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Size-exclusion Chromatography (size-exclusion + chromatography)
Selected AbstractsN -Terminal domain of HTLV-I integrase.JOURNAL OF PEPTIDE SCIENCE, Issue 11 2001Complexation, conformational studies of the zinc finger Abstract The HTLV-I integrase N -terminal domain [50-residue peptide (IN50)], and a 35-residue truncated peptide formed by residues 9,43 (IN35) have been synthesized by solid-phase peptide synthesis. Formation of the 50-residue zinc finger type structure through a HHCC motif has been proved by UV-visible absorption spectroscopy. Its stability was demonstrated by an original method using RP-HPLC. Similar experiments performed on the 35-residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50-residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV-I integrase N -terminal domain is likely to be composed of an helical region (residues 28,42) and a ,-strand (residues 20,23), associated with a HHCC zinc-binding motif. Size-exclusion chromatography showed that the structured zinc finger dimerizes through the helical region. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd. [source] Heparin-binding proteins of human seminal plasma: purification and characterizationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2008Vijay Kumar Abstract Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity,chromatography on Heparin,Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS,PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield. Mol. Reprod. Dev. 75: 1767,1774, 2008. © 2008 Wiley-Liss, Inc. [source] An enigmatic peptide ligation reaction: Protease-catalyzed oligomerization of a native protein segment in neat aqueous solutionPROTEIN SCIENCE, Issue 4 2000Sangaralingam Kumaran Abstract We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free ,-carboxyl and ,-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 °C, producing a gamut of covalent oligomers (dimer through octamer or higher) of a native protein segment TAAAKFE (S39) derived from ribonuclease A (RNAse A). Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases,slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase." [source] Purification, crystallization and preliminary X-ray diffraction studies on human Ca2+ -binding protein S100BACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2005Thorsten Ostendorp S100B, a Ca2+ -binding protein, acts intracellularly as a Ca2+ -signalling protein but is also secreted to the extracellular space, acting in a cytokine-like manner through its receptor RAGE. Recombinant human S100B has been purified and crystallized in the Ca2+ -bound state. Size-exclusion chromatography indicates that S100B can exist as a dimer and as a multimer in solution. Crystals of S100B diffract to 1.9,Ĺ and belong to space group P21, with unit-cell parameters a = 63.4, b = 81.6, c = 71.5,Ĺ, , = 90, , = 107, , = 90°. Preliminary analysis of the X-ray data indicate that there are four homodimers per asymmetric unit. [source] Electrophoresis on a microfluidic chip for analysis of fluorescence-labeled human rhinovirusELECTROPHORESIS, Issue 24 2007Viliam Kolivo Abstract We report the analysis of human rhinovirus serotype 2 (HRV2) on a commercially available lab-on-a-chip instrument. Due to lack of sufficient native fluorescence, the proteinaceous capsid of HRV2 was labeled with Cy5 for detection by the red laser (,ex 630,nm) implemented in the instrument. On the microdevice, electrophoresis of the labeled virus was possible in a BGE without stabilizing detergents, which is in contrast to conventional CE; moreover, analysis times were drastically shortened to the few 10,s range. Resolution of the sample constituents (virions, a contaminant present in all virus preparations, and excess dye) was improved upon adaptation of the separation conditions, mainly by adjusting the SDS concentration of the BGE. Purity of fractions from size-exclusion chromatography after labeling of virus was assessed, and affinity complex formation of the labeled virus with various recombinant very-low-density lipoprotein receptor derivatives differing in the number of concatenated V3 ligand binding repeats was monitored. Virus analysis on microchip devices is of particular interest for experiments with infectious material because of easy containment and disposal of samples. Thus, the employment of microchip devices in routine analysis of viruses appears to be exceptionally attractive. [source] Analysis of poly(amidoamine)-succinamic acid dendrimers by slab-gel electrophoresis and capillary zone electrophoresisELECTROPHORESIS, Issue 15 2005Xiangyang Shi Abstract Ethylenediamine (EDA)-core poly(amidoamine) (PAMAM) succinamic acid dendrimers (Ex.SAH, where x refers to the generation) were synthesized and analyzed by polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), potentiometric acid-base titration, and capillary