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PubMed1989

Reversed-phase HPLC separation of plasma norepinephrine, epinephrine, and dopamine, with three-electrode coulometric detection.

We present a method involving minor modifications of previous techniques, in which we use an ion-pair reversed-phase HPLC separation with three-electrode coulometry. Isocratic baseline separations can be obtained in 5 min. The sample preparation procedure, involving extraction with alumina at low temperature, allows good reproducibility (within-run and between-run CVs less than 10%) and improved sensitivity (less than 5 pg of each catecholamine per extract). This method allows approximately 70 low-cost plasma catecholamine analyses to be done in a working day.

Catecholamines↗

PubMed2006

Aggregation behavior of polypropylene oxide with electric charges at both ends in aqueous solution.

The aggregation behavior of polypropylene oxide (PPO) with positive charges at both ends was investigated in aqueous solution by means of the measurements of solution turbidity, dynamic light-scattering, differential scanning calorimetry, and dye solubilization. The positive charges were produced by protonation of terminal NH(2) groups attached to the polymer composed of 33 PO units. It was found that the aggregation behavior is quite sensitive to temperature. At low temperature, the polymer dissolves in water as a unimer. When temperature is increased, the unimer solution undergoes a phase separation to give a turbid solution. Further increase in temperature produces a transparent micellar solution. The aggregation of the polymer molecules must be induced by the dehydration of PPO chain caused by temperature increase. According to the analysis of heat absorptions associated with the melting of the solid mixture and the phase separation of the unimer solution, it is suggested that approximately 10% dehydration of PPO chain causes the phase separation. The temperature-composition phase diagram of aqueous mixture of this polymer was constructed on the basis of turbidity and DSC experiments, which reveals the aggregation behavior of this polymer in aqueous medium as a function of concentration and temperature.

Journal Article↗

PubMed2000

Characteristics of complexes composed of sodium hyaluronate and bovine serum albumin.

Complexes composed of sodium hyaluronate (NaHA) and bovine serum albumin (BSA) were studied to elucidate the exact composition of the complex, the phase separation, the electrophoretic mobility and the size using dynamic light scattering (DLS) and electrophoretic light scattering (ELS), etc. The phase diagram of the mixed solutions was determined. The complexes were soluble in neutral or weakly acidic pH regions and showed phase separation in the more acidic pH region. From the concentration of Na+ released from NaHA when it binds to BSA, the ratios of BSA to NaHA of the complexes were determined. In the region of soluble complexes, one BSA molecule was determined to bind with 15 carboxylic groups of NaHA and in the region of insoluble complexes to bind with 6 carboxylic groups. At the phase separation point, 117 BSA molecules bound with one NaHA molecule and 17% of the carboxylic groups of NaHA did not contribute to the binding of BSA. The sizes of the complexes decreased from several microm to several hundred nm as the binding ratio of BSA increases. Decreases in the viscosities of the mixed solutions were consistent with the decreases of the sizes. From these results, a model of complex formation is proposed.

Animals↗

PubMed2002

Adhesion of membranes with competing specific and generic interactions.

Biomimetic membranes in contact with a planar substrate or a second membrane are studied theoretically. The membranes contain specific adhesion molecules (stickers) which are attracted by the second surface. In the absence of stickers, the trans-interaction between the membrane and the second surface is assumed to be repulsive at short separations. It is shown that the interplay of specific attractive and generic repulsive interactions can lead to the formation of a potential barrier. This barrier induces a line tension between bound and unbound membrane segments which results in lateral phase separation during adhesion. The mechanism for adhesion-induced phase separation is rather general, as is demonstrated by considering two distinct cases involving: i) stickers with a linear attractive potential, and ii) stickers with a short-ranged square-well potential. In both cases, membrane fluctuations reduce the potential barrier and, therefore, decrease the tendency of phase separation.

Journal Article↗

PubMed2006

Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation.

A combination of protein microarrays and two-dimensional liquid-phase separation of proteins has been used for global profiling of the phosphoproteome in human breast cancer cells. This method has been applied to study changes in phosphorylation profile resulting from treatment of the cancer cells with PD173074, a known receptor tyrosine kinase inhibitor. The proteins separated by 2-D liquid-phase separation were arrayed on epoxy-coated glass slides and first screened for phosphorylation using fluorescent Pro-Q Diamond stain. The candidate proteins were then identified using MALDI/ESI MS/MS analysis. Further, validation was achieved by immunoblot analysis using anti-phosphotyrosine antibodies. A dynamic range of approximately 100 was achieved on the microarray when beta-casein was used as a standard protein for obtaining quantitative data. Importantly, the power of this method lies in its ability to identify a large group of proteins in a single experiment that are coregulated in their posttranslational modifications, upon treatment with the inhibitor. Since proteins are known to form interacting circuits that eventually lead to various signaling events, detection of such global phosphorylation profiles might enable delineation of functional pathways that play an important role during cancer initiation and progression.

