Aqueous Polymer Two-phase Systems Research Articles

Structural properties, antioxidant and hypoglycemic activities of polysaccharides purified from pepper leaves by high-speed counter-current chromatography

In the present study, polysaccharides with antioxidant and hypoglycemic activities were isolated from the crude extract of pepper leaves by high-speed countercurrent chromatography in one step for the first time. An aqueous polymer two-phase system comprising PEG1000-K2HPO4-KH2PO4-H2O (0.8:1.25:1.25:6.5, w/w) was used for separation. During each separation, 2.0 g of the crude sample yielded two homogeneous components of polysaccharides PLP1 (226 mg) and PLP2 (273 mg). The molecular weights of PLP1 and PLP2 were 123 kDa and 243 kDa, respectively. PLP1 primarily consisted of mannose and glucose at a molar ratio of 1:2.4, whereas PLP2 was mainly composed of galactose, glucose, and rhamnose at a molar ratio of 2.6:1:2.2 with 10.3% (w/w) sulphates. Biological activity showed that sulfated PLP2 exhibited stronger antioxidant activity in vitro and hypoglycemic activity in diabetic mice than PLP1. The results demonstrated that sulfated PLP2 possessed potential as natural hypoglycemic agents or antioxidants in food or pharmaceuticals.

Directed assembly of multiplexed single chirality carbon nanotube devices

Herein, we present the fabrication of multiplexed single-walled carbon nanotube (SWCNT) devices, where selected chiralities were separately immobilized on one chip with single-tube precision. Each chirality was subsequently electrically measured individually. Specifically, (6,5) and (7,5) SWCNT species were isolated via aqueous two-phase polymer systems, after which dielectrophoresis was used to precisely control the placement of each chirality, along with a metallic species, separately on prepatterned electrodes on a single chip.

Controlling Macroscopic Phase Separation of Aqueous Two-Phase Polymer Systems in Porous Media.

In previous work, our group discovered a phenomenon in which a mixed polymer-salt or mixed micellar aqueous two-phase system (ATPS) separates into its two constituent phases as it flows within paper. While these ATPSs worked well in their respective studies to concentrate the target biomarker and improve the sensitivity of the lateral-flow immunoassay, different ATPSs can be advantageous for new applications based on factors such as biomarker partitioning or biochemical compatibility between ATPS and sample components. However, since the mechanism of phase separation in porous media is not completely understood, introducing other ATPSs to paper is an unpredictable process that relies on trial and error experiments. This is especially true for polymer-polymer ATPSs in which the characteristics of the two phases appear quite similar. Therefore, our group aimed to develop semiquantitative guidelines for choosing ATPSs that can phase separate in paper. In this work, we evaluated the Washburn equation and its parameters as a potential mathematical framework to describe the flow behavior of polymer-salt and micellar ATPSs in fiberglass paper. We compared bulk phase fluid characteristics and identified the viscosity difference between the phases as a key determinant of the potential for phase separation in paper. We then used this parameter to predict the phase separation capabilities of polyethylene glycol (PEG)-dextran ATPSs in paper and control the composition of the leading and lagging phases. We also, for the first time, successfully demonstrated the phase separation phenomenon in hydrogels, thereby extending its application and potential benefits to an alternative porous medium.

Role of PIP-Aquaporin Phosphorylation in Redox-Dependent Modulation of Osmotic Water Permeability in Plasmalemma from Roots of Pea Seedlings

