Anion Chromatography Research Articles

Quantitative changes in maize cytoplasmic proteins induced by aluminium

Three-day-old maize seedlings were subjected to 100 µM AlCl3 for 24 h. Cytoplasmic proteins were isolated from root tips, root base and from coleoptiles. After fractionation of cytoplasmic proteins on anion chromatography column Bio-Scale Q2 sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to monitor Al-induced changes in polypeptide composition of particular fractions. Four (root) and 7 (coleoptile) fractions were eluted from the column with linear 0 - 1.0 M NaCl gradient. In fraction 1 of cytoplasmic proteins from root tips Al induced accumulation of polypeptide with molecular mass of 16 kD and simultaneous reduction of two polypeptides (67.5 and 60 kD). In fraction 1 isolated from mature zone of maize roots Al-induced accumulation of 22 kD polypeptide and reduction of 67.5, 60, and 14 kD polypeptides. Most pronounced changes were revealed in coleoptile. In three protein fractions increased accumulation of polypeptides with molecular mass of 14, 17.5, 20, 24.5, 28, 30, and 37.5 kD were observed. In the remaining three root or four coleoptile fractions of cytoplasmic proteins, no differences were found between Al-treated and control maize seedlings.

Suppressed ion chromatography of inorganic anions and divalent metal cations with pyromellitic acid as eluent

Suppressed conductimetric detection ion chromatography (IC) using pyromellitate eluent was investigated for the simultaneous determination of common inorganic anions and divalent cations. When pyromellitate and sample anions (Cl −, NO 3 − and SO 4 2−) in the eluent were introduced into a cation-exchange membrane suppressor in the H + form after anion-exchange separation, pyromellitate and the anions were converted to pyromellitic acid and corresponding acids (HCl, HNO 3 and H 2SO 4), respectively. Since these acids are stronger than pyromellitic acid (p K a1=1.92), these acids were detected conductimetrically as an increase of the concentration of H + in the eluent. In contrast, divalent cation–pyromellitate complexes (Mg 2+–, Ca 2+–, Sr 2+–, Ba 2+–, Fe 2+–, Mn 2+–, Co 2+–, Ni 2+–, Zn 2+– and Cd 2+–pyromellitate complexes) were converted to pyromellitic acid by the suppressor and these divalent cations were detected as an increase of the concentration of pyromellitic acid in the eluent. When using both 0.15 m M pyromellitate eluent at pH 5.4 and a 25-cm anion-exchange column (TSKguardgel QAE-SW), both highly sensitive conductimetric detection and excellent simultaneous separation of inorganic anions (Cl −, NO 3 −, I −, SCN −, SO 4 2− and S 2O 3 2−) and divalent cations (Mg 2+, Ca 2+, Mn 2+, Co 2+, Ni 2+, Zn 2+ and Cd 2+) were achieved in 30 min. The detection limits ( S/ N=3; injection volume, 20 μl) for the anions were 0.73–2 μ M and those for the divalent cations were 0.21–4.7 μ M.

Suppression of Inflammatory Responses by Surfactin, a Selective Inhibitor of Platelet Cytosolic Phospholipase A 2

Surfactin inhibits platelet and spleen cytosolic 100 kDa phospholipase A 2 (PLA 2). In contrast, this same compound enhances rat platelet group II PLA 2 activity by ∼2-fold and slightly increases group I PLA 2 activity from porcine pancreas and Naja naja venom in vitro. Surfactin does not affect a Ca 2+-independent PLA 2 partially purified from bovine brain. Thus, this compound inhibits selectively the cytosolic form of PLA 2. Based on in vitro studies utilizing preincubation of surfactin with the enzyme, dialysis, and increased concentrations of substrates, the inhibitory effect of surfactin appears to be due to a direct interaction with the enzyme. Linear regression analysis of the linear portion of a concentration–response curve reveals an ic 50 of 8.5 μM. To further determine the inhibitory pattern, a Dixon plot was constructed to show that the inhibition by surfactin is competitive, but not uncompetitive, with an inhibition constant of K i = 4.7 μM in 50 mM Tris–HCl buffer, pH 8.0, at 37°. Surfactin blocked non-stimulated and calcium ionophore A23187-stimulated release of arachidonic acid from monkey kidney CV-1 cells, which contain a cytosolic 100 kDa PLA 2 as the major activity, as shown in an anionic exchange DEAE-5PW high performance liquid chromatography profile and western blotting analysis. Surfactin ameliorated inflammation induced by several chemicals. That is, it exhibited in vivo anti-inflammatory activity in several tested inflammatory reactions including 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, carrageenan-induced rat paw edema, and acetic acid-induced mouse writhing. These results demonstrate that surfactin is a selective inhibitor for cytosolic PLA 2 and a putative anti-inflammatory agent through the inhibitory effect produced by direct interaction with cytosolic PLA 2, and that inhibition of cytosolic PLA 2 activity may suppress inflammatory responses.