zone electrophoresis (CZE). Various generations (E1.SAH,E7.SAH) PAMAMs and a succinamic acid terminated core-shell tecto(dendrimer) (E5(E3.SAH)n) were first analyzed by PAGE. PAGE results show that the relative mobilities of generation,2 to generation,7 dendrimers decreased with the increasing number of generations. The molecular mass of a generation,5 core generation,3 shell tecto(dendrimer) (denoted as E5(E3.SAH)n) was determined to be between the Mw of E6.SAH and E7.SAH. CZE analysis allowed the evaluation of electrophoretic properties of given-generation dendrimers. The electrophoretic mobilities of individual generations PAMAM polyanions are similar, indicating that the separation mainly depends on their approximately identical charge/mass ratio. The E5(E3.SAH)n tectodendrimer had a lower electrophoretic mobility, which was consistent with its lower charge/mass ratio. The combination of PAGE and CZE analysis provides an alternative and effective way to characterize this group of PAMAM-succinamic acid dendrimers. [source] Trace metal distribution in soluble organic matter from municipal solid waste compost determined by size-exclusion chromatographyENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2002Arno Kaschl Abstract Municipal solid waste (MSW) composts carry high amounts of trace metals and organic complexing agents that may influence metal bioavailability and mobility after application to soils. In order to assess the degree of organic complexation of trace metals in the solution phase of MSW compost and the relevance of organic ligand type, size exclusion chromatography (SEC) was applied to compost-extracted organic ligands. Adjustment of the elution conditions minimized the interaction with the gel matrix for compost humic substances and dissolved organic matter (DOM) fractions. The SEC was then used to separate the aqueous compost extract into samples with distinct differences in chemical constituents. The highest quantities of Cu, Zn, Ni, Mn, and Cd were found to coelute with the main peak of the SEC elution curve, which, as observed by Fourier-transformed infrared (FTIR) spectroscopy, also had the highest density of carboxyl groups. The ratio of aromatic to aliphatic structures was higher for eluates with low retention times, and cations such as Al, Cr, and Fe were preferably associated with these larger organic molecules. All trace metals in the compost solution phase were bound mostly to DOM rather than forming inorganic complexes. [source] Myristyl and palmityl acylation of pI 5.1 carboxylesterase from porcine intestine and liverFEBS JOURNAL, Issue 4 2002Tissue, subcellular distribution Immunoblotting analyses revealed the presence of carboxylesterase in the porcine small intestine, liver, submaxillary and parotid glands, kidney cortex, lungs and cerebral cortex. In the intestinal mucosa, the pI 5.1 enzyme was detected in several subcellular fractions including the microvillar fraction. Both fatty monoacylated and diacylated monomeric (F1), trimeric (F3) and tetrameric (F4) forms of the intestinal protein were purified here for the first time by performing hydrophobic chromatography and gel filtration. The molecular mass of these three enzymatic forms was,estimated to be 60, 180 and 240 kDa, respectively, based on size-exclusion chromatography and SDS/PAGE analysis. The existence of a covalent attachment linking palmitate and myristate to porcine intestinal carboxylesterase (PICE), which was suggested by the results of gas-liquid chromatography (GLC) experiments in which the fatty acids resulting from alkali treatment of the protein forms were isolated, was confirmed here by the fact that [3H]palmitic and [3H]myristic acids were incorporated into porcine enterocytes and hepatocytes in cell primary cultures. Besides these two main fatty acids, the presence of oleic, stearic, and arachidonic acids was also detected by GLC and further confirmed by performing radioactivity counts on the 3H-labelled PICE forms after an immunoprecipitation procedure using specific polyclonal antibodies, followed by a SDS/PAGE separation step. Unlike the F1 and F4 forms, which were both myristoylated and palmitoylated, the F3 form was only palmitoylated. The monomeric, trimeric and tetrameric forms of PICE were all able to hydrolyse short chain fatty acids containing glycerides, as well as phorbol esters. The broad specificity of fatty acylated carboxylesterase is discussed in terms of its possible involvement in the metabolism of ester-containing xenobiotics and signal transduction. [source] Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan-binding protein 1 (HABP1)/p32/gC1qRFEBS JOURNAL, Issue 1 2002Babal Kant Jha Hyaluronan-binding protein 1 (HABP1), a ubiquitous multifunctional protein, interacts with hyaluronan, globular head of complement component 1q (gC1q), and clustered mannose and has been shown to be involved in cell signalling. In