismatch may prevent the formation of the previously reported PPDPC superlattice. With further increase in temperature and starting at (T-T(m)) approximately -1, I(e), tau(R2), and excimer decay times (tau(D)) reach plateaus while increment in trans-->gauche isomerization continues. This behavior is in keeping with an intermediate phase existing in the temperature range -1<(T-T(m))<4 and transforming into the liquid disordered phase as a second order process, the latter being completed when (T-T(m))-->4 and corresponding to approximately 50% of the total transition enthalpy.     

Highly purified mitochondria from rat brain prepared by phase partition

Mitochondria and synaptosomes from adult rat forebrain can easily be separated by counter-current distribution in an aqueous two phase system composed of Dextran T500 and poly(ethylene glycol) 4000. Both particles may also be separated by a batch procedure in which the same phase system is used. Electron micrographs and enzymatic activities show a high purity of the mitochondria obtained from the dextran-rich lower phase. Electron micrographs and enzymatic activities also show that intact synaptosomes can be obtained from the poly (ethylene glycol)-rich upper phase.The mitochondria purified by this method show good ADP/O ratios, respiratory control ratios, and state 3 rates. Synaptosomes showed a state 2-state 3 transition with no recuperation to state 4.