Home About us Contact
|
Home About us Contact | ||
|
|
||
Column Chromatography (column + chromatography)
Kinds of Column Chromatography Selected AbstractsPolyphenol Oxidase from Bean Sprouts (Glycine max L.)JOURNAL OF FOOD SCIENCE, Issue 1 2003T. Nagai ABSTRACT: Polyphenol oxidase (PPO) was purified and characterized from bean sprouts by ammonium sulfate precipitation, DEAE-Toyopearl 650M, CM-Toyopearl 650M, SuperQ-Toyopearl 650S and QAE-Toyopearl 550C column chromatographies. Substrate staining of the crude extract on electrophoresis showed the presence of 2 isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be about 54 kDa. The optimum pH was 9.0 and optimum temperature 40 °C. Heat inactivation occurred about 30 °C. PPO showed activity to catechol, pyrogallol and dopamine. These compounds such as ascorbic acid, L-cysteine, 2-mercaptoethanol, and glutathione used was the effective inhibitor. Enzyme activity was maintained for 7 d at 4 °C but suddenly decreased after 8 d. [source] Verticase: a Fibrinolytic Enzyme Produced by Verticillium sp.JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 11 2007an Endophyte of Trachelospermum jasminoides Abstract Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of Verticillium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pI of 8.5, was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9,10 and 50,60 °C, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease. [source] Effects of salts on protein,surface interactions: applications for column chromatographyJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2007Kouhei Tsumoto Abstract Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein,protein or protein,surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein,protein or protein,surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 1677,1690, 2007 [source] Structure Elucidation and Phytotoxicity of Ecdysteroids from Chenopodium albumCHEMISTRY & BIODIVERSITY, Issue 4 2005Marina DellaGreca The leaves of Chenopodium album were infused in H2O/MeOH. The extract treated with cold acetone gave heavy precipitation, which was removed by centrifugation. Solid material was fractionated into acidic and neutral fractions. The acidic material was subjected to different silica-gel column chromatographies, and then it was purified by reversed-phase HPLC to afford four known ecdysteroids and the new 3,,14, -dihydroxy-5, -pregn-7-ene-2,6,20-trione that were characterized by extensive spectroscopic investigation, especially by 1D- and 2D-NMR. Their effects on germination and growth of Lactuca sativa L. have been studied. The results are reported as percentage differences of germination, root elongation and shoot elongation, from the control at concentrations ranging from 10,4 to 10,7,M. [source] Chenoalbicin, a Novel Cinnamic Acid Amide Alkaloid from Chenopodium albumCHEMISTRY & BIODIVERSITY, Issue 10 2004Francesca Cutillo The roots of Chenopodium album were infused in MeOH, and the extract was partitioned between AcOEt and H2O. AcOEt-Soluble material was subjected to different silica-gel column chromatographies and then purified by reverse-phase HPLC to afford a new cinnamic acid amide alkaloid as a racemic mixture. The new compound, named chenoalbicin (1), was characterized by extensive spectroscopic investigation, especially 1D and 2D NMR spectroscopy. Its effects on the germination and growth of Lactuca sativa L. has been studied. The results are reported as percentage differences of germination, root elongation, and shoot elongation from the control at concentrations ranging from 10,4 to 10,7,M. [source] Phospholipase A2 is present in meconium and inhibits the activity of pulmonary surfactant: an in vitro studyACTA PAEDIATRICA, Issue 4 2001AJJ Schrama Atelectasis, a major contributor to pulmonary dysfunction in meconium aspiration syndrome (MAS), is produced by bronchiolar obstruction and surfactant inactivation. It has been shown that substances in meconium, e.g. fatty acids, inhibit surfactant activity. However, the role of the enzyme phospholipase A2 (PLA2), which hydrolyses surfactant in adult respiratory distress syndrome (ARDS), has not yet been studied. Our objective was to investigate whether PLA2 is present in meconium and inhibits pulmonary surfactant activity in vitro. Therefore, the presence of PLA2 activity in meconium, collected from 10 newborns, was measured by the formation of lysophosphatidylcholine after incubation of meconium with radioactively labelled dipalmitoylphosphati-dylcholine. Meconium was fractionated by Sephadex G-100 column chromatography