vitro, this recombinant protein isolated from human fibroblast exists in different oligomeric forms, as is evident from the results of various independent techniques in near-physiological conditions. As shown by size-exclusion chromatography under various conditions and glutaraldehyde cross-linking, HABP1 exists as a noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers, i.e. a hexamer. The formation of a covalently-linked hexamer of HABP1 through Cys186 as a dimer of trimers is achieved by thiol group oxidation, which can be blocked by modification of Cys186. The gradual structural transition caused by cysteine-mediated disulfide linkage is evident as the fluorescence intensity increases with increasing Hg2+ concentration until all the HABP1 trimer is converted into hexamer. In order to understand the functional implication of these transitions, we examined the affinity of the hexamer for different ligands. The hexamer shows enhanced affinity for hyaluronan, gC1q, and mannosylated BSA compared with the trimeric form. Our data, analyzed with reference to the HABP1/p32 crystal structure, suggest that the oligomerization state and the compactness of its structure are factors that regulate its function. [source] 2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulansFEBS JOURNAL, Issue 12 2001Characterization, comparison of both enzymes In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo -2-methylisocitrate; the erythro -diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (, 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 µm PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved. [source] Design, synthesis and properties of synthetic chlorophyll proteinsFEBS JOURNAL, Issue 11 2001Harald K. Rau A chemoselective method is described for coupling chlorophyll derivatives with an aldehyde group to synthetic peptides or proteins modified with an aminoxyacetyl group at the ,-amino group of a lysine residue. Three template-assembled antiparallel four-helix bundles were synthesized for the ligation of one or two chlorophylls. This was achieved by coupling unprotected peptides to cysteine residues of a cyclic decapeptide by thioether formation. The amphiphilic helices were designed to form a hydrophobic pocket for the chlorophyll derivatives. Chlorophyll derivatives Zn-methylpheophorbide b and Zn-methyl-pyropheophorbide d were used. The aldehyde group of these chlorophyll derivatives was ligated to the modified lysine group to form an oxime bond. The peptide,chlorophyll conjugates were characterized by electrospray mass spectrometry, analytical HPLC, and UV/visible spectroscopy. Two four-helix bundle chlorophyll conjugates were further characterized by size-exclusion chromatography, circular dichroism, and resonance Raman spectroscopy. [source] Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulisFEBS JOURNAL, Issue 16 2000Bingze Xu A cellulase (endo-,-1,4- d -glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication). [source] Vitronectin in clotting factor IX concentratesHAEMOPHILIA, Issue 3 2001D. Josic Highly purified, plasma-derived factor IX (FIX) concentrates are produced in large part by a combination of anion exchange and heparin affinity chromatography. However, the concentrates still contain some accompanying proteins. The main impurity has turned out to be the adhesive glycoprotein, vitronectin. It occurs in concentrates exclusively in its multimeric form, in contrast to the situation in plasma. The multimeric vitronectin can be removed either by nanofiltration with a crossflow system or by size-exclusion chromatography. When these FIX concentrates are used as therapeutic agents, the fact has to be taken into account that considerable amounts of multimeric vitronectin are given to the patient. The physiological consequences of the dosage of this protein have not yet been investigated. Although no thrombogenicity has been reported in connection with the above-mentioned FIX concentrates, we recommend that the impurity should be removed from the preparation with the methods described here. [source] Studies on the association between immunoglobulin E autoreactivity and immunoglobulin E-dependent histamine-releasing factorsIMMUNOLOGY, Issue 2 2002Ilona Kleine Budde Summary It has been reported that serum immunoglobulin E (IgE) from certain atopic patients can sensitize basophils to release histamine in response to IgE-dependent histamine-releasing factors (HRFs). It has also been shown that patients suffering from severe forms of atopy may contain IgE autoantibodies. It was investigated whether HRF-responsive sera contained IgE autoantibodies and if there was an association between IgE autoreactivity and IgE-dependent responsiveness to HRF. The presence of HRF-responsive IgE (IgE+) in serum of patients with respiratory atopy was determined by stimulating stripped human basophils sensitized by serum with peripheral