Breast Neoplasms↗

PubMed1992

Characterization by infrared spectroscopy of the interaction of a cardiotoxin with phosphatidic acid and with binary mixtures of phosphatidic acid and phosphatidylcholine.

The effect of cardiotoxin IIa from Naja mossambica mossambica, a small basic protein extracted from snake venom, on dimyristoylphosphatidic acid (DMPA) and on equimolar mixtures of DMPA and dimyristoylphosphatidylcholine (DMPC) has been studied by Fourier transform infrared spectroscopy. The interaction of cardiotoxin with DMPA dispersions decreases both the cooperativity of the phase transition of the lipid and the molecular order of the lipid acyl chains in the gel phase. This effect increases with the proportion of the toxin in the complexes and leads to the total abolition of the phase transition of DMPA at a lipid-to-protein molar ratio of 5. Small-angle X-ray results demonstrate that the structure of the lipid-protein complexes is poorly ordered and gives rise to broad diffusion peaks rather than to well-resolved diffraction patterns. Infrared spectra of oriented cardiotoxin-DMPA films show that the protein is not homogeneously oriented with respect to the bilayer surface. The destabilization of the gel-phase structure of DMPA by cardiotoxin also results in a deeper water penetration in the interfacial region of the lipid since more carbonyl ester groups appear to be hydrogen bonded in the presence of the toxin. The infrared results on the phosphate group vibrations also indicate clearly that the basic residues of cardiotoxin interact strongly with the phosphate group of DMPA that becomes partly ionized at a pH as low as 6.5. The results obtained on the interaction of cardiotoxin with an equimolar mixture of DMPA and DMPC clearly demonstrate the ability of this toxin to induce lateral phase separation in this mixture with one phase containing DMPA-rich domains perturbed by cardiotoxin while the second phase is composed of regions enriched in DMPC. Comparison of the results of the current study with those obtained on other basic proteins and polypeptides suggests that charge-induced phase separation occurs only when the charge density on certain regions of the protein structure is high enough to lead to efficient electrostatic interactions with anionic phospholipids. This condition occurs only when the conformation of the protein or polypeptide is well-ordered at the lipid interface.

Cobra Cardiotoxin Proteins↗

PubMed2026

Selective targeting of a histone-like silencer Sfx to the R6K conjugal transfer operon.

Conjugative plasmids drive bacterial evolution and antibiotic resistance spread, yet their gene expression must be silenced to protect the host. A histone-like protein H-NS represses many mobile and sedentary xenogenes but fails to silence the conjugal transfer vir operon of R6K, a prototype IncX plasmid. Instead, R6K encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. ChIP-seq reveals that Sfx and H-NS share similar DNA motifs and a preference for negative supercoiling, but occupy reciprocal genomic niches; Sfx is enriched on the R6K vir operon despite weak chromosomal binding, whereas H-NS displays the opposite preference. We show that Sfx binding to vir DNA critically depends on DNA topology and hypothesize that its selective targeting to R6K is mediated by Sfx-vir interactions and phase separation. Our results suggest that Sfx phase separates with R6K to ensure its preferential recruitment to the plasmid DNA and forms stable bridged nucleoprotein filaments that are impermeable to competitors such as H-NS. These findings reveal how histone-like proteins can partition the genome into distinct regulatory niches, a strategy likely mirrored across all life.

Operon↗

PubMed2006

Detrended fluctuation analysis in x-ray photon correlation spectroscopy for determining coarsening dynamics in alloys.

We study the dynamics of precipitate coarsening in phase-separating alloys at late stages of phase separation by x-ray photon correlation spectroscopy (XPCS). For analyzing time series of fluctuating speckle intensities from small-angle scattering of coherent x rays, the method of detrended fluctuation analysis (DFA), which is ideal for determining power-law correlations, is applied. We discuss the application of DFA with respect to XPCS data by means of simulated time series. In particular, the effects of different signal-to-noise ratios are examined. Results from measurements of the two model systems Al-6 at. % Ag at 140 degrees C and Al-9 at. % Zn at 0 degrees C are presented. Since the DFA effectively removes adulterating trends in the data, quantitative agreement with Monte Carlo simulations is obtained. It is verified that two different coarsening mechanisms are predominant in the two systems--coarsening either by diffusion of single atoms or by movement of whole precipitates.