and the fractions were assayed for PLA2 activity. Also, their effect on the surface tension of surfactant (Curosurf) was measured using a pulsating bubble surfactometer (PBS). PLA2 activity was present in all meconium samples. Addition of meconium to surfactant significantly increased surface tension (mean ± SD: 17 ± 1.6 mN/m to 24.3 ± 6.7 mN/m, p= 0.0001) and only the addition of the PLA2 containing fraction from meconium to surfactant also significantly increased surface tension (mean 1.7 ± 1.6mN/m to 19.0 ± 3.58 mN/m, p < 0.0001). Conclusion: PLA2 is present in meconium and inhibits the activity of pulmonary surfactant in vitro. Therefore, PLA2 in meconium may contribute to surfactant inactivation and alveolar ateectasis in MAS. [source] Synthesis and screening of substituted 1,4-naphthoquinones (NPQs) as antifilarial agentsDRUG DEVELOPMENT RESEARCH, Issue 3 2010Nisha Mathew Abstract Eleven amino-substituted 1,4-naphthoquinones were synthesized via the reaction of 1,4-naphthoquinone with different primary and secondary mono- and diamines in the presence of dichloromethane ethanol (1:2) solvent at room temperature. All compounds were purified by flash column chromatography, characterized by TLC, HPLC, 13C-NMR, 1H-NMR, and FT-IR spectral analysis and were evaluated in vitro for antifilarial activity using adult bovine filarial worm Setaria digitata by assessing worm motility and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction. Seven of the 11 compounds had macrofilaricidal activity with compounds 9 (2-[(1,3-dimethylbutyl) amino] naphthalene-1,4-dione) and 11 (2-(4-methylpiperazin-1-yl) naphthalene-1,4-dione) having maximum activity (ED50 values of 0.91 and 1.2,µM, respectively, at 48,h). The effect of different substitutions on antifilarial activity is discussed. Drug Dev Res 2009. © 2009 Wiley-Liss, Inc. [source] Comparative in vitro and in vivo genotoxicities of 7H -benzo[c]fluorene, manufactured gas plant residue (MGP), and MGP fractionsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2004Leslie Cizmas Abstract Manufactured gas plant residue (MGP) is a complex mixture of polycyclic aromatic hydrocarbons (PAHs) that is tumorigenic in the lungs of mice. This study compared the relative genotoxicity of 7H -benzo[c]fluorene (BC), a PAH component of MGP, with MGP and MGP fractions in order to assess the contribution of BC to the genotoxicity of MGP. An MGP sample was separated into seven fractions (F1,F7) using silica gel column chromatography with petroleum ether (PE) followed by PE:acetone (99:1 v/v, then 98:2). PAHs were quantified using gas chromatography/mass spectrometry. An aliquot of F2, the fraction with the highest BC concentration and highest weighted mutagenic activity in Salmonella typhimurium strain TA98, was further separated using silica gel thin-layer chromatography with hexane. The first F2 subfraction, sF2-a, was enriched in BC and coeluting compounds and contained 35,000 ppm BC and 216,109 ppm carcinogenic PAHs (cPAHs, the sum of seven PAHs categorized by the U.S. EPA as class B2 carcinogens). The second F2 subfraction, sF2-b, contained a ninefold lower concentration of BC, with 3,900 ppm BC and 45,216 ppm cPAHs. Female ICR mice received topical application of crude MGP, crude MGP spiked with analytical-grade BC, F2, sF2-a, sF2-b, or analytical-grade BC. DNA adduct levels were analyzed by nuclease P1-enhanced 32P-postlabeling. In lung DNA of mice receiving 0.48 or 3.0 mg/mouse, net total RAL × 109 values were F2, 30.8 and 87.2; sF2-a, 24.8 and 106.7; and sF2-b, 19.6 and 151.0, respectively. Mice dosed with 0.10 mg analytical-grade BC (the mass of BC in 3.0 mg sF2-a) exhibited a net total RAL × 109 value of 7.03 in lung DNA. This was equal to approximately 7% of the total RAL × 109 value produced by 3.0 mg sF2-a. Thus, although BC appears to make an appreciable contribution to pulmonary adduct formation, the results suggest that MGP components other than BC play an important role in lung DNA adduct formation following topical MGP administration. Environ. Mol. Mutagen. 