blood mononuclear cell (PBMC)-derived HRF, and measuring the release of histamine. In parallel, these sera were screened for the presence of IgE autoantibodies to nitrocellulose-blotted human cellular extracts. The capacity of IgE autoantigen-containing preparations to induce histamine release was tested in the stripped basophil assay. Eleven out of 52 sera contained IgE autoantibodies to blotted cellular extracts of human PBMCs or of the human epithelial cell line A431. No significant association was found between IgE autoreactivity and IgE-dependent responsiveness to HRF: 7/26 IgE+ sera contained IgE to human cellular extracts, and 4/26 of the sera without IgE+ did also. IgE autoantigen-containing extracts did not induce histamine release of appropriately sensitized basophils. By size-exclusion chromatography it was shown that a 32,000 MW autoantigen eluted in the >55,000 MW fraction, which indicates that this protein forms polymers or complexes with other macromolecules. This might explain the discrepancy between binding and histamine-releasing activity. A 20,000 MW IgE-defined autoantigen cross-reacted with a shrimp allergen. Our results indicate that IgE-reactivity to immunoblotted human protein and IgE-dependent HRF activity are distinct entities that may co-occur in atopic patients. [source] Hydrothermal processing of rice husks: effects of severity on product distributionJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2008Rodolfo Vegas Abstract BACKGROUND: Treatment in aqueous media (hydrothermal or autohydrolysis reactions) is an environmentally friendly technology for fractionating lignocellulosic materials. Rice husks were subjected to hydrothermal processing under a variety of operational conditions to cause the selective breakdown of xylan chains, in order to assess the effects of reaction severity on the distribution of reaction products. RESULTS: The effects of severity (measured by the severity factor, R0) on the concentrations of the major autohydrolysis products (monosaccharides, xylo- and glucooligosaccharides, xylooligosaccharide substituents, acetic acid, acid-soluble lignin and elemental nitrogen) were assessed. The interrelationship between the severity of treatment and molecular weight distribution was established by high-performance size-exclusion chromatography. Selected samples were subjected to refining treatments as ethyl acetate extraction and ion exchange for refining purposes, and the concentrates were assayed by high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CONCLUSIONS The protein equivalent of the products present in liquors accounted for 43 to 51% of the protein present in the raw rice husks. The concentrations of glucose (derived from starchy material) and arabinose (split from the xylan backbone) were fairly constant with severity. Even in treatments at low severity, high molecular weight compounds derived from xylan accounted for a limited part of the stoichiometric amount. Operating under harsh conditions, about 50% of the total xylan-derived compounds corresponded to fractions with a degree of polymerization (DP) < 9. After refining, saccharides accounted for more than 90% of the non-volatile components of the sample. The refined products showed a series of xylose oligomers up to about DP 13, and a series of acetylated xylose oligomers up to about DP 15. Copyright © 2008 Society of Chemical Industry [source] Purification and Characteristics of Feruloyl Esterase from Aspergillus awamori G-2 StrainJOURNAL OF FOOD SCIENCE, Issue 6 2008M. Kanauchi ABSTRACT:, For food industry production processes and other uses, a mold that produces high levels of feruloyl esterase was obtained from laboratory mold collections and other sources. It was Aspergillus awamori G-2 that produces high levels of feruloyl esterase. The feruloyl esterase was purified using ion-exchange chromatography, size-exclusion chromatography, and HPLC chromatography. The enzyme was identified as a monomer protein using size-exclusion chromatography. Its optimum temperature and pH were, respectively, 40 °C and pH 5. Its activity was stable at pH 3 to 5. The enzyme was combined with xylan and starch, but it was absorbed by cellulose. The km of the feruloyl esterase was 0.0019% (0.01 mM). The enzyme showed stable activity at pH 3 and 50 °C, making this enzyme useful for food production. [source] Mass-transfer effects during separation of proteins in SMB by size exclusionAICHE JOURNAL, Issue 5 2003Joukje Houwing The chromatographic fractionation of proteins by size-exclusion chromatography in a simulated moving bed (SMB) is studied. During experimental fractionation of a mixture of bovine serum albumin (BSA) and myoglobin on Sepharose Big Beads, mass-transfer effects are shown to limit the performance of the SMB. The internal profiles, as well as the extract and raffinate compositions, are described well by a steady-state equivalent