Investigations were directed to study the mechanism based on redox-dependent modulation of osmotic water permeability in plasmalemma from roots of pea (Pisum sativum L.) seedlings. This mechanism may be associated with oxidation of cysteine residues in PIP-aquaporins or involves redox-dependent phosphorylation of these proteins. Plasmalemma was isolated by partitioning of microsomal membranes in the aqueous polymer two-phase system and using the roots homogenization medium additionally contained SH-reagents, dithiothreitol or diamide, and also phenylarsin oxide, an inhibitor of tyrosine protein phosphatases. Water permeability of plasmalemma was estimated by the kinetics of light scattering changes of membrane vesicles due to their osmotic shrinkage and recorded by the stopped flow method. In order to elucidate whether PIP-aquaporins contain cysteines available for oxidation by SH-reagents, isolated plasmalemma was subjected to PEGylation (reaction, based on binding of 5 kD methoxypolyethylene glycol maleimide with available membrane proteins SH-groups) and further western blot analysis was performed to reveal shift of aquaporins molecular mass by 5 kD after their immunodetection with antibodies. Relative content of PIP-aquaporin phosphorylated forms in plasmalemma was determined using fluorescent labeling proteins in gels with ProQ Diamond after membrane proteins separation by two-dimensional electrophoresis (blue native (BN/PAGE) in first dimension and denaturing (SDS/PAGE) in second dimension). It was established that conservative cysteines in molecules of PIP-aquaporins are available for SH-reagents only under denaturing conditions. This fact excludes the possibility of these cysteines participation in modulation of plasmalemma osmotic water permeability. Comparative analysis of phosphorylated PIP-aquaporins pool with different redox status of membrane proteins and osmotic water permeability coefficients and also the inhibitor analysis data showed that redox-dependent modulation of these proteins activity is due to their phosphorylation with involvement of serine-threonine protein phosphatases of PP2C type and/or thyrosine protein phosphatases.

Membrane Protein Identification in Rodent Brain Tissue Samples and Acute Brain Slices.

Acute brain slices are a sample format for electrophysiology, disease modeling, and organotypic cultures. Proteome analyses based on mass spectrometric measurements are seldom used on acute slices, although they offer high-content protein analyses and explorative approaches. In neuroscience, membrane proteins are of special interest for proteome-based analysis as they are necessary for metabolic, electrical, and signaling functions, including myelin maintenance and regeneration. A previously published protocol for the enrichment of plasma membrane proteins based on aqueous two-phase polymer systems followed by mass spectrometric protein identification was adjusted to the small sample size of single acute murine slices from newborn animals and the reproducibility of the results was analyzed. For this, plasma membrane proteins of 12 acute slice samples from six animals were enriched and analyzed by liquid chromatography-mass spectrometry. A total of 1161 proteins were identified, of which 369 were assigned to membranes. Protein abundances showed high reproducibility between samples. The plasma membrane protein separation protocol can be applied to single acute slices despite the low sample size and offers a high yield of identifiable proteins. This is not only the prerequisite for proteome analysis of organotypic slice cultures but also allows for the analysis of small-sized isolated brain regions at the proteome level.

Development of pH-responsive polymer and citrate aqueous two-phase system for extractive bioconversion of cefprozil

A pH-responsive aqueous two-phase system (pH-ATPS) has been developed by sodium citrate and a recyclable pH-responsive polymer PADB6.8 that can response to the change of pH values. Phase separation mechanism is studied through Low field-NMR. All variables affecting the phase separation are evaluated. Phase characteristics (viscosity, density, interfacial tension) and phase separation kinetic are studied for understanding of separation process and operational parameters in applications. This pH-ATPS has the characters of low interfacial tension, high recovery leading efficient mass transfer and low cost. The proposed system can be used as a mild medium for extractive bioconversion with low cost. We applied this pH-ATPS in extractive bioconversion of cefprozil. Cefprozil is partitioned towards the polymer-rich phase while the substrates tended to be partitioned in the salt-rich phase. Extractive bioconversion of cefprozil in this pH-ATPS can improve yield of the enzymatic process and reduce the product hydrolysis in optimal conditions. The maximal conversion yield of cefprozil in the studied system is 91.0%.

PIP1 aquaporins, sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm3) and “heavy” (≥1.146 g/cm3) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.

Comparative proteomic analysis reveals a dynamic pollen plasma membrane protein map and the membrane landscape of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils in rice