Two Subunits of the FoF1-ATPase Are Phosphorylated in the Inner Mitochondrial Membrane

Inside-out submitochondrial particles from potato tuber mitochondria were incubated with [γ-32P]ATP. More than 16 phosphorylated polypeptides were detected by autoradiography on an SDS-gel. Two phosphoproteins, migrating at 22 and 28 kDa, were excised from the SDS-gel, electroeluted, and purified further by anion chromatography. The phosphoproteins were N-terminally sequenced. Over the regions sequenced, the 22 and 28 kDa phosphoproteins had 100% sequence identity with potato proteins identified as the δ’-subunit of the F1-ATPase and theb-subunit of the FoATPase, respectively. We suggest that phosphorylation of these proteins may control the interaction between F1and Foand regulate energy coupling in oxidative phosphorylation.

Fractionation at pilot-plant scale of an haemoglobin hydrolysate by strong anionic exchange chromatography: application to the preparation of an amphiphilic peptide

The development and the scale-up of high performance anion chromatography to obtain 1 milligram to 1 gram yields of a peptide fraction from a complex peptic haemoglobin hydrolysate is described here. The chromatographic conditions were developed using a 1 cm3 Mono Q analytical column and progressively scaled-up to a 6 dm3 Q Sepharose Fast Flow column. For easy recovery of peptide and easy adjustment of conditions for final purification, a volatile buffer, ethanolamine/HCl buffer 20 mmol dm−3, pH 10·5, was employed; desalting was carried out by a pilot-plant scale electrodialysis which permitted the elimination of 99% NaCl without important loss of peptide (less than 15%). A combination of these techniques with reverse phase HPLC proved a useful strategy for fractionation of a complex peptide mixture and enabled pure peptides to be obtained in sufficient quantities for further analyses and biological tests. The example of preparation and purification of an amphiphilic peptide is described. Its ability to solubilize an insoluble photosensitizer, protoporphyrin IX, was determined in order to study its utilization as a carrier for photochemotherapy. © 1998 SCI.

Herpes simplex Virus Type 1 DNase: Functional Analysis of the Enzyme Expressed by Recombinant Baculovirus

Herpes simplex virus type 1 DNase (HSV-1 DNase) was expressed in insect cells by recombinant baculovirus (NPVUL12) and purified by a combination of anionic exchanger chromatography and gel filtration. Two polypeptides of 85 and 75 kD, whose ratio varied during purification, were induced 24 h after infection. The 75-kD protein was isolated and shown to possess catalytic activity. Gel filtration analysis indicated that the active form of the enzyme at an ionic strength of I = 0.3 is a dimeric protein with an apparent molecular weight of 130,000. The recombinant enzyme exhibited the overall characteristics of the native enzyme such as 5′-3′ exonuclease and endonuclease activities with a preferred degradation of DNA. In the absence of extraneously added Mg²⁺, the enzyme was capable of removing mononucleotides from 5′-end-labeled DNA, but not from RNA and 3′-end-labeled DNA. The peculiar mechanism of double-strand DNA degradation suggests a specific role of HSV-1 DNase in DNA recombination processes during viral replication.