true moving-bed (TMB) model that incorporates mass-transfer effects. The selection of the particle size in SMB is a trade-off between productivity and mass transfer. Based on the equivalent TMB model, the optimum particle size and configuration of the SMB can be selected, at which preset performance criteria (purity, recovery) are met at specified flow-rate ratios, total column length, and pressure drop. For the current feed and apparatus, an optimal particle size of approximately 145 ,m is calculated for achievement of purities and overall recoveries of 95%. [source] Synthesis of highly luminescent organoboron polymers connected by bifunctional 8-aminoquinolate linkersJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 16 2010Yuichiro Tokoro Abstract New organoboron aminoquinolate-based polymers linked by ,-conjugated bridge were prepared by Sonogashira,Hagihara coupling of organoboron aminoquinolate-based bisiodo monomers bearing biphenyl or bithiophene moiety with 1,4-diethynylbenzene derivatives. Tetracoordination states of boron atoms in the obtained polymers were confirmed by 11B NMR spectroscopy, and they were also characterized by 1H NMR and IR spectroscopies and size-exclusion chromatography. Their optical properties were studied by UV,vis absorption and photoluminescence spectroscopies. In the region above 400 nm, the polymers prepared from 1,4-diethynyl-2,5-dioctyloxybenzene showed bathochromic shifts when compared with those prepared from 1.4-diethynyl-2-perfluorooctyl-5-trifluoromethylbenzene. The polymers with biphenyl moiety showed higher absolute fluorescence quantum yields (,F = 0.28 and 0.65), whereas those with bithiophene moiety led to decreasing of the low quantum yields (,F = 0.19 and 0.00). The density-functional theory (DFT) and time-dependent,DFT calculations of model compounds corresponding to the polymers were in good agreement with the results from UV,vis properties. The calculations revealed that the electronic structure of the polymer with bithiophene moiety is different from that with biphenyl moiety, and predicted the electron transfer from the bithiophene moiety to the ,-extended quinoline moiety in transition state. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3693,3701, 2010 [source] Controlled synthesis of fluorinated copolymers with pendant sulfonatesJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 23 2008Ivaylo Dimitrov Abstract Novel, fluorinated copolymers with different architectures bearing sulfopropyl groups were synthesized in a three-step procedure. The first step involved atom transfer radical polymerization (ATRP) of aromatic fluorinated monomers followed by two modification reactions performed on the polymer chain: demethylation and sulfopropylation. As a result two types of fluorinated copolymers were obtained. The first one was synthesized by ATRP of 2,3,5,6-tetrafluoro-4-methoxystyrene (TFMS). After the modification steps copolymers with randomly distributed sulfopropyl groups along the backbone were obtained. The second type of copolymers has diblock architecture with one of the blocks being sulfopropylated. They were synthesized via ATRP of 2,3,4,5,6-pentafluorostyrene (FS) initiated by a PTFMS-macroinitiator followed by demethylation and sulfopropylation of the TFMS-block. The copolymers were characterized by size-exclusion chromatography, FTIR, and 1H NMR spectroscopy. Their thermal properties were investigated by differential scanning calorimetry and thermal gravimetric analyses. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7827,7834, 2008 [source] Simultaneous reversible addition fragmentation chain transfer and ring-opening polymerizationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 9 2008Maude Le Hellaye Abstract The simultaneous ring-opening polymerization (ROP) of ,-caprolactone (,-CL) and 2-hydroxyethyl methacrylate (HEMA) polymerization via reversible addition fragmentation chain transfer (RAFT) chemistry and the possible access to graft copolymers with degradable and nondegradable segments is investigated. HEMA and ,-CL are reacted in the presence of cyanoisopropyl dithiobenzoate (CPDB) and tin(II) 2-ethylhexanoate (Sn(Oct)2) under typical ROP conditions (T > 100 °C) using toluene as the solvent in order to lead to the graft copolymer PHEMA- g -PCL. Graft copolymer formation is evidenced by a combination of size-exclusion chromatography (SEC) and NMR analyses as well as confirmed by the hydrolysis of the PCL segments of the copolymer. With targeted copolymers containing at least 10% weight of PHEMA and relatively small PHEMA backbones (ca. 5,000,10,000 g mol,1) the copolymer grafting density is higher than 90%. The ratio of free HEMA-PCL homopolymer produced during the "one-step" process was found to depend on the HEMA concentration, as well as the half-life time of the radical initiator used. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 3058,3067, 2008 [source] Homogeneous phase polymerization of vinylidene fluoride in supercritical carbon dioxideJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 23 2007Sabine Beuermann Abstract For the first time, stabilizer-free vinylidene fluoride (VDF) homopolymerizations were carried out in homogenous phase with supercritical CO2 using the conventional initiator di- tert butyl peroxide (DTBP). In-line FT-NIR spectroscopy showed that complete monomer conversion may be obtained. Molecular weights were determined via size-exclusion chromatography and polymer endgroup analysis by 1H-NMR spectroscopy. The number average molecular weights were below 104 g mol,1 and polydispersities ranged from 3.1 to 5.7 depending on DTBP and VDF concentration. For allowing isothermal reaction, high CO2 contents ranging from 61 to 83 wt % were used. The high-temperature and high-pressure conditions required for homogeneous polymerization did not alter the amount of defects in VDF chaining. Scanning electron microscopy indicated that regular stack-type particles are obtained upon expansion of the homogeneous polymerization mixture. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 5626,5635, 2007 [source] Synthesis of multicyclic and grafted polystyrenesJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 16 2001Bénédicte Lepoittevin Abstract Well-defined multicyclic polystyrenes are prepared in two steps. The first step is the preparation of a cyclic difunctional polystyrene by the reaction of ,,,-dilithiopolystyrene chains with 1,3-bis(phenylethenyl)benzene. Then, this product is covalently grafted to poly(chloromethylstyrene) chains leading to the formation of a high molar mass product containing linear and cyclic parts. As a model reaction and to optimize the previous reaction, a study of coupling of the linear difunctional model polystyrene with poly(chloromethylstyrene) is performed leading to grafted polystyrene. The grafted products are analyzed by size-exclusion chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and liquid chromatography at the exclusion-adsorption transition point. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 2723,2730, 2001 [source] Isolation of inulin-type oligosaccharides from Chinese traditional medicine: Morinda officinalis How and their characterization using ESI-MS/MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 1 2010Zhenmin Yang Abstract Inulin-type oligosaccharides with different DP were prepared by size-exclusion chromatography and purity of each oligosaccharide was determined by HPLC equipped with cyclodextrin-bond column. The purities of obtained inulin-type oligosaccharides with different DP were more than 98% by one-step process. The DP and molecular weight were obtained through ESI-MS in negative mode. The characterization of the inulin-type oligosaccharides with different DP was studied by MS/MS spectra obtained by collision-induced dissociation of molecular ions ([M,H],). When the DP was lower, the fragment ions were formed through cross-ring cleavages of two bonds within the sugar ring and glycosidic cleavages. However, with the increase of DP, the ions resulting from glycosidic cleavages between two sugar residues were predominant. [source] Highly cross-linked polymeric capillary monoliths for the separation of low, medium, and high molecular weight analytesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 15-16 2009Said H. Lubbad Abstract Highly rigid capillary monoliths with low swelling propensity were prepared within the confines of 200 ,m ID fused silica capillaries via thermally induced free radical polymerization of tetrakis(4-vinylbenzyl)silane (TVBS) in the presence of 1-dodecanol and toluene. ,,,,-Azobisisobutyronitrile (AIBN) was used as initiator. The resulting monoliths were optimized for the separation of low, medium, and high molecular weight analytes. The microstructure and porosity of the monoliths prepared were studied by SEM and inverse size-exclusion chromatography (ISEC). The porosity of the monolithic supports was tuned by varying the amount of initiator (i. e. AIBN) between 0.5 and 2 wt%. All monoliths were tested for a series of low molecular weight compounds including alkylbenzenes, amines, carboxylic acids, phenols, carbonyl compounds, and ,-blockers, as well as for the separation of medium molecular weight analytes such as peptides and high-molecular weight analytes such as proteins. Due to the microporous structure, the novel monoliths displayed high efficiency and performance particularly in the separation of low molecular weight analytes. Relevant chromatographic parameters including permeability, swelling propensity, and height equivalents to theoretical plates were determined. [source] A combinatorial approach to studying protein complex composition by employing size-exclusion chromatography and proteome analysisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2007Shi-Sheng