BackgroundThe coordination of pollen tube (PT) growth, guidance and timely growth arrest and rupture mediated by PT-pistil interaction is crucial for the PT to transport sperm cells into ovules for double fertilization. The plasma membrane (PM) represents an important interface for cell–cell interaction, and PM proteins of PTs are pioneers for mediating PT integrity and interaction with pistils. Thus, understanding the mechanisms underlying these events is important for proteomics.ResultsUsing the efficient aqueous polymer two-phase system and alkali buffer treatment, we prepared high-purity PM from mature and germinated pollen of rice. We used iTRAQ quantitative proteomic methods and identified 1,121 PM-related proteins (PMrPs) (matched to 899 loci); 192 showed differential expression in the two pollen cell types, 119 increased and 73 decreased in abundance during germination. The PMrP and differentially expressed PMrP sets all showed a functional skew toward signal transduction, transporters, wall remodeling/metabolism and membrane trafficking. Their genomic loci had strong chromosome bias. We found 37 receptor-like kinases (RLKs) from 8 kinase subfamilies and 209 transporters involved in flux of diversified ions and metabolites. In combination with the rice pollen transcriptome data, we revealed that in general, the protein expression of these PMrPs disagreed with their mRNA expression, with inconsistent mRNA expression for 74% of differentially expressed PMrPs.ConclusionsThis study identified genome-wide pollen PMrPs, and provided insights into the membrane profile of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils. These pollen PMrPs and their mRNAs showed discordant expression. This work provides resource and knowledge to further dissect mechanisms by which pollen or the PT controls PMrP abundance and monitors interactions and ion and metabolite exchanges with female cells in rice.

Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated Pollen.

Pollen germination is an excellent process to study cell polarity establishment. During this process, the tip-growing pollen tube will start elongating. The plasma membrane as the selectively permeable barrier that separates the inner and outer cell environment plays crucial roles in this process. This protocol described an efficient aqueous polymer two-phase system followed by alkaline solution washing to prepare Lilium davidii or Oryza sativa plasma membrane with high purity.

Differentiating Left- and Right-Handed Carbon Nanotubes by DNA.

New structural characteristics emerge when solid-state crystals are constructed in lower dimensions. This is exemplified by single-wall carbon nanotubes, which exhibit a degree of freedom in handedness and a multitude of helicities that give rise to three distinct types of electronic structures: metals, quasi-metals, and semiconductors. Here we report the use of intrinsically chiral single-stranded DNA to achieve simultaneous handedness and helicity control for all three types of nanotubes. We apply polymer aqueous two-phase systems to select special DNA-wrapped carbon nanotubes, each of which we argue must have an ordered DNA structure that binds to a nanotube of defined handedness and helicity and resembles a well-folded biomacromolecule with innate stereoselectivity. We have screened over 300 short single-stranded DNA sequences with palindrome symmetry, leading to the selection of more than 20 distinct carbon nanotube structures that have defined helicity and handedness and cover the entire chiral angle range and all three electronic types. The mechanism of handedness selection is illustrated by a DNA sequence that adopts two distinct folds on a pair of (6,5) nanotube enantiomers, rendering them large differences in fluorescence intensity and chemical reactivity. This result establishes a first example of functionally distinguishable left- and right-handed carbon nanotubes. Taken together, our work demonstrates highly efficient enantiomer differentiation by DNA and offers a first comprehensive solution to achieve simultaneous handedness and helicity control for all three electronic types of carbon nanotubes.

Extraction of natural red colorants from the fermented broth of Penicillium purpurogenum using aqueous two-phase polymer systems.

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two-phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG-rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (KC ) was obtained in the presence of NaCl 0.1 M (KC = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na2 SO4 0.5 M showed the highest KC (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%ηTOP ) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0-3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms.

Evaluation of four different strategies to characterize plasma membrane proteins from banana roots

Plasma membrane proteins constitute a very important class of proteins. They are involved in the transmission of external signals to the interior of the cell and selective transport of water, nutrients and ions across the plasma membrane. However, the study of plasma membrane proteins is challenging because of their poor solubility in aqueous media and low relative abundance. In this work, we evaluated four different strategies for the characterization of plasma membrane proteins from banana roots: (i) the aqueous-polymer two-phase system technique (ATPS) coupled to gelelectrophoresis (gel-based), and (ii) ATPS coupled to LC-MS/MS (gel free), (iii) a microsomal fraction and (iv) a full proteome, both coupled to LC-MS/MS. Our results show that the gel-based strategy is useful for protein visualization but has major limitations in terms of time reproducibility and efficiency. From the gel-free strategies, the microsomal-based strategy allowed the highest number of plasma membrane proteins to be identified, followed by the full proteome strategy and by the ATPS based strategy. The high yield of plasma membrane proteins provided by the microsomal fraction can be explained by the enrichment of membrane proteins in this fraction and the high throughput of the gel-free approach combined with the usage of a fast high-resolution mass spectrometer for the identification of proteins.