Exo- and endo-glucanolytic activity of cellulases purified from Trichoderma reesei

Four cellulases, produced by Trichoderma reesei, have been purified by preparative isoelectric focusing (Rotofor), size exclusion (Sephacryl 100 HR), anionic (Mono Q) and cationic (Mono S) chromatography and chromatofocusing (Mono P). Enzymatic activity with a large number of substrates allowed the proteins to be classified as: cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II. The exo- or endo-glucanase character of these enzymes was analysed by using a technique based on the measurement of the Avicel insoluble fibres reducing power. © Rapid Science Ltd. 1998

Cloning and Characterization of a Novel Peptidase from Rat and Human Ileum

A novel 100-kDa ileal brush border membrane protein (I100) has been purified by anionic glycocholate affinity chromatography. Polyclonal antibodies raised against this protein were utilized to clone and characterize I100 in rats. A partial length human I100 cDNA was identified by hybridization screening. In the rat, the I100 protein is a 746-amino acid glycosylated (calculated core molecular mass of 80 kDa) type II integral membrane protein found on the apical surface of ileal villus enterocytes. Its 2.6-kilobase mRNA is expressed in distal small intestine in rats and in humans. The I100 cDNA is homologous to but distinct from human prostate-specific membrane antigen and rat brain N-acetylaspartylglutamate peptidase. It is expressed on both the basolateral and apical surfaces of stably transfected Madin Darby canine kidney cells. Analysis of these stably transfected Madin Darby canine kidney cells and I100 immunoprecipitates of rat ileal brush border membrane vesicles reveals that it has dipeptidyl peptidase IV activity. Future invesitgations will need to determine the exact substrate specificity of this novel peptidase.

Ion chromatography of anions on stationary phases modified with chondroitin sulfate

Inorganic anions were separated on anion exchangers modified with chondroitin sulfate-C. Retention of anions was not significantly affected by the eluent concentration for the TSKgel IC-Anion-SW anion exchanger modified with chondroitin sulfate-C. Lower concentrations of eluent could be used as the eluent. The system was applied to the determination of UV-absorbing anions in saliva.

Mineralization of Selenium‐Containing Amino Acids in Two California Soils

AbstractOrganic forms of Se as the selenoamino acids, selenomethionine (SeMet) and selenocystine (SeCys), are detected in plants grown on seleniferous soil, yet little is known about the speciation and distribution of Se upon decomposition of SeMet and SeCys in soil. To determine the mineralization rate of selenoamino acids in soil, three concentrations of SeMet, SeCys, methionine (Met), or cystine (Cys) were added to samples of a Panoche (fine‐loamy, mixed [calcareous], thermic Typic Torriorthent) or a Panhill (fine‐silty, mixed, thermic Typic Haplargid) soil and aerobically incubated at 22°C for up to 168 h. The amino acid concentrations were analyzed by high performance anion chromatography. Methionine additions closely followed first‐order reaction kinetics and SeMet additions followed pseudo‐first‐order kinetics, with order dependent on SeMet concentration. The time required for 50% mineralization of SeMet additions in the Panhill and Panoche soils was 23.5 and 3.2 h (5 mg Se kg‐1), 41.6 and 15.5 h (25 mg Se kg‐1), and 47.1 and 36.0 h (50 mg Se kg‐1), respectively. The majority of the SeMet and Met additions were recovered as volatile species (50–80%). In contrast to SeMet, SeCys was rapidly nonextractable (<6 h) from both soils, with little to no volatile Se detected and was initially recovered as phosphate‐soluble selenite and selenide after 6 h of incubation. These results suggest that Se present in seleniferous plant tissue as SeMet will not accumulate in soil due to extensive volatilization. In contrast, additions of seleniferous plant residues rich in SeCys will result in organic Se mineralization to inorganic Se forms in soil.

