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Exchange Chromatography (exchange + chromatography)
Kinds of Exchange Chromatography Selected AbstractsEffect of Conductivity, pH, and Elution Buffer Salinity on Glycomacropeptide Recovery from Whey Using Anion Exchange ChromatographyJOURNAL OF FOOD SCIENCE, Issue 4 2005Hatice N. Tek ABSTRACT: The objective of this study was to investigate the effect of whey conductivity, pH, and the salt concentration of the elution buffer on glycomacropeptide recovery and its extent of contamination using anion exchange chromatography. Glycomacropeptide was isolated from Mozzarella whey. Samples were analyzed for glycomacropeptide and contaminating whey proteins. Mass balances and percent recoveries were calculated from these data. Glycomacropeptide recovery increased substantially with decreasing conductivity and increasing pH of the whey feed stream. Increasing the pH, but not increasing the conductivity, increased contamination of the glycomacropeptide by primarily beta-lactoglobulin. Salt concentration of at least 0.1 M was required for complete elution of bound glycomacropeptide. These data define conditions needed for glycomacropeptide recovery by a process chromatography system that uses food-grade buffers, operates at industrially relevant flow rates, and achieves up to 98% recovery. [source] Biomarker discovery in rat plasma for estrogen receptor-, actionELECTROPHORESIS, Issue 23 2005Tom G. Holt Dr. Abstract To support in vivo screening efforts for estrogen receptor (ER) subtype selective therapeutic agents, we initiated work to discover surrogate markers (biomarkers) in blood plasma that would change in response to ER subtype-specific action. We used a proteomic approach employing strong anion exchange chromatography (SAX), PAGE, and MS to identify potential plasma markers for selective ER-, action. The methodology was used to compare blood from vehicle-treated rats to blood from rats treated with either 17,-estradiol (an ER-,/ER-, agonist) or compound 1 (17,-ethynyl-[3,2-c]pyrazolo-19-nor-4-androstene-17,-ol, an ER-,-selective agonist). Blood samples were first fractionated by SAX to separate fractions containing dominant common plasma proteins from fractions enriched for less-abundant plasma proteins. 1-D PAGE analysis of fractions depleted of dominant plasma proteins revealed treatment-specific changes in protein profiles. Protein bands that changed reproducibly in response to ER-, action were excised from the gel, separated by capillary LC, and identified by microspray ESI-MS. Using this method, the plasma levels of two proteins, transthyretin and apolipoprotein E, were shown to decrease in response to ER-, agonism. The method lacked the sensitivity to identify the known, 1000-fold less-abundant, estrogenic marker prolactin (PRL). However, using a commercial RIA and immunoblots, we showed that PRL levels increase significantly in response to treatment with the ER-, selective agonist, compound 1. [source] Co-inheritance of Hb Hershey [,70(E14) Ala,Gly] and Hb La Pommeraie [,133(H11)Val,Met] in a Sicilian subjectEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2010Antonino Giambona Abstract Objectives:,This report represents the first observation in Sicily of two rare , -globin gene variants, Hb Hershey [,70(E14) Ala,Gly] and Hb La Pommeraie [,133(H11)Val,Met], found in a 35-year-old male patient from Messina, in the north-east of Sicily during population screening for hemoglobinopathies. Methods: The occurrence of the Hb variants was assessed by cation exchange chromatography while complete blood counts were obtained using automatic cell counters. Red cell lysates were analyzed by electrophoresis at alkaline and acid pH. Stability of hemoglobin was checked by the isopropanol precipitation test and by the heat tests while inclusion bodies and reticulocyte count were determined by incubation of blood samples with brilliant cresyl blue. Molecular analysis was performed by DNA sequencing of ,- and , -globin genes. Results: We observed an abnormally high performance liquid chromatography elution with a slight reduction in mean corpuscular volume and mean corpuscular haemoglobin parameters and mutations at codon 70 GCC,GGC (Hb Hershey) and at codon 133 GTG,ATG (Hb La Pommeraie) in , -globin gene. Conclusion: Family analysis of three generations demonstrated the presence of these two mutations in trans. So it was possible to describe the phenotypes of these variants in a heterozygous state and in double heterozygous state. [source] Hev b 9, an enolase and a new cross-reactive allergen from