DNA-Controlled Partition of Carbon Nanotubes in Polymer Aqueous Two-Phase Systems

Sorting single-wall carbon nanotubes (SWCNTs) of different chiralities is both scientifically interesting and technologically important. Recent studies have shown that polymer aqueous two-phase extraction is a very effective way to achieve nanotube sorting. However, works published to date have demonstrated only separation of surfactant-dispersed SWCNTs, and the mechanism of chirality-dependent SWCNT partition is not well understood. Here we report a systematic study of spontaneous partition of DNA-wrapped SWCNTs in several polymer aqueous two-phase systems. We show that partition of DNA-SWCNT hybrids in a given polymer two-phase system is strongly sequence-dependent and can be further modulated by salt and polymer additives. With the proper combination of DNA sequence, polymer two-phase system, and partition modulators, as many as 15 single-chirality nanotube species have been effectively purified from a synthetic mixture. As an attempt to provide a unified partition mechanism of SWCNTs dispersed by surfactants and by DNA, we present a qualitative analysis of solvation energy for SWCNT colloids in a polymer-modified aqueous phase. Our observation and analysis highlight the sensitive dependence of the hydration energy on the spatial distribution of hydrophilic functionalities.

5CN05 partitioning in an aqueous two-phase system: A new approach to the solubilization of hydrophobic drugs

Liquid–liquid extraction for the purification of molecules has been central to many advances in the pharmaceutical industry. These processes were developed based on the property that some polymer and/or micellar solutions present to separate into a concentrated phase and a diluted phase. Based on the differences in the physical and chemical environments of the two coexisting phases, and since both phases contain approximately 60–90% of water, liquid–liquid extraction provides a powerful alternative to both extract and solubilize a molecule. This paper examines the partition behavior of the synthetic drug, 2-[(3,4-dichlorine-benzylidene)-amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (5CN05), in an aqueous two-phase polymer system (ATPPS) and also in an aqueous two-phase micellar system (ATPMS). The results showed that both systems are favorable for extraction the 5CN05 drug high partition coefficient values (K5CN05>200) and yield (Y5CN05>99.48%) in the concentrated phase were achieved with the systems. However, the ATPPS generated a partition coefficient (K5CN05) higher than the one obtained with ATPMS. The results suggest that both processes may be used for the extraction and concentration of molecules with hydrophobic characteristics, such as 5CN05. They also provide an optimal environment for the solubilization of such molecules, allowing for greater efficiency when purifying many classes of drugs.

Specific DNA Sequences for the Purification of Single-Wall Carbon Nanotube Species in Polymer Aqueous Two-Phase Systems

A systematic purification of single-chirality species of single-wall carbon nanotubes (SWCNT) from their synthetic mixture provides foundation for many fundamental studies and technological applications of SWCNT. Previous study demonstrated a robust separation of SWCNT dispersed in bile salt surfactants according to their diameter and metallicity in a polymer aqueous two-phase (ATP) system. In this work, specific DNA sequences were utilized as an effective approach for recognition of specific SWCNT chirality and their subsequent purification using the ATP separation method. We show that many single chirality SWCNTs in a synthetic mixture can be obtained in very high purity. The combination of DNA sequence recognition of specific SWCNT structure and the rapid and tunable ATP separation method open a new route for sorting nanotube into each and every single-chirality species. In addition, the feasibility of volume scale-up in ATP separation allows sufficient materials to be obtained for fundamental studies and application development. Further exploration of DNA recognition sequences in ATP separation method and new polymer ATP systems will be the main focus of our future studies in carbon nanotube sorting.

Separation, enrichment and determination of ciprofloxacin using thermoseparating polymer aqueous two-phase system combined with high performance liquid chromatography in milk, egg, and shrimp samples

Aqueous two-phase systems (ATPS) are potential industrial technology in separation and enrichment areas. However, the main problem is that ATPS could not be recycled, which results in a high cost and pollution of the environment. In this work, a new ATPS which formed by thermoseparating polymer EOPO with phosphate was developed for the separation and enrichment of ciprofloxacin (CIP). In the first procedure, the single factors have been discussed. The results showed that when the amounts of EOPO (80%, w/w) and K2HPO4 (55%, w/w) were 3.0mL, 7.0mL, respectively, pH=11, the extraction efficiency of CIP could reach 97.7%. In the second extraction, with the temperature induced, the extraction efficiency of CIP showed 85.6%. Additionally, the phase components were successfully recycled and reused for more than two times. The new method was successfully applied to the determination of CIP in real samples with detection limits of 6.8ngg−1.