Cyanuric Acid-Based Eluent for Suppressed Anion Chromatography

The performance of cyanuric acid (CA, in the 1,3,5-triazine-2,4,6-trihydroxy form) as eluent for suppressed anion chromatography has been investigated. The distinctive features of this eluent are reported with respect to the others that are now routinely utilized. CA-based eluents for SAC have several of advantages over carbonate and tetraborate buffers: (i) wider range of eluent strength, (ii) lower background conductivities, (iii) good calibration linearities, (iv) improved sensitivity for carboxylic acids, (v) the possibility to perform gradient analysis with limited baseline drifts, and (vi) ease of eluent purification. This makes the CA-based eluent well-suited for the analysis of strongly retained anions like chromate, thiosulfate, iodide, thiocyanate, and perchlorate with good limits of detection and reasonable capacity factors.

Alkanesulfonate homologous series as eluent components in anion chromatography Novel eluent selection system for monovalent analytes

Common inorganic anions can be separated on an anion-exchange column of low capacity using a very dilute solution of alkanesulfonate as eluent components and a conductivity detector. A mathematical relationship has been found between the retention volumes of monovalent analyte using different alkanesulfonates as eluents and quaternary ammonium functionalized styrene-divinylbenzene stationary phase. Retention volumes decrease steadily for the homologous series of alkanesulfonates (methane-, ethane-, propane-, butane- and pentanesulfonates) as the alkyl-chain length increases. This system made it possible to establish the ion-exchange capacity of the separation column and the inter-eluent selectivity coefficients and permitted prediction of retention volume with variation of type and concentration of eluent. The parameters of retention model can be readily derived from elution experiments. The eluent system has been used easily to design separation schemes. Good agreement was obtained between the observed and predicted values for the anions of practical interest (fluoride, chloride, chlorate, nitrite, nitrate, bromide, bromate). Retention of ions can be manipulated by changing either the concentration or carbon number of the alkanesulfonate eluent from homologous series. The response of analyte retention to changing eluent conditions can be illustrated with a retention nomogram.

Simultaneous Separation of Inorganic Cations and Anions by Ion Chromatography Using Strong Anionic/Zwitterionic Mixed Micelles as the Stationary Phase

Mixed micelles obtained by mixing SDS (sodium dodecylsulfate) with Zwittergent-3-14 (N-tetradecyl-N, N-dimethyl- 3-ammonio 1-propanesulfonate) are used as a dynamic stationary phase for the simultaneous ion chromatography of inorganic cations and anions. Analyte cations are separated by the negative charges in the SDS based upon ion-exchange interactions while analyte anions are separated by both the positive and negative charges in Zwittergent-3-14 based upon simultaneous electrostatic attraction/repulsion interactions. The ratio of SDS/Zwittergent-3-14, which constitutes the stationary phase is an important factor in the determination of the retention times and elution orders of the analyte ions. The investigation of the mechanisms involved and the practical applications are discussed in this paper.

Capillary Ion Chromatography with On-Line High-Pressure Electrodialytic NaOH Eluent Production and Gradient Generation

A small inexpensive system is described that allows high-performance suppressed anion chromatography on a capillary scale. A fully computer-controlled stepper motor-driven syringe-type dispenser, equipped with a 500 μL-capacity glass syringe is capable of pumping at pressures up to 1000 psi when equipped with an appropriate inlet check valve. Fused-silica capillary columns ∼50 cm in length and 180 μm i.d., packed in-house with a commercial packing, provide excellent performance, significantly exceeding the efficiencies observed for the same packing in commercially available 2 mm bore format. The system operates with a pressure drop of <800 psi at a flow rate of 2 μL/min. The system utilizes a novel electrodialytic NaOH eluent generator that is deployed on the high-pressure side of the pump and thus requires no special measures for electrolytic gas removal. This device permits both isocratic and gradient operation with excellent eluent purity; the NaOH concentration is generated linearly with applied current with near-Faradaic efficiency, up to a concentration of at least 100 mM.

Application of microcolumnion chromatography using octadecylsilica immobilized with bovine serum albumin as stationary phase for the determination of inorganic anions

Microcolumn ion chromatography of inorganic anions has been studied using octadecylsilica immobilized with bovine serum albumin as a stationary phase. The analytes were monitored with a conductivity detector. The detection limits for chloride, nitrate and thiocyanate at S/N = 3 were 0.8, 1.0 and 3.0 μmol L−1, respectively. The present system was applied to the determination of inorganic anions in tap water and serum samples.