Hevea latex and moldsFEBS JOURNAL, Issue 24 2000Purification, characterization, cloning, expression Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho- d -glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients' IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials. [source] Purification and characterization of a glutathione S -transferase from the fungus Cunninghamella elegansFEMS MICROBIOLOGY LETTERS, Issue 2 2001Chang-Jun Cha Abstract Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S -transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120,000×g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate,polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of Mr 27,000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian ,-, ,-, and ,-class GSTs, although it showed a small degree of cross-reactivity with a ,-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs. [source] Expression and characterization of ,-glucosidase III in the dwarf honeybee, Apis florea (Hymenoptera: Apoidea: Apidae)INSECT SCIENCE, Issue 4 2007CHANPEN CHANCHAO Abstract Alpha-glucosidase is synthesized in the hypopharyngeal glands located in the head of worker bees including Apis florea. To analyze the developmental stage-specific expression of the ,-glucosidase gene in A. florea, total RNA was isolated from eggs, and the heads of nurse and forager bees. By reverse transcription polymerase chain reaction (RT-PCR), it was shown that the highest expression levels of the ,-glucosidase III gene, in the three examined developmental stadia, were found in forager bees, with much lower expression levels in nurse bees and no detectable expression in eggs. A complete ,-glucosidase III cDNA was obtained by RT-PCR and sequenced. The 1 701 bp cDNA nucleotide sequence and the predicted 567 amino acids it encodes were assayed by BLASTn, BLASTp and BLASTx programs and revealed a 95% and 94% similarity to the A. mellifera,-glucosidase III gene at the DNA and amino acid sequence levels, respectively. For purification of the active encoded enzyme, forager bee heads were homogenized in sodium phosphate buffer solution and the crude extract (0.30 U/mg) sequentially precipitated with 95% saturated ammonium sulfate (0.18 U/mg), and purified by DEAE cellulose ion exchange chromatography (0.17 U/mg), and gel filtration on Superdex 200 (0.52 U/mg). After resolution through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single enzymically active band (73 kDa) was identified from renatured substrate gels. Excision of this band, elution of the protein and tryptic peptide digestives identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed six matching masses to the A. mellifera (Q17958) and predicted A. florea,-glucosidase III protein with 12% coverage, supporting the probable purification of the same ,-glucosidase III protein as that encoded by the cloned cDNA. [source] Isolation of DNA from genetically modified oils by fast protein liquid chromatographyINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 7 2010Li Huang Summary In this study, a novel method of fast protein liquid chromatography (FPLC) anion exchange chromatography was developed for isolation of DNA from processed genetically modified (GM) oils. Four kinds of different GM edible oil had been chosen as model sample. Salmon DNA was used as the control sample to determine the pH values and NaCl in mobile phase buffer. Applying pH 8 and NaCl gradient 0.5,2 m were chosen for the DNA isolation. The quality and purity of isolated DNA were tested with agarose gel electrophoresis, scanned with UV absorbance spectra and amplified by polymerase chain reaction (PCR). The result indicated that the quantity of DNA isolated by FPLC was suitable for further PCR analyses. Furthermore, it is more effective and less time-consuming in comparison with cetyltrimethylammonium bromide method and High Pure GMO Sample Preparation Kit method. [source] Inhibition of endive (Cichorium endivia L.) polyphenoloxidase by a Carica papaya latex preparationINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2001David De Rigal When endive polyphenoloxidase (PPO) was incubated with a crude papaya latex extract, it rapidly lost its activity. Inactivation was ascribed to thermostable nonenzymatic factors of low molecular weight. These factors were partially purified by a two step protocol including gel filtration chromatography on Biogel P2 and ion exchange chromatography using DEAE Sephadex A25. The PPO-inactivation rate was first order, when either inactivating agent or proton concentration was evaluated. Inactivation could be