Journal Article↗

PubMed2006

Gradual change or phase transition: characterizing fluid lipid-cholesterol membranes on the basis of thermal volume changes.

Cholesterol has been reported to govern biomembrane permeability, elasticity, and the formation of lipid rafts. There has been a controversy whether binary lipid-cholesterol membranes should better be described in terms of a phase separation (liquid-ordered and liquid-disordered phases) or of gradual changes in largely homogeneous membranes. We present a new approach for detecting and characterizing phase equilibria in colloidal dispersions using pressure perturbation calorimetry (PPC). We apply this to the study of the thermal expansivity of mixtures of 1-palmitoyl-2-oleoyl sn-glycero-3-phosphatidylcholine (POPC) and cholesterol as a function of composition and temperature. We show that cholesterol can condense lipids not only laterally (with respect to interfacial area) but also in volume. A quantitative comparison with expansivity curves simulated assuming either phase separation or random mixing within one phase reveals that the real system shows an intermediate behavior due to submicroscopic demixing effects. However, both models yield consistent system parameters and are thus found to be useful for describing the systems to a similar approximation. Accordingly, one cholesterol may condense 3 +/- 1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine molecules by approximately -(1.4 +/- 0.5) vol % at 2 degrees C; both absolute values decrease with increasing temperature.

Calorimetry↗

PubMed2005

Pre-biotic stage of life origin under non-photosynthetic conditions.

Spontaneous assembling of a simplest bacterial cell even if all necessary molecules are present in a solution seems to be extremely rare event and from the scientific standpoint has to be considered as impossible. Therefore, a predecessor of a living cell has to be very simple for providing its self-assembling and at the same time it should be able of progressive increase in complexity. Now phase-separated particles, first of all micelles, are put forward as possible predecessors of living cell. According to the offered working concept only phase-separated particles possessing autocatalytic properties can be considered as predecessors of living cells. The first stage of evolution of these phase-separated autocatalytic systems is the appearance of pre-biotic metabolism providing synthesis of amphiphiles for formation of capsules of these systems. This synthesis is maintained by the energy of a base reaction being a component of a planet-chemical cycle. Catalytic system providing functioning of pre-biotic metabolism is based on multivariate oligomeric autocatalyst, which reproduces itself from monomers, penetrating the particles from the outside. Since the autocatalyst realizes random polymerization then a collection of other oligomers possessing different catalytic functions is produced. In the paper the functioning of multivariate oligomeric autocatalyst in flow reactor is analyzed.

Catalysis↗

PubMed2001

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line.

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.

Amino Acid Sequence↗

PubMed1994

Expression of recombinant bovine gamma B-, gamma C- and gamma D-crystallins and correlation with native proteins.

Despite the use of bovine gamma-crystallins in numerous biophysical and chemical studies, characterization of these proteins at the molecular level is incomplete. Bovine lenses have at least six gamma-crystallin protein fractions currently assigned as gamma s/I, gamma A/IVb, gamma B/II, gamma C/IIIb, gamma D/IIIa and gamma E/IVa. A lack of primary sequence data for corresponding gamma-crystallin genes and proteins, however, has made these assignments tenuous. To clarify these assignments, we have over-expressed recombinant bovine gamma-crystallin proteins in Escherichia coli using complementary DNAs corresponding to gamma B-, gamma C-, and gamma D-crystallin genes. The recombinant crystallins were characterized by chromatographic and spectroscopic comparisons with native bovine crystallin fractions gamma II, gamma IIIa and gamma IIIb. The elution of recombinant gamma B and native gamma II proteins was identical on cation-exchange chromatography as expected; however, recombinant gamma C coeluted with gamma IIIa and recombinant gamma D co-eluted with gamma IIIb. Sequential Edman degradation through the first 29 residues of the native gamma IIIa and gamma IIIb polypeptides confirmed the colinearity of their sequences with those predicted from the gamma C- and gamma D-crystallin genes, respectively. Absorption and UV circular dichroism (CD) spectra of the recombinant proteins were almost identical to those of their native counterparts, indicating that the secondary and tertiary structures of the recombinant proteins were characteristic for gamma-crystallins. Based on these data the bovine gamma-crystallins proteins and genes are correlated as follows: II/gamma B, IIIa/gamma C and IIIb/gamma D. The assignment of gamma IIIb (previously characterized as having a low critical temperature for phase separation) with gamma D rather than gamma C proves an exception to the hypothesis that the gamma ABC-crystallin group is more resistant to phase separation than the gamma DEF group. These corrected assignments should provide a more substantial base for investigations of residues responsible for phase separation and other biophysical characteristics. Additionally, expression of recombinant gamma-crystallins with structures similar to native proteins may prove to be useful in probing specific structure-function relationships of the gamma-crystallins.