43:159,168, 2004. © 2004 Wiley-Liss, Inc. [source] Metal Effects on the Asymmetric Synthesis of a New Heterobidentate As/P=S LigandEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 12 2010Mengtao Ma Abstract The cycloaddition reaction between 3,4-dimethyl-1-phenylarsole and diphenylvinylphosphane sulfide was promoted by chiral palladium and platinum complexes containingortho -metalated (S)-[1-(dimethylamino)ethyl]naphthalene. They exhibited similar stereoselectivity; the palladium cycloadducts could not be separated via column chromatography and fractional crystallization, however, the corresponding platinum complexes could be successfully converted into their enantiomerically pure counterpart. A formal arsanylidene elimination reaction was observed on the liberated free As/P=S bidentate ligand. [source] On the Photochemical Stability of the 9-Mesityl-10-methylacridinium CationEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 2 2009Andrew C. Benniston Abstract The 9-mesityl-10-methylacridinium cation in aerated deuterated/normal acetonitrile decomposes to give several side products when continuously exposed to white light. The main breakdown product isolated by column chromatography is identified as 3,5-dimethyl-4-(10-methylacridinium)benzaldehyde. This assignment was confirmed by single-crystal X-ray crystallography. In light of these findings it appears that decomposition pathways also need to be considered when discussing the photochemical properties of the 9-mesityl-10-methylacridinium cation, especially in the presence of dioxygen.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] A Lewis Acid Promoted Asymmetric Umpolung Reaction with ChiralN -Sulfinyl Imines as the ElectrophilesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 9 2005Xin Xu Abstract An new asymmetric umpolung reaction has been developed by reacting N -sulfinyl imines with 2-lithio-2-phenyl-1,3-dithiane. The reaction was conducted at between ,20 and,25 °C in THF in the presence of Et2AlCl as the Lewis acid promoter. Excellent diastereoselectivities (up to >95,% de) and chemical yields (64,95,%) have been achieved for nine substrates with all individual isomers separated and characterized. The absolute structure of the chiral products has been unambiguously determined by synthetic conversions to a known sample. 2-Lithio-2-phenyl-1,3-dithiane was found to be much less reactive than its 2-methyl counterpart, which was reported very recently. All individual isomers have been readily separated by column chromatography. The absolute structure of the chiral products has been unambiguously determined by conversion into a known compound. This method provides an easy access to enantiomerically pure ,-amino ketones. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Kinetic study of sn -glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1FEBS JOURNAL, Issue 3 2002Jin-Suk Han A gene having high sequence homology (45,49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94,96 °C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H- dependent dihydroxyacetone phosphate reduction and NAD+ -dependent,glycerol-1-phosphate (Gro1P) oxidation. NADP+ -dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)+ acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi,bi mechanism. [source] Structural and functional studies of cinnamomin, a new type II ribosome-inactivating protein isolated from the seeds of the camphor treeFEBS JOURNAL, Issue 22 2001Liang Xie Cinnamomin is a new type II ribosome-inactivating protein (RIP). Its A-chain exhibits RNA N -glycosidase activity to inactivate the ribosome and thus inhibit protein synthesis, whereas the glycosylated B-chain is a lectin. The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the nucleotide sequences of cDNAs of cinnamomin A- and B-chains. It is composed of a total of 549 amino-acid residues: 271 residues in the A-chain, a 14-residue linker and 264 residues in the B-chain. To explore its biological function, the cinnamomin A-chain was expressed in Escherichia coli with a yield of 100 mg per L of culture, and purified through two-step column chromatography. After renaturation, the recovery of the enzyme activity of the expressed A-chain was 80% of that of native A-chain. Based on the modeling of the three-dimensional structure of the A-chain, the functional roles of five amino acids and the only cysteine residues were investigated by site-directed mutagenesis or chemical modification. The conserved single mutation of the five amino-acid residues led to 8,50-fold losses of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N -glycosidase activity of the A-chain. Most interestingly, the strong electric charge introduced at the position of the single cysteine in A-chain seemed to play a role in enzyme/substrate binding. [source] Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomesFEBS JOURNAL, Issue 10 2000Vasily D. Antonenkov Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacyl-CoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments. The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated. [source] Molecular