partially reversed by CuSO4, which suggested that the inactivating factor(s) bound to the copper site of the enzyme. On a more rapid time scale than inactivation, papaya latex extract acted also as a weak noncompetitive PPO inhibitor. [source] Isolation and preliminary characteristics of ,- N -acetylglucosaminidase in the sperm of Siberian sturgeon (Acipenser baerii) and rainbow trout (Oncorhynchus mykiss)JOURNAL OF APPLIED ICHTHYOLOGY, Issue 4 2008B. Sarosiek Summary The aim of this study was to characterize the enzyme ,- N -acetyglucosaminidase (,-NAGase) in the milt and spermatozoa extracts from Siberian sturgeon and rainbow trout. After ion exchange chromatography one protein peak showed ,-NAGase activity in sturgeon milt plasma and sperm extracts of both species. Surprisingly, two protein peaks showing ,-NAGase activity were found in rainbow trout milt plasma. The molecular mass of ,-NAGase was estimated by gel filtration as 127 kDa for rainbow trout spermatozoa, 271 kDa for sturgeon spermatozoa, and 74 kDa for milt plasma from both species. The kinetic parameters were determined for milt plasma and sperm extracts. The optimum pH of the ,-NAGases was 3.8 for sturgeon milt plasma, 4.4 for sturgeon sperm extract, and 4.4,4.8 for milt plasma and sperm extract from rainbow trout. Km value of the ,-NAGases was 0.212, 0.563, 0.779 mm for sturgeon milt plasma, sturgeon sperm extract or rainbow trout extract, respectively. The ,-NAGase from sperm extracts in both species showed 100% activity even after incubation at 56°C by 20 min, whereas its activity was decreased to 23% in sturgeon milt plasma and to 2% in trout milt plasma. [source] Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffaloJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2001P. Pattnaik Aims:,To characterize a bacteriocin-like factor from Bacillus licheniformis 26 L-10/3RA isolated from buffalo rumen. Methods and Results:,The culture supernatant exhibited the antibacterial activity against a number of indicator organisms in a cut-well agar assay under anaerobic conditions. The inhibitory component was purified by following ammonium sulphate precipitation, gel filtration and ion exchange chromatography and confirmed to be a single peptide. A single band on tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity and having an estimated molecular mass of approximately 1400 dalton. Complete amino acid sequence of the peptide yielded 12 amino acids from the N-terminal end (ISLEICXIFHDN). No homology with previously reported bacteriocins was observed and has been designated as Lichenin. Lichenin was found to be hydrophobic, sensitive to atmospheric oxygen, retained biological activity even after boiling for 10 min and was active over a pH range of 4·0,9·0. Conclusions:,The Lichenin represents the first anaerobiosis specific expression of bacteriocin-like compound isolated from Bacillus licheniformis 26 L-10/3RA of buffalo rumen origin. Significance and Impact of the Study:,Lichenin could be a potential condidate for manipulating the rumen function at molecular level intended for improving the productivity of the ruminant. [source] Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdoseJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2010Bin Li Abstract The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60,84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase. Copyright © 2010 John Wiley & Sons, Ltd. [source] Biochemical characteristics of purified beef liver NADPH,cytochrome P450 reductaseJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2002Emel Arinç Abstract NADPH,cytochrome P450 reductase, an obligatory component of the cytochrome P450 dependent monooxygenase system, was purified to electrophoretic homogeneity from beef liver microsomes. The purification procedure involved the ion exchange chromatography of the detergent-solubilized microsomes on first and second DEAE-cellulose columns, followed by 2,,5,-ADP Sepharose affinity chromatography. Further concentration of the enzyme and removal of Emulgen 913 and 2,-AMP were accomplished on the final hydroxylapatite column. The enzyme was purified 239-fold and the yield was 13.5%. Monomer molecular weight of the enzyme was estimated to be 76000 ± 3000 (N = 5) by SDS-PAGE. The absolute absorption spectrum of beef reductase showed two peaks at 455 and 378 nm, with a shoulder at 478 nm, characteristics of flavoproteins. The effects of cytochrome c concentration, pH, and ionic strength on enzyme activity were studied. Reduction of cytochrome c with the enzyme followed Michaelis,Menten kinetics, and the apparent