Amino Acid Sequence↗

PubMed1989

On studying asymmetric membranes made from polymethylmethacrylate/xylene by the thermal-inversion method.

Both polydisperse and monodisperse PMMA/xylene phase diagrams have been determined successfully by applying the optical density method. The changes in optical density for the polydisperse PMMA system are not as dramatic as the changes for the monodisperse PMMA system. Through the thermal-inversion process, the asymmetric membranes can be successfully made for this PMMA/xylene system. In the later stages of phase separation by spinodal decomposition, the flow properties of the PMMA/xylene system play a vital role in determining the final membrane microstructure. During the very early stage of phase separation by spinodal decomposition, a membrane with lacy structure results. In contrast, during the later stage of phase separation, a membrane structure with small pores dispersed in the polymer matrix results.

Chemical Phenomena↗

PubMed2005

Spatial chemical inhomogeneity and local electronic structure of Mn-doped Ge ferromagnetic semiconductors.

We have investigated the chemical distributions and the local electronic structure of potential diluted magnetic semiconductor Ge0.94Mn0.06 single crystals using scanning photoelectron microscopy (SPEM), x-ray absorption spectroscopy (XAS), and photoemission spectroscopy (PES). The SPEM image shows the stripe-shaped microstructures, which arise from the chemical phase separation between the Mn-rich and Mn-depleted phases. The Mn 2p XAS shows that the Mn ions in the Mn-rich region are in the divalent high-spin Mn2+ states but that they do not form metallic Mn clusters. The Mn 3d PES spectrum exhibits a peak centered at approximately 4 eV below E(F) and the negligible spectral weight near E(F). This study suggests that the observed ferromagnetism in Ge1-xMnx arises from the phase-separated Mn-rich phase.

Journal Article↗

PubMed1987

Physico-chemical properties and organization of lipids in membranes: their possible role in myocardial injury.

Lipids in biological membranes are organized in a bilayer configuration in order to form a semi-permeable barrier. The lipids are freely mobile in the bilayer, which is denoted as "fluid" or liquid-crystalline. For plasma membranes it is assumed that the lipids are not homogeneously distributed over the two leaflets or monolayers. This so-called lipid asymmetry is established for the erythrocyte membrane. There it was found that phosphatidylserine (PS) and phosphatidylethanolamine (PE) are present exclusively and predominantly in the cytoplasmic leaflet, respectively. It is shown that isolated PE at physiological conditions forms a non-bilayer configuration the so-called hexagonal HII phase. Moreover, isolated PS can undergo a transition from the fluid into the solid state upon addition of calcium. In mixtures of PS and PE, calcium is able to induce fusion events, possibly formation of the HII phase and phase separation of solid PS. The physico-chemical behaviour of these phospholipids will be discussed in the light of the structural changes of the sarcolemma of heart muscle cells observed by freeze-fracturing and thin section electron microscopy after ischaemia, ischaemia and reperfusion and the calcium paradox. The lateral phase separation of intramembranous particle aggregation is explained as isothermic phase separation by H+ and calcium. The disruption of the sarcolemma by the formation of blebs (liposomal structures) is interpreted as a destabilization of the bilayer configuration since PE prefers the HII phase and thus induces uncontrolled fusion events. This all leads to an irreversible disruption of the sarcolemma.

Animals↗

PubMed1984

Mobile-phase and temperature effects in the reversed phase chromatographic separation of proteins.