Fluorescent Brightener 28 Removal by Ozonation or Advanced Oxidation Processes (O3/H2O2)

The aim of this work is to study the reaction of ozone and combined ozone/hydrogen peroxide mixtures with the fluorescent brightener 28 in dilute aqueous solution using controlled experimental conditions. The kinetics were also evaluated under various experimental conditions. The main ozonation by-products have been identified by High Pressure Anionic Exchange Chromatography (HPAEC) and Gas Chromatography coupled with Mass Spectrometry (GC-MS) techniques and a reaction pathway is proposed. In order to confirm this mechanism, melamine and s-triazine have been treated under the same reaction conditions and their decomposition pathways were studied.

Comparison of O-Linked Carbohydrate Chains in MUC-1 Mucin from Normal Breast Epithelial Cell Lines and Breast Carcinoma Cell Lines:: DEMONSTRATION OF SIMPLER AND FEWER GLYCAN CHAINS IN TUMOR CELLS

MUC-1 mucin is considered to be aberrantly glycosylated in breast, ovary, and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation, we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1, MTSV1-7, and HB-2) derived from cells cultured from human milk, with three breast cancer cell lines (MCF-7, BT-20, and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1, and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern, with NeuAcalpha2-3Galbeta1-3GalNAc-ol, Galbeta1-3GalNAc-ol, and GalNAc-ol predominating; in contrast, MUC-1 from MTSV1-7 had more complex structures, including a number of disialo, core 2 species, i.e. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler, and fewer, carbohydrate chains than MUC-1 from normal breast epithelial cells, and that these differences, combined or separately, explain the differential tumor specificity of some MUC-1 antibodies and T cells.

Elution delay of fluoride by chromatography of anions on silica gels coated with crown ether dyes

The separation of halide ions by chromatography on silica gel coated with water-insoluble crown ether dyes was investigated. The retention times of fluoride and hydrogencarbonate ions were longer than those of chloride, bromide and iodide ions when distilled water was used as a mobile phase. The order was different from that of traditional anion-exchange selectivity. The elution behaviour is compared with that on silica gel and octadecylsilica gel, and is discussed with regard to the hydrophobicity and the hydrogen-bonding interaction.

Mixed-mode liquid chromatography of carboxylic acids and inorganic anions on a latex-based pellicular stationary phase

The retention behaviour of mono-, dicarboxylate and inorganic anions on latex-based pellicular anion-exchanger was investigated. This paper is focused on the possible retention mechanism providing a retention database on a variety of mobile phase compositions. It has been demonstrated that anions can interact with the cation-exchange layer on the highly crosslinked latex-based pellicular phase as well as with the anion-exchange sites. A hypothesis is proposed to explain a mixed-mode retention of the series of analytes. A combination of two mechanisms has been found responsible for the enhanced selectivity: ion-exchange and ion-exclusion. Applications include the separation of formate, acetate, propionate, lactate, pyruvate, oxalate, malonate, succinate, tartrate, fumarate, maleate , chloride, nitrate and sulphate with NaOH eluent using suppressed conductivity detection. The study effectively characterizes the behaviour of different analytes under elution conditions of practical importance.

Microcolumn ion chromatography of inorganic anions using bovine serum albumin stationary phase with indirect photometric detection

Microcolumn ion chromatography of inorganic anions has been studied using bovine serum albumin immobilized on silica gel as a stationary phase. Several mobile phase solutions were examined, involving sodium iodide, potassium hydrogen phthalate, 2,6-anthraquinone disulfonate (2,6-AQDS) and sodium salicylate. 2,6-AQDS achieved better separation of the analytes tested. Chloride, nitrate, iodide, thiocyanate and sulphate could be separated within 8 min. Detection limits were in the range of 0.9–2.9 μM, corresponding to mass detection limits of 0.18–0.59 pmol. The system was applied to the determination of inorganic anions in environmental water and biological samples.