Li Abstract The genome sequences of numerous organisms are available now, but gene sequences alone do not provide sufficient information to accurately deduce protein functions. Protein function is largely dependent on the association of multiple polypeptide chains into large structures with interacting subunits that regulate and support each other. Therefore, the mapping of protein interaction networks in a physiological context is conducive to deciphering protein functions, including those of hypothetical proteins. Although several high-throughput methods to globally identify protein interactions have been reported in recent years, these approaches often have a high rate of nonspecific or artificial interactions detected. For instance, the fraction of false positives of the protein interactions identified by yeast two-hybrid assay has been predicted to be of the order of 50%. We have developed a strategy to globally map Bacillus subtilis protein,protein interactions in a physiological context by fractionating the cell lysates using size-exclusion chromatography (SEC), followed by proteome analysis. Components of both known and unknown protein complexes, multisubunits and multiproteins, have been identified using this strategy. In one case, the partners of the B. subtilis protein complex have been coexpressed in Escherichia coli, and the formation of the overexpressed protein complex has been further confirmed by a pull-down assay. [source] Oscillatory transverse electric field enhances protein resolution and capacity of size-exclusion chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 5 2006Guo-Min Tan Abstract Protein separations by a novel size-exclusion electrochromatography (SEEC) are presented. The present SEEC, denoted as pSEEC, was established with an oscillatory low-voltage electric field perpendicular to the mobile-phase streamline. Retention experiments with different proteins indicated that the influence of electric field strength on the partition coefficient is different for different proteins as well as for the same protein under different mobile-phase conditions. These results of protein retention led to the experimental design of protein separations with binary mixtures of BSA and immunoglobulin G (IgG), myoglobin (Myo) and lysozyme (Lys), as well as ovalbumin (Oval) and Myo. The separation results for the binary protein systems sufficiently exhibited the applicability of the pSEEC for various separations in terms of their molecular weights (MWs) as well as pIs. For example, it was possible to separate the gel-excluded proteins (BSA/IgG) as well as gel-permeable and similar-molecular-weight proteins (Myo/Lys) by the pSEEC. Moreover, in the cases of Oval/Myo, which could be partially separated by size-exclusion chromatography, the use of the pSEEC greatly improved the resolution and the separation became possible at high sample loading. The results indicate that the pSEEC technology is promising for preparative protein separations. [source] Isolation, structural features and rheological properties of water-extractable ,-glucans from different Greek barley cultivarsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2004Maria Irakli Abstract ,-Glucans were isolated from six Greek barley cultivars (Persefoni, Kos, Thessaloniki, Athinaida, Dimitra and Triptolemos) by water extraction at 47 °C, enzymatic removal of starch and protein and subsequent precipitation of the water-soluble ,-glucans with 37% (w/v) ammonium sulfate saturation. The purity of barley ,-glucans was high (>93% dry basis) with some small contamination by protein (<3.84%). The molecular size of the ,-glucan isolates was determined by high-performance size-exclusion chromatography (HPSEC); the weight-average molecular weights and the intrinsic viscosities ranged between 0.45 × 106 and 1.32 × 106 and 2.77 and 4.11 dl g,1, respectively. Structural features of barley ,-glucans were revealed by 13C NMR spectroscopy and high-performance anion-exchange chromatography (HPAEC) of the oligomers released by the hydrolytic action of lichenase. Lichenase degradation showed that ,-glucans from all barley cultivars consisted of blocks of cellotriosyl and cellotetraosyl units, accounting for 90.6,92.3% of the total oligomers released, with a molar proportion of these units between 2.31 and 2.77. Rheological measurements of aqueous solutions/dispersions of ,-glucans showed the behaviour of non-interacting polysaccharides and a transition from the typical viscoelastic response to gel-like properties after a time period that depended on the molecular size of the polysaccharide. The lowest molecular size ,-glucans from the Triptolemos cultivar showed shorter gelation times than their higher molecular weight counterparts. The effect of sugar incorporation (glucose, fructose, sucrose, xylose and ribose), at a concentration of 30% (w/v), to the ,-glucans