Study on the Role of Surfactants to Aqueous Two-Phase Synthesizing CPAM

In this paper,cationic polyacrylamide (CPAM) was synthesized by using aqueous two-phase polymerization. The Role of surfactants to aqueous two-phase synthesizing CPAM was studied, including the effect of the type,the composition and the concentration of the surfactants. The results showed that non-ionic surfactants had a better stabilizing impact on the polymer aqueous two-phase system. And the effect of the compound surfactant was better than that of a single surfactant. Take the chemical compound of Tween80 and OP10 as the polymerization auxiliaries, the optimum reaction condition of synthesis were as follows:wTween80:ѡOP10=1.5,w=1.6%, 50°C.In this process conditions, the distribution coefficient was the most appropriate in the continuous phase, the total monomer conversion rate and the intrinsic viscosity number of the product reached the maximum.

Purification of a novel protease enzyme from kesinai plant (Streblus asper) leaves using a surfactant–salt aqueous micellar two-phase system: a potential low cost source of enzyme and purification method

Serine protease from kesinai leaves was purified for the first time by a surfactant–polymer aqueous micellar two-phase system. The effectiveness of different types and concentrations of non-ionic surfactants (Pluronic series and X-114) on the partitioning behaviour of the protease was evaluated. The results showed that the enzyme preferentially partitioned into the bottom surfactant-rich phase, while the hydrophilic amino acid preferred the top aqueous phase. This distribution of the enzyme is due to the hydrophobic interaction of the serine protease with the hydrophobic lid of the micelle core in the bottom phase. The influence of different types of salts (K2SO4, KH2PO4, KCl and KNO3) on the purification and selectivity of the enzyme was determined. The protease partitioning in the bottom phase increased in the presence of KNO3, which confirmed that the salt was able to improve the protein solubility in bottom phase and increase the hydrophobic interaction between the two phases. In addition, the protease from the bottom phase was re-extracted to a new aqueous phase solution to remove and recycle the surfactant. Addition of potassium thiocyanate led to the partitioning of the enzyme in top aqueous phase due to high ionic strength of SCN−, which forced the lighter micellar phase toward the upper position of the system. A high purification factor (10.3) and yield of 92 % of the enzyme were achieved in a solution of 31 % of Pluronic L61 using 0.3 % KNO3 and 50 % crude feedstock at pH 7.0.

Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized metal affinity chromatography, and TiO2 were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized with a confidence level of 99% or higher. 41.4% of the identified proteins were allocated to the plasma membrane and about half of the phosphorylation sites have not been reported previously. A bioinformatic screen for 12 consensus sequences identified putative kinases for 642 phosphorylation sites. In summary, the protocol deployed here identified several hundred novel phosphorylation sites of cerebellar proteins. Furthermore, it provides a valuable tool to monitor the plasma membrane proteome from any small brain samples of interest under differing physiological or pathophysiological conditions.

Phosphatidic Acid Binds and Stimulates Arabidopsis Sphingosine Kinases

Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) have both been identified as lipid messengers mediating plant response to abscisic acid (ABA). To determine the relationship of these messengers, we investigated the direct interaction of PA with Arabidopsis sphingosine kinases (SPHKs) that phosphorylate phytosphingosine to generate phyto-S1P. Two unique SPHK cDNAs were cloned from the annotated At4g21540 locus of Arabidopsis, and the two transcripts are differentially expressed in Arabidopsis tissues. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs) and are associated with the tonoplast. They both interact with PA as demonstrated by lipid-filter binding, liposome binding, and surface plasmon resonance (SPR). SPHK1 and SPHK2 exhibited strong binding to 18:1/18:1, 16:0/18:1, and 16:0/18:2 PA, but poor binding to 16:0/16:0, 8:0/8:0, 18:0/18:0, and 18:2/18:2 PA. Surface dilution kinetics analysis indicates that PA stimulates SPHK activity by increasing the specificity constant through decreasing K(m)(B). The results show that the annotated At4g21540 locus is actually comprised of two separate SPHK genes. PA binds to both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. The PA-SPHK interaction depends on the PA molecular species. The data suggest that these two Arabidopsis SPHKs are molecular targets of PA, and the PA stimulation of SPHK is part of the signaling networks in Arabidopsis.