analysis of the inhibitory effects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases from Streptococcus mutans MT8148FEMS MICROBIOLOGY LETTERS, Issue 1 2003M Matsumoto Abstract An oolong tea polyphenol (OTF6) has been shown to possess a strong anti-glucosyltransferase (GTF) activity and inhibit experimental dental caries in rats infected with mutans streptococci. The effects of OTF6 on the functional domains of GTFs of Streptococcus mutans, an N-terminal catalytic domain (CAT), and a C-terminal glucan-binding domain (GBD), were examined. The maximum velocity of glucan synthesis by recombinant GTFB (rGTFB) and GTFD (rGTFD) became significantly slower in the presence of OTF6, however, Km values remained stable when compared in their absence. These results suggest that OTF6 reduces glucan synthesis by non-competitively inhibiting the GBD of S. mutans GTFB and GTFD. Further, the recombinant proteins of CAT (rCAT) and GBD (rGBD) were expressed using Escherichia coli, and purified by affinity column chromatography. rGBD but not rCAT was found to possess dextran-binding activity, which was shown to be inhibited by OTF6. These results indicate that OTF6, a polymeric polyphenol specific for oolong tea is able to reduce glucan synthesis by inhibiting the GBD of S. mutans GTFB. [source] Chemical composition and antibacterial activity of the essential oil of Michelia foveolata Merryll ex Dandy from VietnamFLAVOUR AND FRAGRANCE JOURNAL, Issue 4 2007Dominique Lesueur Abstract The chemical composition of the volatile oil extracted from aerial parts (twigs, leaves and flowers) of Michelia foveolata growing wild in Vietnam was investigated by combination of column chromatography, GC (retention indices), GC,MS and 13C-NMR spectroscopy. Fifty-eight components were identified, with sabinene (32.4%) and terpinen-4-ol (13.7%) as the main constituents. The essential oil was tested against 11 bacteria using the agar disc diffusion method, and exhibited a significant antibacterial activity against Salmonella enterica, Staphylococcus epidermidis, Staphylococcus aureus and Bacillus cereus (MICs = 2,4 µl/ml). Copyright © 2007 John Wiley & Sons, Ltd. [source] Characterization of the major odorants found in the peel oil of Citrus reticulata Blanco cv. Clementine using gas chromatography,olfactometryFLAVOUR AND FRAGRANCE JOURNAL, Issue 4 2003Mary G. Chisholm Abstract Gas chromatography,olfactometry (GC,O), gas chromatography,mass spectrometry (GC,MS) and preparative column chromatography were used to identify the key odorants present in laboratory-extracted clementine oil from Spain. Almost 50 odorants were identi,ed using GC,O, many of which were unsaturated aldehydes with high odour spectrum values (OSV). , - and , -sinensal, trans -4,5-epoxy-(E)-2-decanal, (E,Z)-2,6-dodecadienal and linalool were found to dominate clementine oil aroma. Enrichment of the oxygenates using preparative column chromatography provided further identi,cation of a total of 50 aldehydes, not all of which were present in the oil at concentrations high enough to produce a response using GC,O. Aldehydes contributed approximately 80% of the total aroma of clementine oil. New odorants not previously reported in clementine oil include many unsaturated aldehydes, trans -4,5-epoxy-(E)-2-decenal, trans -4,5-epoxy-(E)-2-dodecenal, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (furaneol), 3-hydroxy-4,5-dimethyl-3(2H)-furanone (sotolon) and 1,8-cineole. No single odorant emerged as being characteristic of clementine oil aroma. Copyright © 2003 John Wiley & Sons, Ltd. [source] Mass spectrometric analysis of microtubule co-sedimented proteins from rat brainGENES TO CELLS, Issue 4 2008Tatsuhiko Sakamoto Microtubules (MTs) play crucial roles in a variety of cell functions, such as mitosis, vesicle transport and cell motility. MTs also compose specialized structures, such as centrosomes, spindles and cilia. However, molecular mechanisms of these MT-based functions and structures are not fully understood. Here, we analyzed MT co-sedimented proteins from rat brain by tandem mass spectrometry (MS) upon ion exchange column chromatography. We identified a total of 391 proteins. These proteins were grouped into 12 categories: 57 MT cytoskeletal proteins, including MT-associated proteins (MAPs) and motor proteins; 66 other cytoskeletal proteins; 4 centrosomal proteins; 10 chaperons; 5 Golgi proteins; 7 mitochondrial proteins; 62 nucleic acid-binding proteins; 14 nuclear proteins; 13 ribosomal proteins; 28 vesicle transport proteins; 83 proteins with diverse