gels (6% w/v) on compression parameters seemed to be related to the type of sugar used; the pentose sugars substantially reduced gel firming. Copyright © 2004 Society of Chemical Industry [source] PLP-SEC Study into the Free-Radical Propagation Rate Coefficients of Partially and Fully Ionized Acrylic Acid in Aqueous SolutionMACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 8 2004Igor Lacík Abstract Summary: Propagation rate coefficients, kp, for acrylic acid (AA) polymerization at 6,°C in aqueous solution were measured via pulsed laser polymerization (PLP) with the degree of ionization, ,, varied over the entire range between 0 and 1. These measurements were carried out in conjunction with aqueous-phase size-exclusion chromatography (SEC). Strictly speaking, the reported kp's are "apparent" propagation rate coefficients deduced from the PLP-SEC data under the assumption that the local monomer concentration at the radical site is identical to overall monomer concentration. At an AA concentration of 0.69 mol,·,L,1, the apparent kp decreases from 111,000 L,·,mol,1,·,s,1 at ,,=,0 to 13,000 L,·,mol,1,·,s,1 at ,,=,1.0. The significant lowering of kp with higher , is attributed to the repulsion between both monomer molecules and macroradicals becoming negatively charged. Addition of up to 10 mol-% (with respect to AA) sodium hydroxide to the fully ionized aqueous AA solution leads to an enhancement of kp up to 57,000 L,·,mol,1,·,s,1. Dependence of apparent kp values on the degree of ionization of acrylic acid (a) and on pH (b) for aqueous polymerizations of acrylic acid. [source] Cross-Linked Poly(, -caprolactone/D,L -lactide) Copolymers with Elastic PropertiesMACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 18 2002Antti O. Helminen Abstract Cross-linked , -caprolactone (CL) and D,L -lactide (DLLA) copolymers with elastic properties were synthesized in three steps. First, the monomers were copolymerized in ring-opening polymerization to obtain telechelic star-shaped oligomers with almost completely random monomer distribution. The oligomers were methacrylated with methacrylic anhydride in the second step and cured in a third. Molar CL/DLLA compositions of 30/70, 50/50, 70/30, 90/10, and 100/0 were used to obtain elastic structures with a wide range of properties. The effect of the average length of the copolymer block on the properties of the networks was evaluated with three different co-initiator contents (0.5, 1.0, and 2.0/100) in the oligomer synthesis. The oligomers were characterized by 13C NMR spectroscopy, size-exclusion chromatography (SEC), and differential-scanning calorimetry (DSC). The formation of elastic networks was confirmed by the absence of a flow region in dynamic mechanical analysis (DMA), the increase in Tg in DSC, and the full recovery of the sample dimensions after tensile testing. In addition, gel contents were high and the samples swelled in CH2Cl2. The networks possessed break stresses from 0.7,9.7 MPa with elongations from 80,350%. Networks with 100 or 90% of , -caprolactone retained their form in vitro for 12 weeks, but an increase in lactide content made the networks more vulnerable to hydrolysis. Water absorption of the polymers during hydrolysis. [source] Standardization of allergen products: 1.ALLERGY, Issue 7 2009Detailed characterization of GMP-produced recombinant Bet v 1.0101 as biological reference preparation Background:, Standardization of allergen extracts requires the availability of well-characterized recombinant allergens, which can be used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101, which shall be used in a ring trial within the framework of the Biological Standardization Programme BSP090 of the European Directorate for Quality of Medicines and Healthcare. Methods:, Recombinant Bet v 1.0101 Y0487 was produced under good manufacturing practice conditions and analysed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, as well as biological activity. Results:, Batch Y0487 was shown to contain monomeric and well-folded protein being identical with rBet v 1.0101, as determined by mass spectrometry. SDS-PAGE, isoelectric focusing, deamidation analysis and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4°C batch Y0487 retained the monomeric state up to 3 months. Protein quantification determined by amino acid analysis was found coinciding with half-maximal inhibition of serum IgE in ELISA. Biological activity of batch Y0487 was shown to be comparable to natural Bet v 1 by IgG and IgE immunoblotting, as well as basophil and T-cell activation. Conclusion:, Recombinant Bet v 1.0101 Y0487 was characterized extensively by physicochemical and immunological methods. It was shown highly stable, monomeric and immunologically equivalent to its natural counterpart. Thus, it represents an appropriate candidate reference standard for Bet v 1. [source] | ||||||||||||||||||||||||||||||||||