Km of the purified enzyme was found to be 47.7 ,M for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer (pH 7.7). Stability of cytochrome c reductase activity was examined at 25 and 37°C in the presence and absence of 20% glycerol. The presence of glycerol enhanced the stability of cytochrome c reductase activity at both temperatures. Sheep lung microsomal cytochrome P4502B and NADPH,cytochrome P450 reductase were also purified by the already existing methods developed in our laboratory. Both beef liver and sheep lung reductases were found to be effective in supporting benzphetamine and cocaine N-demethylation reactions in the reconstituted systems containing purified sheep lung cytochrome P4502B and synthetic lipid, phosphatidylcholine dilauroyl. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:286,297, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10054 [source] Purification and characterization of heparan sulfate from human primary osteoblastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2009Sadasivam Murali Abstract Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts,soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non-identical by a number of parameters, and that these differences have significant ramifications for their ligand-binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS-dependent factors between the ECM and the cell surface. J. Cell. Biochem. 108: 1132,1142, 2009. © 2009 Wiley-Liss, Inc. [source] Bone-specific heparan sulfates induce osteoblast growth arrest and downregulation of retinoblastoma proteinJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2006Kerry J. Manton The heparan sulfate (HSs) sugars of the extracellular matrix (ECM) play a key role during both development and wound repair in regulating the flow of growth and adhesive factors across their cell surface receptors. The aim of this study was to assess the structural and functional differences of HS chains extracted from the conditioned media (soluble), cell surface, and ECM of primary human osteoblast cultures, and to analyze their effects on osteoblast cell growth. HS chains from these compartments were characterized through a combination of enzymatic degradation, anion exchange chromatography, and molecular sieving. Although the chains were all approximately the same size, they varied systematically in their sulfate content, suggesting differences in their protein-binding domains. When added to pre-confluent hFOB1.19 osteoblast cultures, HS doses exceeding 500 ng/ml inhibited proliferation, without affecting viability, irrespective of their origin. Furthermore, HS doses of 500 ng/ml also downregulated retinoblastoma, Cyclin A and CDK1 protein expression, indicating that high doses of osteoblast HS negatively regulate cell cycle, resulting in growth arrest; when high doses of HS were withdrawn after a prolonged period, linear cell growth was reestablished. Thus, despite differences in sulfation, HS from either the soluble, cell surface, or matrix compartments of primary human osteoblast cultures are functionally similar with respect to their effects on growth. Binding assays revealed that the HS chains bound TGF,1, a known inhibitor of osteoprogenitor growth, at higher affinity than a suite of other bone-related, heparin-binding growth factors. Overcoming such sugar-mediated inhibition may prove important for wound repair. J. Cell. Physiol. 209: 219,229, 2006. © 2006 Wiley-Liss, Inc. [source] Purification and characterization of an organic solvent and detergent-tolerant novel protease produced by Bacillus sp.JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2008Abstract BACKGROUND: Purification and characterization of a novel protease produced by Bacillus sp. RKY3, has been investigated, with special emphasis on the stability of the enzyme in the presence of different oxidizing and reducing agents as well as organic solvents. The enzyme was purified in two steps through concentration of the crude enzyme by ammonium sulfate precipitation, followed by anion exchange chromatography. RESULTS: The purified protease had a molecular mass of approximately 38 kDa, which was highly active over a broad range of pH between 7.0 and 9.0 and was also stable over a wide pH range from 5.0 to 11.0. Although the optimum temperature for enzyme activity was found to be 60 °C, it was rapidly deactivated at temperatures above 60 °C. It also showed good stability at 50 °C, with a 70 min half-life. Ca2+ ions did not greatly enhance the activity or the stability of the enzyme. PMSF (1 mmol L,1) completely inhibited the protease activity, and