This paper explores the influence of mobile-phase and temperature effects on the gradient elution reversed phase chromatographic behavior of proteins. Using LiChrospher SI 500, bonded with n-butyl groups and a gradient in 1-propanol, 10 mM H3PO4, rapid separation and high mass recovery were obtained for a series of globular proteins. This protein separation and recovery are compared to those obtained when acetonitrile and acetonitrile plus 10 mM H3PO4 are used as eluting gradient solvents. In general, acetonitrile yielded lower recovery than 1-propanol, particularly for the more hydrophobic proteins, e.g., ovalbumin. For all three gradient solvents, little difference was observed in retention or recovery when the n-alkyl chain of the bonded phase varied. On the other hand, relative to the n-alkyl phases, a significantly lower retention of all proteins was found on more hydrophilic phases, e.g., cyano and nonendcapped n-butyl, when acetonitrile was the organic modifier, while in the case of 1-propanol, no retention difference was observed. Thus, column comparisons depend on the protein/mobile-phase combinations examined. The role of column temperature was also studied, and it was found that for certain proteins dramatic changes in peak shape occurred as a function of temperature. The influences of ionic strength and salt type were also studied. Protein mass recovery was shown to decrease with an increase in salt concentration; moreover, perchlorate was shown to have a larger effect in this regard than phosphate. In addition, salt concentration and type were found to influence peak shape greatly in certain cases. The results indicate the strong influences of mobile phase and temperature on chromatographic behavior, and some of the options available when this behavior is not satisfactory. Several protein separations are presented illustrating the power of the reversed phase approach.

Chemical Phenomena↗

PubMed2004

Pressure-induced formation of diblock copolymer "micelles" in supercritical fluids. A combined study by small angle scattering experiments and mean-field theory. II. Kinetics of the unimer-aggregate transition.

We developed a simple time-dependent mean-field theory to describe the phase separation kinetics of either homopolymers or AB-diblock copolymers in supercritical (SC) fluids. The model, previously used to describe the phase behavior of AB-block copolymers under the assumption of strong solvent selectivity for just one copolymer chain, has been extended to study the kinetics of the phase separation process. Time resolved small angle x-ray scattering (TR-SAXS) measurements have been performed on different AB-diblock copolymers containing a perfluorinated chain and dissolved in SC-CO2. The data obtained over a wide range of pressure and temperature confirm our theoretical predictions. Particularly interesting is the presence of two relaxation frequencies for the homogeneous solution --> spherical aggregate transition, where the two relaxation processes depend on the depth of the pressure jump and on temperature. The whole phenomenon could be explained as an initial SC solvent/polymer phase separation followed by a slow reorientation process to form spherical aggregates driven by the copolymer solvophilic moiety.

Journal Article↗

PubMed1982

Effect of pH, mono- and divalent cations on the mixing of phosphatidylglycerol with phosphatidylcholine. A monolayer (pi, delta V) and fluorescence study.

The mixing of various molecular species of phosphatidylglycerol and phosphatidylcholine differing in their acyl chain lengths has been studied both in monolayers (pi, delta V), and in water dispersions (fluorescence polarization) with varying pH and ionic strength of the aqueous phase and in the presence of the divalent cations Mg2+ and Ca2+. In dilauroylphosphatidylglycerol/dipalmitoylphosphatidylcholine mixtures, both in monolayers and in water dispersions, no phase separation was detected at pH 2.9 where phosphatidylglycerol was protonated. With dipalmitoylphosphatidylglycerol/dipalmitoylphosphatidylcholine mixtures, in monolayers and at the same pH, no phase separation was detected for surface pressures below pi = 40 mN.m-1. In monolayers, and under ionic conditions such that phosphatidylglycerol was ionized (pH 5.6, 10 mM NaCl) miscibility was observed with dilauroylphosphatidylglycerol and dipalmitoylphosphatidylcholine and also with dipalmitoylphosphatidylglycerol and dilauroylphosphatidylcholine. Varying the ionic strength did not alter the miscibility of these lipids. The divalent cations Mg2+ and Ca2+ did not modify that of dilauroylphosphatidylglycerol with dilauroylphosphatidylcholine or with dipalmitoylphosphatidylcholine. Both in monolayers and in water dispersions, dipalmitoylphosphatidylglycerol and dilauroylphosphatidylcholine appeared to be at least partly miscible, in the presence of magnesium. Only in the presence of calcium and at high surface pressure might the monolayer data account for phase separation between these two lipids. The data presented demonstrate the existence of strong cohesive forces between phosphatidylcholine and phosphatidylglycerol with a marked influence of the former on the physical state of the latter. From an analysis of the delta V data, it is suggested that intrafacial hydrogen bonds may play a significant role in stabilizing phosphatidylcholine/phosphatidylglycerol mixtures.

Calcium↗