function and/or localization; and 42 uncharacterized proteins. Of these uncharacterized proteins, six proteins were expressed in cultured cells, resulting in the identification of three novel components of centrosomes and cilia. Our present method is not specific for MAPs, but is useful for identifying low abundant novel MAPs and components of MT-based structures. Our analysis provides an extensive list of potential candidates for future study of the molecular mechanisms of MT-based functions and structures. [source] Oligo(triacetylene) Derivatives with Pendant Long Alkyl ChainsHELVETICA CHIMICA ACTA, Issue 6 2004Jean-François Nierengarten Substituted (E)-2-(ethynyl)but-2-ene and (E)-hex-3-ene-1,5-diyne derivatives 6 and 10, respectively, were prepared by dicyclohexylcarbodiimide(DCC)-mediated esterification of tris(dodecyloxy)benzoic acid (4) with (E)-2-[(triisopropylsilyl)ethynyl]but-2-ene-1,4-diol (3) and (E)-2,3-bis[(trimethylsilyl)ethynyl]but-2-ene-1,4-diol (8), respectively, followed by deprotection with Bu4NF in wet THF (Schemes,1 and 2). Oligomerization reactions of diyne derivative 10 were attempted by treatment with the Hay catalyst in the presence of mono-alkyne 6 as an end-capping reagent. Under these conditions, only compound 7 resulting from the homocoupling of 6 (Scheme,1), and polymers of 10 were obtained due to the difference in reactivity of the alkyne groups in 6 and 10. In contrast, when phenylacetylene was used as the stopper, the oligomerization of 10 afforded a mixture of end-capped oligomers, from which 11,13 were isolated by column chromatography (Scheme,3). The poly(triacetylenes) (PTA) 16,18 were prepared in a similar manner starting from diol 8 and stearic acid (Schemes,4 and 5). Whereas the end-capped monomers and dimers 11, 12, 16, and 17 with pendant long alkyl chains do not exhibit any liquid-crystalline behavior, the trimeric derivatives 13 and 18 show mesomorphic properties, thus demonstrating that the poly(triacetylene) backbone can behave as a mesogenic unit. [source] Synthesis and conformational study of P -heterocyclic androst-5-ene derivativesHETEROATOM CHEMISTRY, Issue 1 2008Éva Frank The reactions of (20R)-3,-acetoxy-21-hydroxymethylpregn-5-en-20-ol (2) and (20R)-3,-acetoxypregn-5-ene-20,21-diol (11) with phenylphosphonic dichloride 3 and aryl dichlorophosphates 4,6 afforded novel types of P -heterocyclic androst-5-ene derivatives 7,10 and 12 as epimeric pairs. The diastereomers were separated by column chromatography and were characterized by NMR spectroscopy. Estimation of the stereostructures of the corresponding epimers by B3LYP/631G(d) DFT ab initio calculations suggested that the six-membered hetero ring in compounds 7b and 8a,10a adopts predominantly a chair conformation, with the P -substituents in their preferred orientation. The cyclic phosphonate moiety in 7a or 8b,10b, however, seems to exist as an equilibrium mixture of chair,distorted- boat or chair,chair forms. The theoretical calculations indicate that the conformational equilibrium is shifted toward the distorted- boat conformer for 7a, with a pseudoequatorial P -phenyl substituent, whereas for 8b,10b the chair conformer with an equatorial P -phenoxy group predominates. © 2008 Wiley Periodicals, Inc. Heteroatom Chem 19:7,14, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20372 [source] Tyrosinase inhibitors isolated from the roots of Paeonia suffruticosaINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 2 2010H. -Y. J. Cosmet. Sci., 60, 347,352 (May/June 2009) Accepted for publication November 6, 2008. Synopsis The inhibition of mushroom tyrosinase by Paeonia suffruticosa root-derived materials was evaluated. Six tyrosinase inhibitors were isolated by ethanol extraction, n -hexane, ethyl acetate, n -BuOH, and water partition, silica gel column chromatography, Sephadex LH-20, Lobar PR-8, and high-performance liquid chromatography methods, and they were identified as kaempferol (I), quercetin (II), mudanpioside B (III), benzoyloxypaeoniflorin (IV), mudanpioside H (V), and pentagalloyl-,-D-glucose (VI) on the basis of spectroscopic evidence. The inhibitory activities of compounds I to VI against mushroom tyrosinase were determined with IC50 values of 0.120, 0.108, 0.368, 0.453, 0.324, and 0.063 mM, respectively. The kinetic study indicated that all purified inhibitors acted competitively for the L-dopa binding site of the enzyme, with an exception of compound VI, which acted non-competitively. [source] Partial purification and characterisation of banana [Musa (AAA Group) ,Gros Michel'] polyphenol oxidaseINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 4 2009Chitsuda Chaisakdanugull Summary Polyphenol oxidase (PPO) from pulp of banana [Musa (AAA Group) ,Gros Michel'] was extracted and precipitated with 80% saturated ammonium sulphate followed by conventional column chromatography on Sephacryl S-200 HR and fast protein liquid chromatography on Mono Q column. The lyophilised PPO obtained from Sephacryl S-200 HR column was used for characterisation and inhibition studies. The partially purified PPO obtained from the Mono Q column exhibited at least three isoenzymes. The banana PPO had optimum pH for activity at 7 and it was stable around the same pH. Only 48% of initial enzyme activity was lost after heating at 70 °C for 30 min. The enzyme was completely inhibited by 2 mm sodium metabisulphite, 2 mm l -cysteine, 4 mm ascorbic acid, and 100 mm 4-hexylresorcinol. The Km and Vmax of banana PPO for dopamine were 2.08 mm and 0.124 mm min,1 respectively. [source] Partial purification of proteases that are generated by processing of the Northern shrimp Pandalus borealis and which can tenderize beefINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 5 2004Hitoshi Aoki Summary The crude extracts obtained from the heads of Northern shrimps, Pandalus borealis, (adapted to cold), showed considerable collagenolytic activities. When tested for beef tenderization, resulted in an overdegradation of meat proteins, which was detected organoleptically. Subsequently, four fractions with proteolytic activity were partially purified from the crude extracts by hydroxyapatite followed by MonoQ or Superdex 200 column chromatography. Warner-Bratzler shear force values of steaks treated with three protease fractions (Q, S2, S3) at 10 °C were significantly lower (P < 0.01) than that of the control and the enzyme preparations were completely inactivated after mild heat treatment. These results suggest that the potential for Northern shrimp enzymes to be used in industrial processes, particularly in the food industry, is quite large, where working at lower temperatures to prevent undesirable chemical reactions is necessary. [source] Facile Synthesis of Enantiopure 4-Substituted 2-Hydroxy-4- butyrolactones using a Robust Fusarium LactonaseADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 17 2009Bing Chen Abstract A facile chemo-enzymatic process has been developed for producing stereoisomers of 4-substituted 2-hydroxy-4-butyrolactones with good to excellent enantioselectivity. This process involves an easy separation of the diastereoisomers by column chromatography and efficient enzymatic resolution by whole cells of Escherichia coli JM109 expressing Fusarium proliferatum lactonase gene. This biocatalyst shows strong tolerance towards different substrate structures and at least three out four possible isomers could be obtained in excellent enantiomeric purity. Different substrate concentrations (10,mM,200,mM) were examined, giving a substrate to catalyst ratio of up to 26:1. This general and efficient enzymatic process provides access to stereoisomers of 4-substituted 2-hydroxy-4-butyrolactones readily and cost-effectively. The stereochemical assignments were conducted systematically based on NMR, X-ray diffraction and circular dichroism, leading to further understanding of the enzyme's stereoselectivity. [source] IDENTIFICATION OF PROCYANIDIN A2 AS POLYPHENOL OXIDASE SUBSTRATE IN PERICARP TISSUES OF LITCHI FRUITJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2007JIAN SUN ABSTRACT Postharvest browning of litchi fruit results in short shelf life and reduced commercial value. Experiments were conducted to separate, purify and identify polyphenol oxidase (PPO ) substrates that cause litchi fruit to brown. PPO and its substrate were extracted from the pericarp tissues of litchi fruit. The litchi PPO substrate was purified using polyamide column, silica gel column and Sephadex LH-20 column chromatography. The browning substrate was selected by a 0.5% FeCl3 solution and then identified using a partially purified litchi PPO. Analyses of ultraviolet spectrometry, nuclear magnetic resonance and electrospray ionization mass spectrometry indicated that the PPO substrate was procyanidin A2. The substrate can be oxidized to , -quinones by litchi PPO and then form brown-colored by-products, resulting in pericarp browning of harvested litchi fruit. [source] PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp.JOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2004ABSTRACT Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. ,-CD yield of