thus the purified protease was considered to be serine protease. The purified protease was stable with oxidants (H2O2, 2%), reducing agents (sodium dodecyl sulfate, 2%), and organic solvents (25%) such as benzene, hexane, and toluene. CONCLUSION: The purified enzyme, protease, seems to possess potential applications in protease-based detergent and bleaching industries. The enzymatic activity against a wide variety of substrates suggests that the purified enzyme should be investigated for a range of commercial applications, especially for soy protein and gelatin hydrolysis in the food processing industry. Copyright © 2008 Society of Chemical Industry [source] CHARACTERIZATION OF POLYPHENOL OXIDASE FROM ROOSTER POTATO (SOLANUM TUBEROSUM CV ROOSTER)JOURNAL OF FOOD BIOCHEMISTRY, Issue 1 2010D. NI EIDHIN ABSTRACT The isolation and purification of polyphenol oxidase from potatoes (Solanum tuberosum cv. Rooster) is described. A 64-fold purified preparation has been obtained with 10% yield by a procedure involving (NH4)2SO4 precipitation, phenyl sepharose chromatography, ion exchange chromatography and hydroxyapatite chromatography. The partially purified enzyme has both cresolase and catecholase activity. Activity was lower toward monophenols than diphenols. Enzyme activity was optimal at pH 6.0,6.5 and at 30C. Greater than 50% activity was retained during storage for 72 h at pH 6.0,7.5. Residual activity was greater than 50% after incubation at 20C for 72 h, 30C for 48 h, 40C for 24 h, 50C for 2 h and 60C for 15 min. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid. Sodium dodecyl sulphate appeared to activate the enzyme. The enzyme was capable of cross-linking casein but did not increase gel-strengths in acidified milk gels. PRACTICAL APPLICATIONS Rooster is the most important potato cultivar grown in Ireland and data on its isolation and characterization has not been reported previously. This work describes a method to isolate polyphenol oxidase and characterization of the enzyme. Information on characterization of the enzyme could be valuable in relation to control of enzymatic browning during current processing and in minimum processing. There is potential for use of the enzyme in the emerging cross-linking area, as the results show some success and there may be potential of more cross-linking as the field develops and as interest in natural methods of cross-linking for food texture grows. This could lead to an important use for potato waste. Food product applications are given. [source] PARTIAL PURIFICATION AND CHARACTERIZATION OF NEUTRAL TREHALASE FROM COMMERCIAL BAKER'S YEAST, SACCHAROMYCES CEREVISIAEJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2000SANIYE YARAR ABSTRACT The neutral trehalase of a commercial baker's yeast (S. cerevisiae) strain has been partially purified using ammonium sidfate fractionation and DEAE-cellulose column chromatography techniques. Trehalase was precipitated between 35,50% ammonium sulfate saturation and approximately 5,8 fold purification was achieved. The yeast cAMP-dependent protein kinase was also precipitated in the same fraction and these two proteins were separated by DEAE-cellulose column chromatography. Trehalase became totally inactive after ion exchange chromatography, "cryptic trehalase" (tre-c), but was later activated with the addition of partially purified protem kinase together with cAMP and ATP. A 215 fold purification was obtained after DEAE-ceUulose column chromatography. One mM EDTA caused complete inhibition of the enzyme in crude extract, however the inhibition levels in ammonium sulfate and DEAE-cellulose fractions were 73.5% and 50%, respectively. Optimal pH range and temperature of the enzyme were determined as pH 6,6.8 and 30C, respectively. The kinetic parameters, Km and Vmax, were estimated as 11.78 mM trehalose and 12.47 ,mole glucose/min-mg protein, respectively. [source] Polyphenol Oxidase from Apple (Malus domestica Borkh. cv Bramley's Seedling): Purification Strategies and CharacterizationJOURNAL OF FOOD SCIENCE, Issue 1 2006Deirdre M. Ni Eidhin ABSTRACT Polyphenol oxidase (PPO) was isolated from Bramley's Seedling apples with 75.7-fold purification and 26.5% recovery by ammonium sulfate precipitation, phenyl sepharose chromatography, ion exchange chromatography, and hydroxyapatite chromatography. Molecular weight was estimated to be about 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Optimum PPO activity was at pH 6.5 and greater than 50% activity was retained during storage for 72 h at pH 5.5 to 6.5. Optimum temperature for activity was 30 °C and the enzyme had residual activity of greater than 50% during storage for 72 h at 20 °C to 30 °C and for 24 h at 40 °C to 50 °C. Of the substrates tested, activity was greatest with 4-methylcatechol followed by catechol, pyrogallol, and (,)epicatechin. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid. [source] Effect of Conductivity, pH, and Elution Buffer Salinity on Glycomacropeptide Recovery from Whey Using Anion Exchange ChromatographyJOURNAL OF FOOD SCIENCE, Issue 4 2005Hatice N. Tek ABSTRACT: The objective of this study was to investigate the effect of whey conductivity, pH, and the salt concentration of the elution buffer on glycomacropeptide recovery and its extent of contamination using anion exchange chromatography. Glycomacropeptide was isolated from Mozzarella whey. Samples were analyzed for glycomacropeptide and contaminating whey proteins. Mass balances and percent recoveries were calculated from these data. Glycomacropeptide recovery increased substantially with decreasing conductivity and increasing pH of the whey feed stream. Increasing the pH, but not increasing the conductivity, increased contamination of the glycomacropeptide by primarily beta-lactoglobulin. Salt concentration of at least 0.1 M was required for complete elution of bound glycomacropeptide. These data define conditions needed for glycomacropeptide recovery by a process chromatography system that uses food-grade buffers, operates at industrially relevant flow rates, and achieves up to 98% recovery. [source] Stabilization and Partial Purification of a Protease from Ginger Rhizome (Zingiber offinale Roscoe)JOURNAL OF FOOD SCIENCE, Issue 3 2005Pitaya Adulyatham ABSTRACT: Ginger protease (GP) or zingibain is of interest as a meat tenderizing agent. The objective of this research was to investigate food-compatible methods for stabilizing GP during storage or enzyme fractionation. Crude GP extracted from fresh ginger had a half-life (t1/2) of 2.1 (±0.16) d at 5°C decreasing to 20 min at 30°C. Addition of ascorbate (0.2% w/v) increased the t1/2 for GP from 2 to 20 d at 5°C. Dithiothreitol or Ethylenediaminetetraacetic acid (EDTA) had no effect on GP stability. Acetone powder preparations from ginger yielded GP with t1/2 of 18 mo at 5°C. Crude GP extracted from acetone powder was sufficiently stabilized to allow fractionation by ion exchange chromatography without the addition of toxic or expensive additives. GP was partially purified 252-fold with a recovery of 61%. The nomimal molecular weight of GP was 34.8 kDa compared with 25.1 kDa for papain. This work shows that the stability of GP can be greatly improved, increasing its attractiveness as a commercial product. Some possible routes of GP deactivation and stabilization are discussed. [source] A Lipoprotein-derived Antimicrobial Factor from Hen-egg Yolk is Active Against Streptococcus SpeciesJOURNAL OF FOOD SCIENCE, Issue 8 2002D. Brady ABSTRACT: Oral administration of hen-egg yolk provides protection against specific pathogens. We examined the antibacterial activity of fractionated egg yolk against 2 pathogenic Streptococcus strains, using an in vitro assay. A water-soluble protein fraction (WSPF) of egg yolk consistently inhibited the growth of S. mutans by 25%. The WSPF treated with pancreatin demonstrated > 80% inhibition of bacterial growth. Growth of S. sanguis was completely inhibited. Gel filtration and ion exchange chromatography established that anti-Streptococcal activity resided with lipoproteins. Antibacterial activity was released by crude lipase or a combination of lipase and protease treatment of egg lipoproteins. Thus, hen-egg yolk lipoproteins are important molecules for lipid-mediated antimicrobial activity. [source] Clarification of Citrus Juice is Influenced by Specific Activity of Thermolabile Pectinmethylesterase and Inactive PME-Pectin ComplexesJOURNAL OF FOOD SCIENCE, Issue 7 2002J. Ackerley ABSTRACT: Thermolabile pectinmethylesterase (PME) from Valencia orange pulp was extracted, partially purified by cation exchange chromatography (IEX), and added to reconstituted orange juice at 2 units/ml. Of the juices that clarified, %T increased, cloud particle size increased and % degree of esterification (DE) decreased in the 15 d storage study. The rate of clarification was most rapid for juices with added PME extracts that never bound Hi-Trap SP and contained 36 and 27 kDa peptide, intermediate for crude extracts of PME not applied to IEX, and lowest for PME extracts that bound Hi-Trap SP and contained 36 and 13 kDa peptide. These results suggest that PME-pectin complexes and low peptides moderate PME activity and juice clarification. [source] Purification of Angularin, A Novel Antifungal Peptide from Adzuki BeansJOURNAL OF PEPTIDE SCIENCE, Issue 3 2002Dr X. Y. Ye Abstract An antifungal peptide was isolated from the adzuki bean with a procedure involving affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein designated angularin was adsorbed on both types of chromatographic media and possessed a molecular weight of 8 kDa. Angularin exhibited antifungal activity against a variety of fungal species including Mycospharella arachidiocola and Botrytis cinerea. It inhibited mycelial growth in B. cinerea with an IC50 of 14.3 µM. Fusarium oxysporum and Rhizoctonia solani were not inhibited. Angularin demonstrated inhibitory activity on translation in the rabbit reticulocyte lysate system (IC50 = 8.0 µM) but did not affect proliferation of splenocytes. The activity of HIV-1 reverse transcriptase was inhibited in the presence of angularin. Its N -terminal sequence was GEPGQKE. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Compounds from rose (Rosa rugosa) flowers with human immunodeficiency virus type 1 reverse transcriptase inhibitory activityJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 9 2006M. Fu The aqueous extracts and ethanol precipitates of aqueous extracts of 18 medicinal herbs traditionally used in China were screened for their ability to inhibit human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) in-vitro. Among the samples screened at a concentration of 500 ,g mL,1, dried rose (Rosa rugosa) flowers showed the strongest inhibition. The ethanol precipitate of the aqueous extract of R. rugosa was processed and two components (P1 and P2) were obtained after ion exchange chromatography on DEAE-cellulose. Then, P1-a (Mr 150 kDa) and P1-b (Mr 8 kDa) were isolated from P1 by gel filtration on Sephadex G-200. They inhibited the activity of HIV-1 RT with an IC50 of 158 nm and 148.16 ,g mL,1 (18.5 ,m), respectively. Further structural analyses revealed that P1-a was a polysaccharide-peptide complex, and P1-b was a polymer consisting of acteoside and acteoside derivatives identified by Fourier transform infrared spectroscopy, nuclear magnetic resonance, assays of carbohydrate and protein contents and high-performance liquid chromatography electrospray ionization mass spectrometry. [source] STRUCTURE-ACTIVITY RELATIONSHIPS OF OLIGOAGAR ELICITORS TOWARD GRACILARIA CONFERTA (RHODOPHYTA)JOURNAL OF PHYCOLOGY, Issue 3 2001Florian Weinberger Agar oligosaccharides in the neoagarobiose series were prepared by partial enzyme hydrolysis, separated on Biogel P2 and P4, and analyzed by high-performance anion exchange chromatography with pulsed amperometric detection, yielding neoagarosaccharide fractions with a disaccharide repetition degree ranging from 1 (neoagarobiose) to more than 8 (neoagarohexadecaose). These fractions were analyzed for their biological activity toward the marine red alga Gracilaria conferta (Schousboe ex Montagne) J. et G. Feldmann in terms of increase of oxygen consumption, release of hydrogen peroxide, elimination of epiphytic bacteria, and induction of thallus tip bleaching. The structure,activity and dose,response relationships of neoagarosaccharides were very similar in the respiratory and oxidative burst responses and in their bactericidal properties, with neoagarosaccharides consisting of 6 to 8 disaccharide repeating units being the most active. All these responses were competitively inhibited by the reduced form of neoagarohexaose, neoagarohexaitol. In contrast, the tip-bleaching response was light dependent, required much higher concentrations of neoagarosaccharides, and was not inhibited by neoagarohexaitol, suggesting that it is an unspecific oxidative stress reaction. Putative structural effects on the recognition of endogenous agar-oligosaccharide elicitors by G. conferta are discussed. [source] Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 13 2008Rosa Cabrera Abstract Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on ImmobilineTM strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock. [source] Assessing a novel microfluidic interface for shotgun proteome analysesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2007An Staes Abstract Microfluidic interfaces coupled to ESI mass spectrometers hold great potential for proteomics as they have been shown to augment the overall sensitivity of measurements and require only a minimum of operator manipulations as compared to conventional nano-LC interfaces. Here, we evaluated a new type of HPLC-Chips holding larger enrichment