CGTase from alkalophilic Bacillus species is usually much lower than ,-CD, while from alkalophilic Bacillus sp. 7-12. ,-CD yield is close to ,-CD. A CGTase from alkalophilic Bacillus sp. 7-12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and Sepharose CL-6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS-PAGE. The enzyme showed a Kmof 1.24 mg/mL and Vmax0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6,10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag+, Cu2+, Mg2+, Al3+, Co2+, Zn2+, Fe2+and slightly inhibited by Sn2+, Mn2+. The ions Ca2+and K+, EDTA and DTT had no influence on the enzyme activity. [source] PHENOLIC COMPOUNDS IN THE FLESH OF KOREAN APPLE CULTIVAR, BUSAJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2002HEA-JEUNG WHANG ABSTRACT Phenolic compounds were purified from Busa, the most widely cultivated apple cultivar in Korea, by a sequential separation employing PVPP, Ambertite XAD-2 and Sephadex LH-20 column chromatography. Phenolic compounds in some fractions were farther identified by directly comparing with corresponding phenolic standards after separation on HPLC and GC-MS. The phenolic compounds identified were p-hydoxybenzoic, protocatechuic, 4-hydroxymetyl-benzonic, caffeic, p-coumaric, o-coumaric, ferulic, sinapic and chlorogenic acids and (±)-catechin. Among them, 4-hydroxymethylbenzoic, protocatechuic, o-coumaric and sinapic acids were ascertained as new members of a family of phenolic acids present-in apples. Also the presence of rutin, quercetin and phlorizin in apples were demonstrated using HPLC. [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] Effect of Actinidin on the Protein Solubility, Water Holding Capacity, Texture, Electrophoretic Pattern of Beef, and on the Quality Attributes of a Sausage ProductJOURNAL OF FOOD SCIENCE, Issue 3 2009M. Aminlari ABSTRACT:, The objective of this study was to study the effect of actinidin, a sulfhydryl protease from kiwi fruit, on the protein solubility (nitrogen solubility index [NSI]), water holding capacity (WHC), texture, and SDS,PAGE pattern of beef and to evaluate the effect of pretreatment of beef with actinidin on the quality attributes of a sausage product. Actinidin was partially purified by precipitation with ammonium sulfate, followed by DEAE-Sephadex column chromatography. Actinidin significantly (P < 0.05) increased NSI and WHC of beef; the highest NSI and WHC (approximately 20% and 8% increase, respectively) was observed when beef was incubated with 0.9 unit enzyme/g beef. Texture analysis indicated increased tenderization (10% decrease in shear force) when slices of cattle beef were treated with actinidin at 37 °C for 2 h. SDS,PAGE results indicated appearance of several low molecular weight bands (<10 kDa) after treating beef with different levels of actinidin for 30 or 60 min. Slight changes in protein band in the range of 100 to 120 kDa and 13 to 25 kDa were also observed. Use of actinidin-tenderized beef significantly improved emulsion stability, texture, and organoleptic properties of the sausage product. [source] N -Cyanomethyl- N -Methyl-1-(3,,4,-methylenedioxyphenyl)-2-propylamine: An MDMA Manufacturing By-ProductJOURNAL OF FORENSIC SCIENCES, Issue 5 2008Helen Salouros B.Sc. Abstract:, This paper describes the structural elucidation of a compound produced during the synthesis of 3,4-methylenedioxymethylamphetamine (MDMA) via the reductive amination of 3,4-methylenedioxyphenyl-2-propanone (3,4-MDP-2-P) with methylamine and sodium cyanoborohydride. The compound was isolated from MDMA by column chromatography, proton and carbon nuclear magnetic resonance spectroscopy, LC/mass spectrometry, and total synthesis were used to identify the compound as N -cyanomethyl- N -methyl-1-(3,,4,-methylenedioxyphenyl)-2-propylamine. This compound has been identified as a potential synthetic route marker for the reductive amination of 3,4-MDP-2-P with methylamine and sodium cyanoborohydride and as such it should prove valuable to forensic scientists engaged in profiling illicit drugs. Profiling MDMA can provide useful information to law enforcement agencies relating to synthetic route, precursor chemicals and reagents employed and may be used for comparative analyses of different drug seizures. This paper also describes the structural elucidation of the analogous methylamphetamine synthetic route marker compound, N -cyanomethyl- N -methyl-1-phenyl-2-propylamine, produced during the reductive amination of phenyl-2-propanone using methylamine and sodium cyanoborohydride. [source] | |||||||||||||||||||||||||||||||||||||