columns (thus an increased sample loading capacity) for gel-free proteome studies. A tryptic digest of a human T-cell proteome was fractionated by strong cation exchange chromatography and selected fractions were analyzed by MS/MS on an IT mass spectrometer using both the new HPLC-Chip as well as a conventional nano-LC-MS/MS interface. Our results indicate that the HPLC-Chip is capable of handling very complex peptide mixtures and, in fact, leads to the identification of more peptides and proteins as compared to when a conventional interface was used. The HPLC-Chip preferentially produced doubly charged tryptic peptides. We further show that MS/MS spectra of doubly charged tryptic peptide ions are more readily identified by MASCOT as compared to those from triply charged precursors and thus argue that besides the improved chromatographic conditions provided by the HPLC-Chip, its peptide charging profile might be a secondary factor leading to an increased proteome coverage. [source] Analysis of nuclear proteome in C57 mouse liver tissue by a nano-flow 2-D-LC,ESI-MS/MS approachJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17 2006Jie Zhang Abstract The analysis of whole cell or tissue extracts is too complex for current protein identification technology and not suitable for the study of proteins with low copy levels. To concentrate and enrich low abundance proteins, organelle proteomics is a promising strategy. This approach can not only reduce the protein sample complexity but also provide information about protein location in cells, organs, or tissues under analysis. Nano-flow two-dimensional strong-cation exchange chromatography (SCX),RPLC,ESI-MS/MS is an ideal platform for analyzing organelle extracts because of its advantages of sample non-bias, low amounts of sample required, powerful separation capability, and high detection sensitivity. In this study, we apply nano-scale multidimensional protein identification technology to the analysis of C57 mouse liver nuclear proteins. Organelle isolation has been optimized to obtain highly pure nuclei. Evaluation of nucleus integrity and purity has been performed to demonstrate the effectiveness of the optimized isolation procedure. The extracted nuclear proteins were identified by five independent nano-flow on-line SCX,RPLC,ESI-MS/MS analyses to improve the proteome coverage. Finally, a total of 462 proteins were identified. Corresponding analyses of protein molecular mass and pI distribution and biological function categorization have been undertaken to further validate our identification strategy. [source] Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymersJOURNAL OF SEPARATION SCIENCE, JSS, Issue 1 2006Yinmao Wei Abstract A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column , such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability , were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-,) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-, in only one chromatographic step were obtained to be 93% and 7.8×107 IU/mg, respectively. [source] Purification and biochemical characterisation of a novel glutamate decarboxylase from rice branJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 6 2010Li Wang Abstract BACKGROUND: Glutamate decarboxylase (GAD) is a useful enzyme whose main function is to catalyse the irreversible ,-decarboxylation of L -glutamate to produce ,-aminobutyric acid. The cheap and abundant rice-processing by-product rice bran contains a high amount of GAD, the purification and characterisation of which have not yet been reported. In this study, research on rice bran GAD was initiated. RESULTS: Rice bran GAD was purified to homogeneity via a combined purification protocol of ammonium sulfate fractionation, ion exchange chromatography and two gel filtrations, with a purification fold of 128.6 and an activity recovery of 21.3%. The enzyme was active at pH 5.5 and 40 °C and retained 80% of its original activity in the pH range 5,9 and the temperature range 30,50 °C. GAD activity was significantly enhanced in the presence of Ca2+ but strongly inhibited by Ag+, Hg2+, sodium dodecyl sulfate and CH3COOH. Kinetic determination of the apparent Km for L -glutamate and pyridoxal 5,-phosphate gave values of 27.4 mmol L,1 and 1.16 µmol L,1 respectively. CONCLUSION: Considering that rice bran is cheap and commercially available and that rice bran GAD is relatively stable, the development of cost-effective rice bran GAD-related functional foods would seem to be feasible. Copyright © 2010 Society of Chemical Industry [source] | |||||||||||||||||||||||||||||||